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Lactic Acid Bacteria Isolated from Japanese Fermented Fish (Funa-Sushi) Inhibit Mesangial Proliferative Glomerulonephritis by Alcohol Intake with Stress

Lactic Acid Bacteria Isolated from Japanese Fermented Fish (Funa-Sushi) Inhibit Mesangial... Hindawi Journal of Nutrition and Metabolism Volume 2018, Article ID 6491907, 8 pages https://doi.org/10.1155/2018/6491907 Research Article Lactic Acid Bacteria Isolated from Japanese Fermented Fish (Funa-Sushi) Inhibit Mesangial Proliferative Glomerulonephritis by Alcohol Intake with Stress 1 2 3 4 Yumiko Yamada, Masumi Endou, Shunichi Morikawa, Jun Shima, and Noriko Komatshzaki Nodakamada Gakuen, 389-1 Noda, Noda, Chiba 278-0037, Japan Department of Human Nutrition, Seitoku University, 550 Iwase, Matsudo, Chiba 271-8555, Japan Department of Anatomy and Developmental Biology, Tokyo Women’s Medical University, 8-1 Kawada-Cho, Shinjuku, Tokyo 162-8666, Japan Faculty of Agriculture, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194, Japan Correspondence should be addressed to Noriko Komatshzaki; norikoma@seitoku.ac.jp Received 10 August 2017; Revised 25 November 2017; Accepted 12 December 2017; Published 11 February 2018 Academic Editor: Stan Kubow Copyright © 2018 Yumiko Yamada et al. ,is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ,e aim of this study was to examine the effect of heat-killed Lactobacillus paracasei NFRI 7415 on kidney and bone in mice fed an ethanol-containing diet with stress. Eight-week-old Cril : CD1 mice were fed a control diet (CD), an alcohol diet (AD) (35.8% of total energy from ethanol), or an alcohol diet containing 20% heat-killed Lb. paracasei NFRI 7415 (10 CFU/g) (LD) for 4 weeks. Mice in the AD and LD groups also underwent restraint stress for two weeks from 13 days. ,e mice were placed in a 50 mL plastic tube, which had a small hole drilled around its base to allow ventilation, and restrained for 1 h every day. High final body weight was in the following order: CD, LD, and AD (p< 0.05). ,e heat-killed Lb. paracasei NFRI 7415 lowered liver total cholesterol concentration and plasma glutamic-oxaloacetic transaminase (GOT) level. In addition, fecal bile acids of the LD group were higher than in the AD group (p< 0.05). ,e glomerulus of the kidney in the AD group was observed to be more fibrotic than in the CD and LD groups with azan stain. Immunostaining confirmed that brown areas indicating the existence of mesangial cells were increased in the AD group, but not in the CD and LD groups. ,ese results indicated that the heat-killed Lb. paracasei NFRI 7415 inhibited mesangial proliferative glomerulonephritis by alcohol intake with stress. food fermentation, and typical examples can be found in the 1. Introduction dairy industry for the production of cheese, yogurt, and People are subjected to many stressors in modern life, in- other fermented milk products [7, 8]. cluding both physical and mental stresses. Excessive stress Recent studies have indicated that several LAB are ef- can cause psychosomatic disorders, and sometimes death fective as probiotics for the prevention of osteoporosis and from overwork [1]. In recent years, lifestyle-related diseases chronic nephritis. For example, Bifidobacterium longum are in most cases caused by overwork, excessive stress, sleep alleviated bone loss in ovariectomized rats and enhanced deprivation, smoking, and drinking [2]. It is well known that bone mass density [9]. It was reported that yogurt con- chronic ethanol consumption induces osteoporosis and sumption retarded chronic kidney disease progression [10]. chronic nephritis [3–5]. Lactobacillus paracasei NFRI 7415, isolated from tradi- Lactic acid bacteria (LAB) have been utilized as a natural tional Japanese fermented fish (funa-sushi), showed high health food since ancient times, and the health-promoting c-aminobutyric acid (GABA)-producing ability [11]. We effects of LAB are well recognized [6]. Some LAB are used in reported that Lb. paracasei NFRI 7415 removed cholesterol 2 Journal of Nutrition and Metabolism Table 1: Composition of experimental diets. from the plasma and liver of rats fed an ethanol-containing diet [12]. Moreover, it was shown that this strain reduced the 1 2 3 Ingredient (g/L) CD AD LD content of liver lipids in C57BL/6J mice fed a high-fat diet Casein 41.4 41.4 41.4 [13]. We speculated that Lb. paracasei NFRI 7415 might have L-cystine 0.5 0.5 0.5 improved liver function in the abovementioned clinical DL-methionine 0.3 0.3 0.3 study by somehow reducing hepatic lipid content. To the Corn oil 8.5 8.5 8.5 best of our knowledge, no study has investigated the effect of Lb. paracasei in the kidney of rat with stress and chronic Olive oil 28.4 28.4 28.4 ethanol consumption. Safflower oil 2.7 2.7 2.7 ,e aim of the present study was to examine the effect of Vitamin mixture 2.5 2.5 2.5 heat-killed Lb. paracasei NFRI 7415 on the kidney and bone Mineral mixture 8.75 8.75 8.75 in mice fed an ethanol-containing diet with stress. We in- Maltose dextrin mixture 115.2 25.6 25.6 vestigated body and fat tissue weight, as well as calcium in Cellulose 10.0 10.0 10.0 the bone and tissues of the kidney in mice. Symptoms as- Choline bitartrate 0.53 0.53 0.53 sociated with glomerulonephritis are hyperlipidemia and proteinuria [14]. It has been reported that supply of amino Xanthan gum 3.0 3.0 3.0 acid-fortified low-protein diets to nephritic rats improved Ethanol — 50.0 50.0 their symptoms, and fecal bile acid excretion was enhanced Lb. paracasei extract — — (10 CFU/g) [15]. ,us, we focused on the effect of fecal bile acid ex- 1 2 3 CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol cretion by Lb. paracasei NFRI 7415 and investigated the fecal diet; vitamin mixture (g/kg of mix): retinal, 4.8; cholecalciferol, 0.4; thi- lipids of mice. amine, 24.0; riboflavin, 0.6; pantothenic acid, 0.6; pyridoxine, 0.7; co- balamin, 0.01; menadione, 0.05; nicotinic acid, 3.0; D-calcium pantothenic acid, 1.6; folic acid, 0.2; biotin, 0.02; para-aminobenzoic acid, 5.0; inositol, 2. Materials and Methods 10.0; glucose, 949.02; mineral mixture (g/kg of mix): CaHPO , 500.0; NaCl, 74.0; K C H O ·H O, 220.0; K SO , 52.0; MGO, 24.0; MnSO ·5H O, 6.77; 3 6 5 7 2 2 4 4 2 2.1. Preparation of Extract. A preculture of Lb. paracasei FeSO ·7H O, 4.95; ZnCO , 1.6; CuCO Cu(OH) H O, 0.3; KlO , 0.01; 4 2 3 3 2 2 3 ° NaSeO , 0.01; CrK(SO ) ·12H O, 0.55; NaF, 0.06; sucrose, 115.75. 3 4 2 2 NFRI 7415 was grown to the stationary phase at 37 C for 20 h in MRS agar medium (Difco Laboratories, Detroit, MI). ,e medium was prepared by mixing an alcohol diet (AD) and sterilized water at a ratio of 1 : 3. ,e precultures (10 CFU/g) were inoculated in 0.4 liters of AD at 37 C for 48 h. ,e medium was then heated at 100 C for 30 min and used in the animal experiments. 