| Introduction With the prevalence of obesity, hyperlipidemia, type II diabetes, and metabolic syndrome, the morbidity of non-alcoholic fatty liver disease(NAFLD) is constantly rising worldwide. Recent studies indicate that about 50 % of NAFLD patients may develop into non-alcoholic steatohepatitis(NASH) and 40 % may progress to liver fibrosis after 4-13 years. The pathophysiological process of NAFLD is extremely complicated and the pathogenesis is unclear. No effective cures have been found currently with the prognosis of NASH being pessimistic. Therefore, there is an urgent need to develop effective remedies for patients suffering NAFLD. NAFLD patients are usually accompanied with obesity and insulin resistance, but not all obese people develop into NAFLD, the intestinal factors may play a key role in the pathogenesis of NAFLD. Studies have shown that the lipopolysaccharide(LPS) of gram-negative bacteria in the intestine can be transported to the liver through the portal vein. Under condition of dysbiosis, a large amount of Gram-negative bacteria may over proliferate and produce endotoxins, results in metabolic disorders, obesity, diabetes, NALD and NASH. Changes of intestinal microbiota in the acute liver injured mice resulted in increased intestinal permeability and bacterial translocation. In addition, the reaction of the mouse to high-fat diet(HFD) can be determined by the different composition of the intestinal microbiota and levels of circulating endotoxin level, which are significantly higher in NAFLD patients with excessive growth of intestinal bacteria and enhanced intestinal permeability. Since the intestinal microbiota is closely related to obesity, insulin resistance, NAFLD and NASH, it is possible to prevent the occurrence of NAFLD by adjusting the intestinal microbial structure. Probiotics and Chinese herb medicine may be promising approaches to affect intestinal microbiota. There have been studies on the capacity of probiotics to attenuate the unbalanced homeostasis and cholesterol levels in the body. Many probiotics were made into yogurt using as food supplements for patients with hyperlipidemia and NAFLD. On the other hand, Cassia obtusifolia L. as natural functional food additives with the characteristic of inhibiting fat accumulation can result in satisfactory effect on the improvement of obesity, insulin resistance and other metabolic diseases, which provides a new direction for the treatment of NAFLD. Cassia obtusifolia L. belongs to a leguminous annual herb in tropical countries in Asia, its main active ingredients are anthraquinone compounds, including obtusin, emodin and aloe-emodin. Its herbal ingredients are popular as a kind of functional beverage with the effects of reducing serum levels of fat and cholesterol, anti-oxidation, anti-fungal, and neuroprotection. Cassia obtusifolia L. can also protect the liver function in rats with liver injury, alleviate obesity, insulin resistance and NAFLD by up-regulating the AMP-dependent protein kinase. Although both probiotics and anthraquinone of Cassia obtusifolia L.(AC) have beneficial effects for meliorating metabolic diseases such as obesity, insulin resistance and NAFLD, it is still unclear whether application of them together as a symbiotic formulation could enhance the NAFLD-preventing effect. We hypothesized that by combining the probiotics and AC, the preventive or improving effects on NAFLD can be enlarged, and this may associated with the improvement of the intestinal microbial structure and metabolism of energy by the liver. To test the hypothesis, the screening and characterization of lipid-lowering probiotics were conducted, and with the anthraquinone from Cassia obtusifolia L. joint training, verify the efficiency of lipid changes. Then, by treating the selected probiotics together with AC, the effects of the symbiotic formulation on HFD-induced NAFLD rats were tested.Part I Screening and characterization of lipid-lowering probiotics in vitroObjectives: The cholesterol degradation strains isolated from fermented food or healthy human intestine were screened by MRS-cholesterol broth with the Ammonium ferric sulfate method(In vitro cholesterol degradation rate> 50%). on this basis, The ability of strains triglycerides-lowering, and tolerance to acid, bile, pepsin and trypsin, cell adhesion ability were further evaluated. They were co-cultured with anthraquinone from Cassia obtusifolia L.