Protective Effect Of Kang Brick Tea Compound On Liver And Kidney In Diet-Induced Obesity Mice

Objective The morbidity of obesity is increasing year by year.It is not only a risk factor of cardiovascular disease,but also a cause of nonalcoholic fatty liver disease.In addition,studies have shown that the occurrence and development of chronic kidney disease is also associated with obesity.Lipotoxicity and lipid metabolic disorders can induce renal injury by activating inflammation,endoplasmic reticulum stress,fibrosis and apoptosis.At present,the treatments for obesity-related renal damage are not ideal.As a traditional drink in China,tea has been proved to have many effects,such as antioxidation,anti-inflammation and so on.Kang brick tea is a kind of Tibetan tea.Many studies have shown that Kang brick tea plays a certain role in regulating lipid metabolism.The purpose of this study is to observe whether Kang brick tea complex can protect liver and kidney injury induced by high fat diet and to explore its specific protective mechanism.Methods4-week-old male C57BL/6 mice were divided into four groups with 8 mice in each group.The control group(CON)was fed with normal feed and pure water.The other three groups were fed with high-fat diet for 12 weeks to establish a diet-induced obesity(DIO)mouse model,with fat accounting for 60% of HFD.After that,the model group(DIO group)were fed with high-fat diet and pure water sequentially,while the other two groups(Qing and Yi)continued to be fed with high-fat diet and were given two different formulations of Kang brick tea(Qing group and Yi group)at the dose of400mg/(kg·d),for 12 weeks.During this period,the body weight of mice was recorded every week,fasting blood glucose was measured every other week.The mice were killed 12 weeks later(28 weeks old).The intestinal contents were extracted and the differences of gut microbiota and their metabolites were analyzed by 16 Sr RNA and metabonomics.RNA was extracted from mouse liver tissue and the gene expression profiles of control group and model group were compared by high-throughput sequencing technique,and the differentially expressed genes were analyzed by GO and KEGG enrichment analysis.The serum levels of triglyceride,total cholesterol,aspartate aminotransferase,alanine aminotransferase,albumin,creatinine and uric acid were measured to evaluate the function of liver and kidney.HE,PAS and oil red O staining were used to evaluate the pathological damage of liver and kidney.q PCR and Western blot were used to detect the levels of fatty acid β oxidation and lipid synthesis related proteins(FASN,SREBP-1,ACC1,CPT1 A,PPARγ,etc.)in liver and kidney tissues.Western blot was used to detect the levels of inflammatory cytokines(IL-6,TNF-α and COX2)in kidney.In vitro,mouse renal tubular cells(TCMK1)were stimulated with 150 μmol/L palmitic acid,and the treatment group was treated with isovaleric acid with final concentration of 1mmol/L.After cultured for 24 h,the cells were extracted from each group.The expression of fatty acid β oxidation and lipid synthesis related proteins in the cells were detected by Western blot.The statistical method was that continuous variables were expressed by mean ± standard deviation,t-test was used for comparison between two groups,one-way ANOVA was used for comparison between three groups and more than three groups,and Dunnett’s multiple comparisons test was used between each group and DIO group.When p<0.05,the difference was considered to be statistically significant.The data were statistically analyzed by SPSS26.0.ResultsThe analysis of serum biochemical indexes showed that HFD could significantly increase the levels of ALT,AST,TC and TG in mice.The use of Kang brick tea complex Yuanqing or Yuanyi could reduce the increase of blood lipid and the level of transaminase induced by HFD.The results of HE staining showed that the hepatocytes in the DIO group had typical steatosis and obvious inflammatory cell infiltration between the lobules;the number of lipid droplets and vacuoles in the cytoplasm of Qing and Yi groups decreased significantly,and the balloon degeneration of hepatocytes caused by HFD was significantly improved.Renal PAS staining showed that the volume of glomerular mesangial area increased,cells proliferated and renal tubular cells proliferated in DIO group,and mesangial proliferation was effectively inhibited in Qing and Yi groups.The results of oil red O staining showed that the positive area of oil red O staining in liver and kidney of DIO group was significantly higher than that of control group.For the treatment group,the number of red lipid droplets in hepatocytes and renal tubules of mice treated with Yuanqing and Yuanyi decreased significantly.The results of q PCR and WB showed that Kang brick tea can significantly promote the expression of fatty acid oxidation-related proteins(CPT1A,PPARγ and PGC1α)and inhibit the expression of lipid synthesis-related proteins(FASN,ACC1,SREBP-1)in liver and kidney,and significantly reduce the levels of inflammatory factors(IL-6,TNF-α and COX2)in kidney.Liver transcriptome analysis showed that the DIO group had 2201 differentially expressed genes compared with the CON group.The differentially expressed genes were annotated by GO.The results showed that most of the differentially expressed genes were enriched in metabolism-related pathways,and there were 22 differentially expressed genes related to lipid metabolism.In KEGG enrichment analysis,it was found that the PPAR pathway was located in Top20,and the differentially expressed genes in this pathway were FABP4,PPARγ and so on.There was a significant difference in the structure of gut microbiota between the DIO group and the CON group,and HFD could induce the decrease of intestinal flora diversity.The dominant bacteria among the four groups were Bilophila,Eisenbergiella,Akkermansia,Ruminiclostridium-9 and Muribaculaceae.The differences in the contents of short-chain fatty acids in feces among the four groups were butyric acid and isovaleric acid.Besides,isovaleric acid was located in the center of the network regulation map between short-chain fatty acids and gut microbiota.Immunofluorescence and immunohistochemical staining showed that GPR41 and 43 receptors were mainly expressed in renal tubular epithelial cells.In vitro,isovaleric acid treatment can significantly regulate the expression of TCMK1 lipid synthesis-related proteins and reduce the level of inflammatory factors in mouse renal tubular epithelial cells.ConclusionKang brick tea complex can regulate lipid metabolism and protect liver and kidney function by regulating the content of gut microbiota and its metabolites short-chain fatty acids,promoting fatty acid oxidation,inhibiting lipid production and reducing inflammation.