2.2. Animals and Diets. Twenty-four 8-week-old Cril : CD1 mice were purchased from Charles River Japan (Yokohama, Japan). All animals were housed individually in plastic cages in a controlled environment of 22± 1 C at 50% relative humidity under a 12 h dark/light cycle (19:00–7:00). ,e animals were randomly divided into three dietary treatment groups with equal mean body weight: the control diet (CD) group (n � 8), the alcohol diet (AD) group (n � 8), and the heated medium with Lb. paracasei NFRI 7415 (10 CFU/g) Figure 1: Restraint stress. Mice were placed in a 50 ml plastic tube, and alcohol diet bended at a ratio of 1 : 4 (LD) group (n � 8). which had a small hole drilled around its base to allow ventilation, Composition of the liquid diets (CD, AD, and LD) is and restrained for 1 h every day. given in Table 1; diets were formulated with reference to the Lieber–DeCarli diet [16]. All liquid diets were freshly pre- pared on alternate days. ,e mice were fed the CD, AD, or tissue. ,e blood was collected by heart puncture with LD for 4 weeks. Food intake was recorded daily, and body a heparinized syringe. ,e blood was maintained at 4 C and weight was measured on alternate days. ,e mice of AD and centrifuged at 1,000 g for 15 min; the plasma and liver were LD group were subjected to restraint stress for two weeks stored at −80 C until analysis. All procedures were per- from day 13 after feeding. Restraint stress was made with formed in accordance with the Animal Experimentation reference to the method of De Francesco et al. [17]. ,e mice Guidelines of the Laboratory Animal Care Committee of were placed in a 50 mL plastic tube, which had a small hole Seitoku University (approval number 179). drilled around its base to allow ventilation, and restrained for 1 h every day (Figure 1). ,en, experimental mice were returned to their home cages. After the feeding period, the 2.3. Biochemical Assays of Plasma and Liver. Liver lipids mice were fasted for 16 h and sacrificed humanely under were extracted by the method described in the work of Folch ether anesthesia to collect the liver, kidney, and perirenal fat et al. [18]. Triacylglycerol (TG), total cholesterol (T-cho), Journal of Nutrition and Metabolism 3 HDL-cholesterol, glutamic-oxaloacetic transaminase (GOT), solution, and Mallory’s aniline blue orange G stain solution). and glutamic-pyruvic transaminase (GPT) in plasma and liver Samples were then examined under a microscope (Olympus extracts were measured using test kits (Triacylglycerol E-Test, CH20 with Shimazu Moticam 580). Cholesterol E-Test, HDL-cholesterol E-Test, and Transaminase CII-Test, Wako Pure Chemical Industries, Osaka, Japan). 2.7. Immunostaining. Deparaffinized and rehydrated 3 μm paraffin sections of formaline-fixed kidneys were enzyme- treated by protease. ,e primary antibody used for this 2.4. Assays of Ash, Calcium, and Phosphorus. ,e right femur study was desmin (West Grove, PA, USA). Secondary anti- was taken and weighed after removing the meat. Ash content ° bodies used peroxidase-conjugated AffiniPure Donkey Anti- in the femur was first treated by drying at 105 C for 48 h, Rabbit IgG (H + L), purchased from Jackson ImmunoResearch then defatted by absolute ether for 3 days. Defatted bone was (West Grove, PA, USA). Liquid DAB + Substrate Chromogen treated by ashing process at 550 C overnight in an electric System (Dako, Tokyo, Japan) were used for the color reaction, furnace. ,e ash in the femur was measured with the weight after counterstaining with hematoxylin. For confirmation of the method [19]. Ash was then added to 10 mL of 2 N HCl, and mesangial proliferative glomerulonephritis by inducing ethanol calcium and phosphorus in ash were measured with test kits intake and restraint stress, the number of the mesangial cells was (Calcium E-Test and Phospha C-Test, Wako Pure Chemical counted in the 65 glomerulus of the kidney. Industries, Osaka, Japan). 2.8. Statistical Analysis. Values were expressed as mean ±SD. 2.5. Assay of Fecal Lipids and Fecal Cholesterol. Here, 0.1 g of Repeated-measures analysis of variance (ANOVA) was used to homogenized dry fecal matter was added to 4 mL of con- evaluate the effects of groups. Differences in mean values centrated sulfuric acid in test tubes for 30 min at room between groups were tested by Scheffe multiple-range test. A p temperature. Diethyl ether was added to reach 25 mL, and value of less than 0.05 was considered statistically significant. mixed. ,e diethyl ether-containing layer was moved to a flask, and the diethyl ether was evaporated. ,e fecal lipid 3. Results in the flask was then weighed. ,e T-cho concentration in fecal matter was determined in the same way as in the liver 3.1. Food Intake, Total Energy Intake, Body Weight, Liver, T-cho concentration. Kidney, and Perirenal Fat Tissue Weight. No significant Fecal bile acids were measured by the procedure de- differences in liver and kidney weights were observed among scribed in the work of Iwami et al. [20]; 10 mg of the sample the three groups. Total energy intake and perirenal fat tissue was mixed with 0.2 mL of 90% ethanol during vortex mixing weight of the AD and LD groups were lower than in the CD and incubated for 1 h at 65 C. ,e mixture was subjected to group (p< 0.05) (Table 2). Figure 2 shows the body weight of centrifugation at 5,000 rpm for 3 min. ,e supernatant was mice during the experiment. ,e final body weight of mice was transferred to a 1.5 mL tube, and the ethanol was evaporated. high in the following order: CD, LD, and AD (p< 0.05). After ,en, 0.2 mL of 90% ethanol was added in the precipitate for the mice in the AD and LD groups underwent restraint stress vortex mixing. ,e sample was dissolved in 1 mL of 90% from 13 days, weight between the AD and LD groups increased. ethanol and measured using test kits (total bile acid test by enzyme colorimetric method, Wako pure Chemical In- dustries, Osaka, Japan). 