(AC) to test the cholesterol-lowering and triglycerides-lowering activity. The optimal strains were screened out for further research.Materials and Methods: 1. The cholesterol-lowering strains co-cultured with AC were compared of cholesterol-lowering efficiency by MRS-cholesterol broth with the Ammonium ferric sulfate method. 2. The cholesterol-lowering strains co-cultured with AC were compared of triglycerides-lowering efficiency by MRS-triglycerides broth. 3. The properties of selected candidate strains, including tolerance to acid, bile, pepsin and trypsin, cell adhesion ability were evaluated. 4. The strains were identified by 16 S r DNA sequencing and detected plasmid.Results: 1. The cholesterol-lowering efficiency of DM9054, DM9073 and 86066 was improved significantly, when co-cultured with AC(P <0.05).2. The triglycerides-lowering efficiency of 8503, 84034 and 86066 was improved significantly, when co-cultured with AC(P <0.05). 3. DM9054, 86066 survived at p H 2 and pepsin, and strains DM9007, DM9054 and 86066 were able to survive in 0.3% bile salts, all strains tolerance to trypsin, while DM9007, DM9054 and 86066 had the best cell adhesion abilities. 4. DM9054(Lactobacillus Rhamnosus GG, LGG) and 86066(Lactobacillus plantarum WCFS1, LP) no plasmid were selected as the optimal strains by 16 S r DNA sequencing.Conclusions: DM9054(Lactobacillus Rhamnosus GG, LGG) and 86066(Lactobacillus plantarum WCFS1, LP) were selected as probiotics, and co-cultured with AC the cholesterol-lowering and triglycerides-lowering efficiency was improved significantly. It’s synergistic effect. The strains survived at digestive juice, also had the best cell adhesion abilities and without a plasmid.Part II Co-administration of lipid-lowering probiotics and anthraquinone from Cassia obtusifolia L. effect on lipid metabolism and related gene expression of non-alcoholic fatty liver in ratsObjectives: The selected lipid-lowering probiotics DM9054 and 86066 together with anthraquinone from Cassia obtusifolia L.(AC)the effects of the symbiotic formulation on HFD-induced NAFLD rats were analyzed the expression of related genes and proteins of lipid metabolism. In order to found out lipid-lowering probiotics combined AC could in the role of lipid metabolic pathways targets, further explore the mechanism of improvement of NAFLD.1. Male Sprague–Dawley rats were divided randomly into 5 experimental groups. Group 1 received a normal diet(ND). Group 2 received a high-fat diet(HFD) to establish NAFLD models. Group 3 received HFD containing AC(0.2g/kg per day). Group 4 received HFD containing lipid-lowering probiotics(P)(2×1010 CFU/ml). Group 5 received HFD containing AC and P. Rats were euthanized on day 150 after the diet regimens were completed. 2. All group rats general condition, body weight, Lee’s index and liver index were evaluated. 3. All group rats blood lipid level, insulin resistance, serum endotoxin, hepatic TNF-α level were tested. HE and oil red O staining to observed rats liver pathological. 4. The expression levels of CYP7A1, HMGCR, LDL-R m RNA in liver and FXR m RNA in the intestine were analyzed by q PCR. The expression levels of PPAR-α, PPAR-γ, SREBP-1c protein in liver were analyzed by Western-Bloting. Materials and Methods:Results: 1. Fed on HFD for 150 consecutive days to build NAFLD animal models. 2. All group rats vital signs were stable, compared with other group, the levels of body weight, Lee’s index and liver index of HFD Group increased significantly(P <0.05). In the three intervention groups, the levels of body weight, Lee’s index and liver index decreased greatly(P <0.05). 3. Compared with other group, the serum levels of TC, TG, LDL, FFA, insulin resistance, serum endotoxin and hepatic TNF-α level of HFD Group increased significantly, while serum HDL decreased greatly(P <0.05). By HE staining and oil red O, it was found that the hepatic steatosis and scattered chronic inflammatory cells were seen in the infiltration. Compared with the Group HFD, the serum TC, TG, LDL, FFA, insulin resistance, serum endotoxin and hepatic TNF-α level of Group HFD+AC,Group HFD+P and HFD+AC+P decreased, while serum HDL increased(P <0.05). While the fatty degeneration and inflammatory cells infiltration of the other three intervention groups were improved. 