3.2. Plasma Lipids Profiles and Liver Lipids. Although no significant differences in plasma TG concentration were observed between the three groups, the plasma T-cho and 2.6. Kidney Histology. Kidneys of mice from the three di- HDL-cholesterol concentrations of the AD group were lower etary treatment groups were compared histologically. Under than those of the CD and LD groups (p< 0.05) (Table 3). No deep anesthesia with ether, the chest of a mouse from the differences were observed in liver total lipids and liver TG three groups was opened rapidly and the vasculature was concentrations between groups, but liver T-cho concen- perfused with 50 mL of a fixative (4% paraformaldehyde in trations of the CD and LD groups were lower than those of 0.01 M sodium phosphate-buffered saline (PBS: pH 7.4)) at the AD group (p< 0.05) (Table 3). ,e plasma GOT level of a pressure of 120 mmHg from a 18-gauge cannula inserted the AD group was higher than those of the CD and LD into the aorta via an incision in the left ventricle. Imme- groups (p< 0.05) (Figure 3). In addition, the plasma GPT diately after fixative perfusion, the kidney was removed, cut level of the AD group was higher than that of the CD group into small pieces, and immersed in the same fixative (p< 0.05) (Figure 3). overnight at 4 C. Kidney pieces were then washed with PBS, dehydrated in an ascending series of ethanol aqueous so- lutions (70%, 80%, 90%, and 100%), cleared in xylene, and 3.3. Bone Contents, Plasma Calcium, and Fecal Lipids. ,e embedded in paraffin wax. ,ree-micrometer-thick sections ash content in the bone of the AD group was lower than of paraffin-embedded kidneys were then subjected to he- those in the CD and LD groups, although there were no matoxylin and eosin (H&E) staining by a routine procedure significant differences in right femur weight, calcium, or (Meyer’s hematoxylin staining, followed by eosin Y staining) phosphorus in the bone between the groups (p< 0.05) and azan staining by a routine procedure (Mordant, Mal- (Table 4). ,ere was also no difference in plasma calcium lory’s azocarmine G solution, 5% phosphotungstic acid between groups. No significant differences were observed 4 Journal of Nutrition and Metabolism Table 2: Food intake, total energy intake, body weight, and liver, kidney, and perirenal fat tissue weight. 1 2 3 Group CD AD LD a b b Food intake (g/day) 17.2± 0.66 10.9± 0.95 11.0± 0.91 a b b Total energy intake (Kcal) 2040± 780 1382± 121 1400± 116 a b b Total energy intake (Kcal/day) 75.5± 2.89 51.2± 4.48 51.9± 4.29 a c b Final body weight (g) 40.2± 1.39 29.8± 2.73 34.5± 3.41 Liver weight (g/100 g BW) 4.45± 1.54 4.85± 1.01 5.05± 1.86 Kidney weight (g/100 g BW) 0.59± 0.08 0.74± 0.08 0.68± 0.22 a b b Perirenal fat tissue weight (g/100 g BW) 0.88± 0.19 0.33± 0.12 0.34± 0.21 1 2 3 CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol diet. Values represent mean±SD, n � 8. Values not sharing a common superscript letter are significantly different at p< 0.05. tissue weights of the AD and LD groups were lower than that of the CD group (p< 0.05) (Table 2). It is known that chronic alcohol intake and excessive stress accelerate energy meta- bolism in the body [21]. ,e final body weight of the AD group demonstrated frequently observed symptoms such as weight loss compared to be in the LD group. Intake of Lb. paracasei NFRI 7415 may have prevented enhanced energy metabolism by alcohol intake and excessive stress. Fermented dairy products such as yogurt utilizing LAB have been reported to lower serum cholesterol concentrations in animals [22]. We also reported that live Lb. paracasei NFRI 0 5 10 15 20 25 30 7415 reduced the plasma T-cho and hepatic T-cho concen- Feeding period (day) tration in rats fed an ethanol-containing diet [12]. In this study, LAB were not found to lower plasma lipids. However, in terms Control diet of cholesterol-reducing activity, effects against liver lipids and Alcohol diet Lb. paracasei-containing alcohol diet the plasma GOT level were observed in the LD group receiving heat-killed Lb. paracasei NFRI 7415. Plasma GOTand GPTare Figure 2: Body weight of mice during the experiment (n � 8). enzymes recognized as indicators of hepatitis, liver cirrhosis, Arrow: start of restraint stress. and cardiac infarction [23]. Levels of these enzymes increase with liver cell damage, as in hepatitis. ,us, these results in fecal T-cho concentrations, but the fecal bile acids of the indicate that heat-killed Lb. paracasei NFRI 7415 decreased the AD group were lower than those of the CD and LD groups plasma GOT level and liver cholesterol concentration caused (p< 0.05) (Table 4). by chronic alcohol consumption. As described in our previous paper, the live Lb. paracasei NFRI 7415 has the capacity to lower fecal T-cho excretion, 3.4. Kidney Histology. ,ere were no differences in kidney and it effectively reduced plasma T-cho concentration [24]. tissue between the three groups in H&E staining (data not Although no significant difference in fecal T-cho concen- shown). However, more fibrosis was observed in the glo- trations were observed here, the bile acid level of feces in the merulus of the kidney in the AD group than in the other two LD group increased (Table 4). ,is increase in the bile acid groups in azan stain (Figures 4(a)–4(c)). Immunostaining level appears to have been due to bile acid adsorption by LAB results confirmed that the brown areas indicating the ex- in the intestine [25, 26]. Hence, it suggested that heat-killed istence of mesangial cells were increased in the AD group Lb. paracasei NFRI 7415 had strong bile acid adsorption but was almost nonexistent in the CD and LD groups ability in feces. (Figures 4(d)–4(f)). ,e average of the number of mesangial Restraint stress load causes osteoporosis, due to an in- cells in the glomerulus of the CD, AD, and LD groups was crease in the corticosteroid hormone secreted by the cortex 3.08± 1.02, 4.06± 1.07, and 3.39± 0.90, respectively. ,e of the adrenal gland, and increases bone resorption [27]. number of mesangial cells in the AD group was significantly Heavy alcohol consumption has been associated with in- higher than those of the CD and LD groups (p< 0.001). creased risk of bone fracture [3]. It is known that osteo- porotic bones are more likely to fracture, and it is important 4. Discussion to determine the bone content in mice fed alcohol- We found that live Lb. paracasei NFRI 7415 is beneficial for containing diets with restraint stress. improving liver damage due to chronic alcohol intake [12]. In this experiment, the ash content in the bone of the AD In the current study, we investigated whether heat-killed Lb. group was lower than those of the other two groups (p< 0.05) paracasei NFRI 7415 influenced kidney and bone in mice fed (Table 4). Although there were no significant differences in an ethanol-containing diet with stress. ,e perirenal fat right femur weight, or in calcium and phosphorus in bone, Body weight (g) Journal of Nutrition and Metabolism 5 Table 3: Plasma lipids and liver lipids. 1 2 3 Group CD AD LD Plasma lipids Triacylglycerol (mg/dL) 143.5± 23.8 141.0± 42.0 176.3± 91.6 a b a Total cholesterol (mg/dL) 167.4± 33.8 126.2± 14.8 179.2± 25.5 a b a HDL-cholesterol (mg/dL) 127.9± 18.7 90.9± 12.9 120.8± 23.8 Liver lipids Total fat (mg/g) 75.4± 18.4 76.9± 25.3 79.0± 16.4 Triacylglycerol (mg/g) 27.5± 12.4 28.6± 12.6 33.3± 12.0 ab a b Total cholesterol (mg/g) 8.69± 3.46 11.1± 3.60 6.37± 1.80 1 2 3 CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol diet. Values represent mean±SD, n � 8. Values not sharing a common superscript letter are significantly different at p< 0.05. 80 80 70 70 60 60 ab 50 b 50 40 40 30 30 20 20 10 10 0 0 CD AD LD CD AD LD Figure 3: Plasma GOT level and plasma GPT level concentration of mice fed experimental diets. CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol diet. Values represent mean ±SD; n � 8. Values not sharing a common superscript letter are significantly different at p< 0.05. Table 4: Bone content, plasma calcium, and fecal lipids. 1 2 3 Group CD AD LD Bone contents Right femur weight (dry) (mg) 57.4± 6.23 53.0± 7.85 56.1± 5.56 Right femur weight (defatted) (mg) 53.6± 6.0 49.3± 6.76 52.3± 5.30 a b a Ash (%) 53.0± 1.44 50.9± 2.13 54.0± 2.67 Calcium (mg/g) 13.3± 0.85 12.7± 1.07 13.0± 0.77 Phosphorus (mg/g) 15.7± 1.23 14.6± 1.34 15.4± 0.92 Plasma calcium (mg/g) 3.09± 0.58 4.06± 0.62 3.54± 1.46 Fecal lipids a b ab Total fat (mg/g) 48.2± 9.30 29.8± 10.9 39.9± 9.77 a b a Bile acid (μg/g) 3.54± 0.99 2.38± 0.56 3.91± 0.74 Total cholesterol (mg/g) 1.87± 0.43 1.89± 0.56 1.90± 0.41 1 2 3 CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol diet. Values represent mean±SD, n � 8. Values not sharing a common superscript letter are significantly different at p< 0.05. Plasma GOT level (mg/dL) Plasma GPT level (mg/dL) 6 Journal of Nutrition and Metabolism (a) (b) (c) 10μm (d) (e) (f) Figure 4: Mesangiolysis in mice. (a)–(c) Azan staining of the glomerulus in the kidney. (a) ,e glomerulus in the kidney from a normal mouse fed with control diet (CD). Blue regions (white arrows) show collagen fiber. (b) ,e glomerulus in the kidney from a mouse fed with alcohol diet (AD). Blue regions (white arrows) show collagen fiber, which are increased compared to (a). (c) ,e glomerulus in the kidney from a mouse fed with AD containing Lb. paracasei. Blue regions (white arrows) show collagen fiber, which are reduced compared to (b). (d)–(f) Immunostaining of the glomerulus in the kidney. (d) ,e glomerulus in the kidney from a normal mouse fed CD. A mesangial cell (black arrow) is stained by desmin. (e) ,e glomerulus in the kidney from a mouse fed with AD. ,e number of mesangial cells (black arrows) is higher than in the normal kidney (d). (f) ,e glomerulus in the kidney from a mouse fed AD containing Lb. paracasei. ,e number of mesangial cells (black arrow) is fewer than that of animals fed AD (e). each average value of the AD group was lower than those of It was observed that the AD group had lower urine the CD and LD groups. For example, the right femur weight volume than the CD group during the experimental period. (dry) of the CD, AD, and LD groups was 57.4± 6.23 mg, Kidney function weakness was therefore suspected in the AD 53.0± 7.85 mg, and 56.1± 5.56 mg, respectively. Moreover, group. Fibrosis of the organization occurs to the organ that the plasma calcium of CD, AD, and LD groups were 3.09± the whole body is approximately important except the brain. 0.58 mg, 4.06± 0.62 mg, and 3.54± 1.46 mg, respectively. ,e organ that caused the fibrosis will eventually mal- ,ese results indicate that some minerals were eluted from the function. ,is means that renal function decreases as bone of AD group, promoting bone resorption. On the other kidney fibrosis advances [29]. We therefore performed hand, the plasma calcium of the LD group tended to be lower, azan staining to observe fibrosis of the kidney. ,e suggesting that bone resorption was inhibited by LAB. It was mesangium domain in the glomerulus of the kidney reported that probiotic yogurt containing strains of Lacto- consists of mesangium cells and mesangium substrates, bacillus casei, Lactobacillus reuteri, and Lactobacillus gasseri including type IV collagen. Collagen fiber is stained blue by increased apparent calcium absorption and bone mineral azan staining. An increase of the mesangium domain was content in rats [28]. ,ese LAB strains produce certain confirmed, by the increase in blue in the glomerulus of the prebiotics such as oligosaccharides that help new bone tissue AD group. We then attempted immunostaining using the to grow. Further investigation of the effects of live Lb. par- desmin antibody, which can specifically stain mesangial acasei NFRI 7415 as probiotics is needed. cells. Many more mesangium cells, colored brown by Journal of Nutrition and Metabolism 7 resulting from ovariectomy,” BioMed Research International, immunostaining, were observed in the AD group com- vol. 2015, Article ID 897639, 10 pages, 2015. pared to the CD and LD groups. [10] R. Yacoub, D. Kaji, S. N. Patel et al., “Association between ,ere are always mesangial cells in the glomerulus of the probiotic and yogurt consumption and kidney disease: in- kidney. ,ey maintain capillary vessels of the glomerulus of sights from NHANES,” Nutrition Journal, vol. 15, p. 10, 2016. the kidney and work to regulate its ability to filter the blood. [11] N. Komatsuzaki, J. Shima, S. Kawamoto, H. Momose, and Most chronic glomerulonephritis involves mesangial pro- T. Kimura, “Production of γ-aminobutyric acid (GABA) by liferative glomerulonephritis (MesPGN), for which the char- Lactobacillus paracasei isolated from traditional fermented acteristic is an increase of mesangial cells [30]. ,e causes of foods,” Food Microbiology, vol. 22, no. 6, pp. 497–504, 2005. MesPGN development remain to be elucidated. It is thought, [12] N. Komatsuzaki and J. 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Lactic Acid Bacteria Isolated from Japanese Fermented Fish (Funa-Sushi) Inhibit Mesangial Proliferative Glomerulonephritis by Alcohol Intake with Stress

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Copyright © 2018 Yumiko Yamada et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2018/6491907