4. Compared with the Group HFD, the expression levels of CYP7A1, LDL-R m RNA, PPAR-α protein in liver and FXR m RNA in the intestine of Group HFD+AC,Group HFD+P and HFD+AC+P up-regulated, while HMGCR m RNA, SREBP-1c、PPAR-γ protein in liver down-regulated(P <0.05).Conclusions: The cholesterol-lowering probiotics alone and in combination with AC protect against HFD-induced NAFLD. The underlying mechanisms involve reducing intestinal endotoxemia and decreasing the release of inflammatory cytokines, regulation of lipid metabolism and improving IR through increasing the expression levels of both CYP7A1 and LDL-R in the liver, decreasing the expression levels of HMG-CR, up regulating PPAR-α levels while down regulating PPAR-γ and SREBP-1C levels. Therefore, an optimized blend of the lipid-lowering probiotics and AC could be exploited as a potential biotherapeutic remedy to decrease cholesterol levels and lower the risk of NAFLD.Part III Co-administration of lipid-lowering probiotics and anthraquinone from Cassia obtusifolia L. effect on intestinal microbiota and intestinal barrier function of non-alcoholic fatty liver in ratsObjectives: Intestinal microbiota and intestinal barrier function play crucial roles in the pathogenesis of NAFLD. Therefore, based on the second part, to further explore the co-administration of lipid-lowering probiotics and anthraquinone from Cassia obtusifolia L. ameliorate intestinal microbiota and intestinal barrier function of non-alcoholic fatty liver in rats.Materials and Methods: 1. Microbial genomic DNA was extracted from fecal samples using Stool DNA Kit and detected the quality of DNA Sample.The V4 hypervariable region of 16 S r RNA gene were amplified using the primers and agarose gel electrophoresis to detected the amplification. 2. Caecal samples from receiver rats on day 50, day 100 and day 150 were sequenced by Illumina Miseq with a 6 cycle index read. Each sample’s trimmed sequence was compared to sequences were assigned to different taxonomic levels, high-quality sequences were analyzed. 3. According to sequencing results, the rats in each group consisting of intestinal microbiota composition, species abundance and intestinal microbiota similarity were analyzed. 4. HE staining to observed rats intestinal mucosa pathological. The expression levels of tight-junction proteins Occludin and ZO-1 in intestinal were analyzed by Western-Bloting.Results: 1. The quality of microbial genomic DNA was detected, OD260 / OD280 values were generally between 1.8 and 2.0 and used the V4 region primers successfully amplified bands. 2. A total of 7,583,846 sequences was obtained by Illumina Miseq and after trimming, 5,740,971 sequences were further analyzed. The proportion of high-quality sequence of 75.7% and an average 63,788 each sample, sequence length were between 225 ~ 226 bp. And the sequence could be sufficient to covered 97% of the intestinal microbial strains, and uniform distribution of abundance. 3. The levels of the commensal microbes of rats in the five groups were detected, it turned out that the Firmicutes of Group HFD increased obviously while the Bacteroidetes decreased. When analyzing the intestinal microbial taxa of rats, we found that the Bacteroides, Lactobacillus and Parabacteroides of Group HFD decreased greatly compared with Group ND, while Oscillospira increased obviously(all P<0.05), and the intervention of probiotics or AC effectively increased Bacteroides, Lactobacillus and Parabacteroides and decreased Oscillospira. Among the intervention groups, the effect of AC+P was the most obvious. PCo A was performed based on their microbial composition, and the cluster analysis was performed. Results showed that Group HFD+AC+P was closer to the normal group. The heat map analysis further confirmed our results. 4. The intestinal mucosal villi of rats in HFD Group appeared rupture, missing, and the epithelial cell necrosis were founded. Through the intervention of probiotics or AC, the arrangement of the villi of the three intervention groups had recovered, in which the effect of AC+P was the most obvious. Western-blotting results showed that compared with Group ND, the expression of ZO-1 and occludin of Group HFD decreased significantly, and increased in the three intervention groups, in which the effect of AC+P was the most obvious(P<0.05).Conclusions: The lipid-lowering probiotics alone and in combination with AC keeping a homeostasis of the intestinal microbiota and making dysbiosis intestinal microbiota tend to be normal, modulation of intestinal barrier function through increased the expression of occludin and ZO-1 protein. Therefore play a role in improved NAFLD. |
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