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Hindawi Journal of Nutrition and Metabolism Volume 2018, Article ID 6491907, 8 pages https://doi.org/10.1155/2018/6491907 Research Article Lactic Acid Bacteria Isolated from Japanese Fermented Fish (Funa-Sushi) Inhibit Mesangial Proliferative Glomerulonephritis by Alcohol Intake with Stress 1 2 3 4 Yumiko Yamada, Masumi Endou, Shunichi Morikawa, Jun Shima, and Noriko Komatshzaki Nodakamada Gakuen, 389-1 Noda, Noda, Chiba 278-0037, Japan Department of Human Nutrition, Seitoku University, 550 Iwase, Matsudo, Chiba 271-8555, Japan Department of Anatomy and Developmental Biology, Tokyo Women’s Medical University, 8-1 Kawada-Cho, Shinjuku, Tokyo 162-8666, Japan Faculty of Agriculture, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194, Japan Correspondence should be addressed to Noriko Komatshzaki; norikoma@seitoku.ac.jp Received 10 August 2017; Revised 25 November 2017; Accepted 12 December 2017; Published 11 February 2018 Academic Editor: Stan Kubow Copyright © 2018 Yumiko Yamada et al. ,is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ,e aim of this study was to examine the effect of heat-killed Lactobacillus paracasei NFRI 7415 on kidney and bone in mice fed an ethanol-containing diet with stress. Eight-week-old Cril : CD1 mice were fed a control diet (CD), an alcohol diet (AD) (35.8% of total energy from ethanol), or an alcohol diet containing 20% heat-killed Lb. paracasei NFRI 7415 (10 CFU/g) (LD) for 4 weeks. Mice in the AD and LD groups also underwent restraint stress for two weeks from 13 days. ,e mice were placed in a 50 mL plastic tube, which had a small hole drilled around its base to allow ventilation, and restrained for 1 h every day. High final body weight was in the following order: CD, LD, and AD (p< 0.05). ,e heat-killed Lb. paracasei NFRI 7415 lowered liver total cholesterol concentration and plasma glutamic-oxaloacetic transaminase (GOT) level. In addition, fecal bile acids of the LD group were higher than in the AD group (p< 0.05). ,e glomerulus of the kidney in the AD group was observed to be more fibrotic than in the CD and LD groups with azan stain. Immunostaining confirmed that brown areas indicating the existence of mesangial cells were increased in the AD group, but not in the CD and LD groups. ,ese results indicated that the heat-killed Lb. paracasei NFRI 7415 inhibited mesangial proliferative glomerulonephritis by alcohol intake with stress. food fermentation, and typical examples can be found in the 1. Introduction dairy industry for the production of cheese, yogurt, and People are subjected to many stressors in modern life, in- other fermented milk products [7, 8]. cluding both physical and mental stresses. Excessive stress Recent studies have indicated that several LAB are ef- can cause psychosomatic disorders, and sometimes death fective as probiotics for the prevention of osteoporosis and from overwork [1]. In recent years, lifestyle-related diseases chronic nephritis. For example, Bifidobacterium longum are in most cases caused by overwork, excessive stress, sleep alleviated bone loss in ovariectomized rats and enhanced deprivation, smoking, and drinking [2]. It is well known that bone mass density [9]. It was reported that yogurt con- chronic ethanol consumption induces osteoporosis and sumption retarded chronic kidney disease progression [10]. chronic nephritis [3–5]. Lactobacillus paracasei NFRI 7415, isolated from tradi- Lactic acid bacteria (LAB) have been utilized as a natural tional Japanese fermented fish (funa-sushi), showed high health food since ancient times, and the health-promoting c-aminobutyric acid (GABA)-producing ability [11]. We effects of LAB are well recognized [6]. Some LAB are used in reported that Lb. paracasei NFRI 7415 removed cholesterol 2 Journal of Nutrition and Metabolism Table 1: Composition of experimental diets. from the plasma and liver of rats fed an ethanol-containing diet [12]. Moreover, it was shown that this strain reduced the 1 2 3 Ingredient (g/L) CD AD LD content of liver lipids in C57BL/6J mice fed a high-fat diet Casein 41.4 41.4 41.4 [13]. We speculated that Lb. paracasei NFRI 7415 might have L-cystine 0.5 0.5 0.5 improved liver function in the abovementioned clinical DL-methionine 0.3 0.3 0.3 study by somehow reducing hepatic lipid content. To the Corn oil 8.5 8.5 8.5 best of our knowledge, no study has investigated the effect of Lb. paracasei in the kidney of rat with stress and chronic Olive oil 28.4 28.4 28.4 ethanol consumption. Safflower oil 2.7 2.7 2.7 ,e aim of the present study was to examine the effect of Vitamin mixture 2.5 2.5 2.5 heat-killed Lb. paracasei NFRI 7415 on the kidney and bone Mineral mixture 8.75 8.75 8.75 in mice fed an ethanol-containing diet with stress. We in- Maltose dextrin mixture 115.2 25.6 25.6 vestigated body and fat tissue weight, as well as calcium in Cellulose 10.0 10.0 10.0 the bone and tissues of the kidney in mice. Symptoms as- Choline bitartrate 0.53 0.53 0.53 sociated with glomerulonephritis are hyperlipidemia and proteinuria [14]. It has been reported that supply of amino Xanthan gum 3.0 3.0 3.0 acid-fortified low-protein diets to nephritic rats improved Ethanol — 50.0 50.0 their symptoms, and fecal bile acid excretion was enhanced Lb. paracasei extract — — (10 CFU/g) [15]. ,us, we focused on the effect of fecal bile acid ex- 1 2 3 CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol cretion by Lb. paracasei NFRI 7415 and investigated the fecal diet; vitamin mixture (g/kg of mix): retinal, 4.8; cholecalciferol, 0.4; thi- lipids of mice. amine, 24.0; riboflavin, 0.6; pantothenic acid, 0.6; pyridoxine, 0.7; co- balamin, 0.01; menadione, 0.05; nicotinic acid, 3.0; D-calcium pantothenic acid, 1.6; folic acid, 0.2; biotin, 0.02; para-aminobenzoic acid, 5.0; inositol, 2. Materials and Methods 10.0; glucose, 949.02; mineral mixture (g/kg of mix): CaHPO , 500.0; NaCl, 74.0; K C H O ·H O, 220.0; K SO , 52.0; MGO, 24.0; MnSO ·5H O, 6.77; 3 6 5 7 2 2 4 4 2 2.1. Preparation of Extract. A preculture of Lb. paracasei FeSO ·7H O, 4.95; ZnCO , 1.6; CuCO Cu(OH) H O, 0.3; KlO , 0.01; 4 2 3 3 2 2 3 ° NaSeO , 0.01; CrK(SO ) ·12H O, 0.55; NaF, 0.06; sucrose, 115.75. 3 4 2 2 NFRI 7415 was grown to the stationary phase at 37 C for 20 h in MRS agar medium (Difco Laboratories, Detroit, MI). ,e medium was prepared by mixing an alcohol diet (AD) and sterilized water at a ratio of 1 : 3. ,e precultures (10 CFU/g) were inoculated in 0.4 liters of AD at 37 C for 48 h. ,e medium was then heated at 100 C for 30 min and used in the animal experiments. 2.2. Animals and Diets. Twenty-four 8-week-old Cril : CD1 mice were purchased from Charles River Japan (Yokohama, Japan). All animals were housed individually in plastic cages in a controlled environment of 22± 1 C at 50% relative humidity under a 12 h dark/light cycle (19:00–7:00). ,e animals were randomly divided into three dietary treatment groups with equal mean body weight: the control diet (CD) group (n � 8), the alcohol diet (AD) group (n � 8), and the heated medium with Lb. paracasei NFRI 7415 (10 CFU/g) Figure 1: Restraint stress. Mice were placed in a 50 ml plastic tube, and alcohol diet bended at a ratio of 1 : 4 (LD) group (n � 8). which had a small hole drilled around its base to allow ventilation, Composition of the liquid diets (CD, AD, and LD) is and restrained for 1 h every day. given in Table 1; diets were formulated with reference to the Lieber–DeCarli diet [16]. All liquid diets were freshly pre- pared on alternate days. ,e mice were fed the CD, AD, or tissue. ,e blood was collected by heart puncture with LD for 4 weeks. Food intake was recorded daily, and body a heparinized syringe. ,e blood was maintained at 4 C and weight was measured on alternate days. ,e mice of AD and centrifuged at 1,000 g for 15 min; the plasma and liver were LD group were subjected to restraint stress for two weeks stored at −80 C until analysis. All procedures were per- from day 13 after feeding. Restraint stress was made with formed in accordance with the Animal Experimentation reference to the method of De Francesco et al. [17]. ,e mice Guidelines of the Laboratory Animal Care Committee of were placed in a 50 mL plastic tube, which had a small hole Seitoku University (approval number 179). drilled around its base to allow ventilation, and restrained for 1 h every day (Figure 1). ,en, experimental mice were returned to their home cages. After the feeding period, the 2.3. Biochemical Assays of Plasma and Liver. Liver lipids mice were fasted for 16 h and sacrificed humanely under were extracted by the method described in the work of Folch ether anesthesia to collect the liver, kidney, and perirenal fat et al. [18]. Triacylglycerol (TG), total cholesterol (T-cho), Journal of Nutrition and Metabolism 3 HDL-cholesterol, glutamic-oxaloacetic transaminase (GOT), solution, and Mallory’s aniline blue orange G stain solution). and glutamic-pyruvic transaminase (GPT) in plasma and liver Samples were then examined under a microscope (Olympus extracts were measured using test kits (Triacylglycerol E-Test, CH20 with Shimazu Moticam 580). Cholesterol E-Test, HDL-cholesterol E-Test, and Transaminase CII-Test, Wako Pure Chemical Industries, Osaka, Japan). 2.7. Immunostaining. Deparaffinized and rehydrated 3 μm paraffin sections of formaline-fixed kidneys were enzyme- treated by protease. ,e primary antibody used for this 2.4. Assays of Ash, Calcium, and Phosphorus. ,e right femur study was desmin (West Grove, PA, USA). Secondary anti- was taken and weighed after removing the meat. Ash content ° bodies used peroxidase-conjugated AffiniPure Donkey Anti- in the femur was first treated by drying at 105 C for 48 h, Rabbit IgG (H + L), purchased from Jackson ImmunoResearch then defatted by absolute ether for 3 days. Defatted bone was (West Grove, PA, USA). Liquid DAB + Substrate Chromogen treated by ashing process at 550 C overnight in an electric System (Dako, Tokyo, Japan) were used for the color reaction, furnace. ,e ash in the femur was measured with the weight after counterstaining with hematoxylin. For confirmation of the method [19]. Ash was then added to 10 mL of 2 N HCl, and mesangial proliferative glomerulonephritis by inducing ethanol calcium and phosphorus in ash were measured with test kits intake and restraint stress, the number of the mesangial cells was (Calcium E-Test and Phospha C-Test, Wako Pure Chemical counted in the 65 glomerulus of the kidney. Industries, Osaka, Japan). 2.8. Statistical Analysis. Values were expressed as mean ±SD. 2.5. Assay of Fecal Lipids and Fecal Cholesterol. Here, 0.1 g of Repeated-measures analysis of variance (ANOVA) was used to homogenized dry fecal matter was added to 4 mL of con- evaluate the effects of groups. Differences in mean values centrated sulfuric acid in test tubes for 30 min at room between groups were tested by Scheffe multiple-range test. A p temperature. Diethyl ether was added to reach 25 mL, and value of less than 0.05 was considered statistically significant. mixed. ,e diethyl ether-containing layer was moved to a flask, and the diethyl ether was evaporated. ,e fecal lipid 3. Results in the flask was then weighed. ,e T-cho concentration in fecal matter was determined in the same way as in the liver 3.1. Food Intake, Total Energy Intake, Body Weight, Liver, T-cho concentration. Kidney, and Perirenal Fat Tissue Weight. No significant Fecal bile acids were measured by the procedure de- differences in liver and kidney weights were observed among scribed in the work of Iwami et al. [20]; 10 mg of the sample the three groups. Total energy intake and perirenal fat tissue was mixed with 0.2 mL of 90% ethanol during vortex mixing weight of the AD and LD groups were lower than in the CD and incubated for 1 h at 65 C. ,e mixture was subjected to group (p< 0.05) (Table 2). Figure 2 shows the body weight of centrifugation at 5,000 rpm for 3 min. ,e supernatant was mice during the experiment. ,e final body weight of mice was transferred to a 1.5 mL tube, and the ethanol was evaporated. high in the following order: CD, LD, and AD (p< 0.05). After ,en, 0.2 mL of 90% ethanol was added in the precipitate for the mice in the AD and LD groups underwent restraint stress vortex mixing. ,e sample was dissolved in 1 mL of 90% from 13 days, weight between the AD and LD groups increased. ethanol and measured using test kits (total bile acid test by enzyme colorimetric method, Wako pure Chemical In- dustries, Osaka, Japan). 3.2. Plasma Lipids Profiles and Liver Lipids. Although no significant differences in plasma TG concentration were observed between the three groups, the plasma T-cho and 2.6. Kidney Histology. Kidneys of mice from the three di- HDL-cholesterol concentrations of the AD group were lower etary treatment groups were compared histologically. Under than those of the CD and LD groups (p< 0.05) (Table 3). No deep anesthesia with ether, the chest of a mouse from the differences were observed in liver total lipids and liver TG three groups was opened rapidly and the vasculature was concentrations between groups, but liver T-cho concen- perfused with 50 mL of a fixative (4% paraformaldehyde in trations of the CD and LD groups were lower than those of 0.01 M sodium phosphate-buffered saline (PBS: pH 7.4)) at the AD group (p< 0.05) (Table 3). ,e plasma GOT level of a pressure of 120 mmHg from a 18-gauge cannula inserted the AD group was higher than those of the CD and LD into the aorta via an incision in the left ventricle. Imme- groups (p< 0.05) (Figure 3). In addition, the plasma GPT diately after fixative perfusion, the kidney was removed, cut level of the AD group was higher than that of the CD group into small pieces, and immersed in the same fixative (p< 0.05) (Figure 3). overnight at 4 C. Kidney pieces were then washed with PBS, dehydrated in an ascending series of ethanol aqueous so- lutions (70%, 80%, 90%, and 100%), cleared in xylene, and 3.3. Bone Contents, Plasma Calcium, and Fecal Lipids. ,e embedded in paraffin wax. ,ree-micrometer-thick sections ash content in the bone of the AD group was lower than of paraffin-embedded kidneys were then subjected to he- those in the CD and LD groups, although there were no matoxylin and eosin (H&E) staining by a routine procedure significant differences in right femur weight, calcium, or (Meyer’s hematoxylin staining, followed by eosin Y staining) phosphorus in the bone between the groups (p< 0.05) and azan staining by a routine procedure (Mordant, Mal- (Table 4). ,ere was also no difference in plasma calcium lory’s azocarmine G solution, 5% phosphotungstic acid between groups. No significant differences were observed 4 Journal of Nutrition and Metabolism Table 2: Food intake, total energy intake, body weight, and liver, kidney, and perirenal fat tissue weight. 1 2 3 Group CD AD LD a b b Food intake (g/day) 17.2± 0.66 10.9± 0.95 11.0± 0.91 a b b Total energy intake (Kcal) 2040± 780 1382± 121 1400± 116 a b b Total energy intake (Kcal/day) 75.5± 2.89 51.2± 4.48 51.9± 4.29 a c b Final body weight (g) 40.2± 1.39 29.8± 2.73 34.5± 3.41 Liver weight (g/100 g BW) 4.45± 1.54 4.85± 1.01 5.05± 1.86 Kidney weight (g/100 g BW) 0.59± 0.08 0.74± 0.08 0.68± 0.22 a b b Perirenal fat tissue weight (g/100 g BW) 0.88± 0.19 0.33± 0.12 0.34± 0.21 1 2 3 CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol diet. Values represent mean±SD, n � 8. Values not sharing a common superscript letter are significantly different at p< 0.05. tissue weights of the AD and LD groups were lower than that of the CD group (p< 0.05) (Table 2). It is known that chronic alcohol intake and excessive stress accelerate energy meta- bolism in the body [21]. ,e final body weight of the AD group demonstrated frequently observed symptoms such as weight loss compared to be in the LD group. Intake of Lb. paracasei NFRI 7415 may have prevented enhanced energy metabolism by alcohol intake and excessive stress. Fermented dairy products such as yogurt utilizing LAB have been reported to lower serum cholesterol concentrations in animals [22]. We also reported that live Lb. paracasei NFRI 0 5 10 15 20 25 30 7415 reduced the plasma T-cho and hepatic T-cho concen- Feeding period (day) tration in rats fed an ethanol-containing diet [12]. In this study, LAB were not found to lower plasma lipids. However, in terms Control diet of cholesterol-reducing activity, effects against liver lipids and Alcohol diet Lb. paracasei-containing alcohol diet the plasma GOT level were observed in the LD group receiving heat-killed Lb. paracasei NFRI 7415. Plasma GOTand GPTare Figure 2: Body weight of mice during the experiment (n � 8). enzymes recognized as indicators of hepatitis, liver cirrhosis, Arrow: start of restraint stress. and cardiac infarction [23]. Levels of these enzymes increase with liver cell damage, as in hepatitis. ,us, these results in fecal T-cho concentrations, but the fecal bile acids of the indicate that heat-killed Lb. paracasei NFRI 7415 decreased the AD group were lower than those of the CD and LD groups plasma GOT level and liver cholesterol concentration caused (p< 0.05) (Table 4). by chronic alcohol consumption. As described in our previous paper, the live Lb. paracasei NFRI 7415 has the capacity to lower fecal T-cho excretion, 3.4. Kidney Histology. ,ere were no differences in kidney and it effectively reduced plasma T-cho concentration [24]. tissue between the three groups in H&E staining (data not Although no significant difference in fecal T-cho concen- shown). However, more fibrosis was observed in the glo- trations were observed here, the bile acid level of feces in the merulus of the kidney in the AD group than in the other two LD group increased (Table 4). ,is increase in the bile acid groups in azan stain (Figures 4(a)–4(c)). Immunostaining level appears to have been due to bile acid adsorption by LAB results confirmed that the brown areas indicating the ex- in the intestine [25, 26]. Hence, it suggested that heat-killed istence of mesangial cells were increased in the AD group Lb. paracasei NFRI 7415 had strong bile acid adsorption but was almost nonexistent in the CD and LD groups ability in feces. (Figures 4(d)–4(f)). ,e average of the number of mesangial Restraint stress load causes osteoporosis, due to an in- cells in the glomerulus of the CD, AD, and LD groups was crease in the corticosteroid hormone secreted by the cortex 3.08± 1.02, 4.06± 1.07, and 3.39± 0.90, respectively. ,e of the adrenal gland, and increases bone resorption [27]. number of mesangial cells in the AD group was significantly Heavy alcohol consumption has been associated with in- higher than those of the CD and LD groups (p< 0.001). creased risk of bone fracture [3]. It is known that osteo- porotic bones are more likely to fracture, and it is important 4. Discussion to determine the bone content in mice fed alcohol- We found that live Lb. paracasei NFRI 7415 is beneficial for containing diets with restraint stress. improving liver damage due to chronic alcohol intake [12]. In this experiment, the ash content in the bone of the AD In the current study, we investigated whether heat-killed Lb. group was lower than those of the other two groups (p< 0.05) paracasei NFRI 7415 influenced kidney and bone in mice fed (Table 4). Although there were no significant differences in an ethanol-containing diet with stress. ,e perirenal fat right femur weight, or in calcium and phosphorus in bone, Body weight (g) Journal of Nutrition and Metabolism 5 Table 3: Plasma lipids and liver lipids. 1 2 3 Group CD AD LD Plasma lipids Triacylglycerol (mg/dL) 143.5± 23.8 141.0± 42.0 176.3± 91.6 a b a Total cholesterol (mg/dL) 167.4± 33.8 126.2± 14.8 179.2± 25.5 a b a HDL-cholesterol (mg/dL) 127.9± 18.7 90.9± 12.9 120.8± 23.8 Liver lipids Total fat (mg/g) 75.4± 18.4 76.9± 25.3 79.0± 16.4 Triacylglycerol (mg/g) 27.5± 12.4 28.6± 12.6 33.3± 12.0 ab a b Total cholesterol (mg/g) 8.69± 3.46 11.1± 3.60 6.37± 1.80 1 2 3 CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol diet. Values represent mean±SD, n � 8. Values not sharing a common superscript letter are significantly different at p< 0.05. 80 80 70 70 60 60 ab 50 b 50 40 40 30 30 20 20 10 10 0 0 CD AD LD CD AD LD Figure 3: Plasma GOT level and plasma GPT level concentration of mice fed experimental diets. CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol diet. Values represent mean ±SD; n � 8. Values not sharing a common superscript letter are significantly different at p< 0.05. Table 4: Bone content, plasma calcium, and fecal lipids. 1 2 3 Group CD AD LD Bone contents Right femur weight (dry) (mg) 57.4± 6.23 53.0± 7.85 56.1± 5.56 Right femur weight (defatted) (mg) 53.6± 6.0 49.3± 6.76 52.3± 5.30 a b a Ash (%) 53.0± 1.44 50.9± 2.13 54.0± 2.67 Calcium (mg/g) 13.3± 0.85 12.7± 1.07 13.0± 0.77 Phosphorus (mg/g) 15.7± 1.23 14.6± 1.34 15.4± 0.92 Plasma calcium (mg/g) 3.09± 0.58 4.06± 0.62 3.54± 1.46 Fecal lipids a b ab Total fat (mg/g) 48.2± 9.30 29.8± 10.9 39.9± 9.77 a b a Bile acid (μg/g) 3.54± 0.99 2.38± 0.56 3.91± 0.74 Total cholesterol (mg/g) 1.87± 0.43 1.89± 0.56 1.90± 0.41 1 2 3 CD, control diet; AD, alcohol diet; LD, Lb. paracasei-containing alcohol diet. Values represent mean±SD, n � 8. Values not sharing a common superscript letter are significantly different at p< 0.05. Plasma GOT level (mg/dL) Plasma GPT level (mg/dL) 6 Journal of Nutrition and Metabolism (a) (b) (c) 10μm (d) (e) (f) Figure 4: Mesangiolysis in mice. (a)–(c) Azan staining of the glomerulus in the kidney. (a) ,e glomerulus in the kidney from a normal mouse fed with control diet (CD). Blue regions (white arrows) show collagen fiber. (b) ,e glomerulus in the kidney from a mouse fed with alcohol diet (AD). Blue regions (white arrows) show collagen fiber, which are increased compared to (a). (c) ,e glomerulus in the kidney from a mouse fed with AD containing Lb. paracasei. Blue regions (white arrows) show collagen fiber, which are reduced compared to (b). (d)–(f) Immunostaining of the glomerulus in the kidney. (d) ,e glomerulus in the kidney from a normal mouse fed CD. A mesangial cell (black arrow) is stained by desmin. (e) ,e glomerulus in the kidney from a mouse fed with AD. ,e number of mesangial cells (black arrows) is higher than in the normal kidney (d). (f) ,e glomerulus in the kidney from a mouse fed AD containing Lb. paracasei. ,e number of mesangial cells (black arrow) is fewer than that of animals fed AD (e). each average value of the AD group was lower than those of It was observed that the AD group had lower urine the CD and LD groups. For example, the right femur weight volume than the CD group during the experimental period. (dry) of the CD, AD, and LD groups was 57.4± 6.23 mg, Kidney function weakness was therefore suspected in the AD 53.0± 7.85 mg, and 56.1± 5.56 mg, respectively. Moreover, group. Fibrosis of the organization occurs to the organ that the plasma calcium of CD, AD, and LD groups were 3.09± the whole body is approximately important except the brain. 0.58 mg, 4.06± 0.62 mg, and 3.54± 1.46 mg, respectively. ,e organ that caused the fibrosis will eventually mal- ,ese results indicate that some minerals were eluted from the function. ,is means that renal function decreases as bone of AD group, promoting bone resorption. On the other kidney fibrosis advances [29]. We therefore performed hand, the plasma calcium of the LD group tended to be lower, azan staining to observe fibrosis of the kidney. ,e suggesting that bone resorption was inhibited by LAB. It was mesangium domain in the glomerulus of the kidney reported that probiotic yogurt containing strains of Lacto- consists of mesangium cells and mesangium substrates, bacillus casei, Lactobacillus reuteri, and Lactobacillus gasseri including type IV collagen. Collagen fiber is stained blue by increased apparent calcium absorption and bone mineral azan staining. An increase of the mesangium domain was content in rats [28]. ,ese LAB strains produce certain confirmed, by the increase in blue in the glomerulus of the prebiotics such as oligosaccharides that help new bone tissue AD group. We then attempted immunostaining using the to grow. Further investigation of the effects of live Lb. par- desmin antibody, which can specifically stain mesangial acasei NFRI 7415 as probiotics is needed. cells. Many more mesangium cells, colored brown by Journal of Nutrition and Metabolism 7 resulting from ovariectomy,” BioMed Research International, immunostaining, were observed in the AD group com- vol. 2015, Article ID 897639, 10 pages, 2015. pared to the CD and LD groups. [10] R. Yacoub, D. Kaji, S. N. Patel et al., “Association between ,ere are always mesangial cells in the glomerulus of the probiotic and yogurt consumption and kidney disease: in- kidney. ,ey maintain capillary vessels of the glomerulus of sights from NHANES,” Nutrition Journal, vol. 15, p. 10, 2016. the kidney and work to regulate its ability to filter the blood. [11] N. Komatsuzaki, J. Shima, S. Kawamoto, H. Momose, and Most chronic glomerulonephritis involves mesangial pro- T. Kimura, “Production of γ-aminobutyric acid (GABA) by liferative glomerulonephritis (MesPGN), for which the char- Lactobacillus paracasei isolated from traditional fermented acteristic is an increase of mesangial cells [30]. ,e causes of foods,” Food Microbiology, vol. 22, no. 6, pp. 497–504, 2005. MesPGN development remain to be elucidated. It is thought, [12] N. Komatsuzaki and J. Shima, “Effects of live Lactobacillus however, that immunoreaction is the factor that causes this paracasei on plasma lipid concentration in rats fed ethanol- condition. Mesangium proliferative glomerulonephritis de- containing diet,” Bioscience, Biotechnology, and Biochemistry, veloped in the AD group by alcohol intake with stress. vol. 76, no. 2, pp. 232–237, 2012. [13] N. Komatsuzaki, Y. Yamada, Y. Ueki, J. Shima, and In conclusion, the present investigation shows that heat- S. Morikawa, “Lactobacillus paracasei NFRI 7415 reduces liver killed Lb. paracasei NFRI 7415 inhibits mesangial pro- lipid contents in C57BL/6J mice fed a high-fat diet,” In- liferative glomerulonephritis by alcohol intake with stress. ternational Journal of Clinical Nutrition and Dietetics, vol. 2, Further studies are needed to investigate in more detail with no. 1, p. 108, 2016. five dietary treatment groups: a control diet (CD) group, an [14] K. Yagasaki, “Nutritional and bromacological studies of food alcohol diet (AD) group, the CD diet with restraint stress factors using in vitro and in vivo disease models,” Journal of group, the AD with restraint stress group, and the AD with Japan Society of Nutrition and Food Sciences, vol. 62, no. 2, restraint stress containing LAB group. ,is is needed to pp. 61–74, 2009. clarify the relationship between restraint stress and risk of [15] K. Fujisawa, K. Yagasaki, Y. Miura, and R. Funabiki, “Im- MesPGN. provement of hyperlipidemia and proteinuria without no- ticeable growth retardation by feeding a methionine and threonine supplemented low-casein diet to nephritic rats,” Conflicts of Interest Bioscience, Biotechnology, and Biochemistry, vol. 59, no. 10, pp. 1896–1900, 1995. ,e authors have no conflicts of interest to report. [16] C. S. Lieber and L. M. DeCarli, “Liquid diet technique of ethanol administration,” Alcohol and Alcoholism, vol. 24, no. 3, pp. 197–211, 1989. 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