Effects Of Different Dietary Patterns On Serum Short-chain Fatty Acids And Hepatic Inflammatory Factors In NAFLD Rats

Objective Based on the high fat diet-induced NAFLD rat model,four dietary diets were given,including common diet,high fat diet,high carbohydrate diet and high protein diet,for grouping studies.To investigate the changes of serum short-chain fatty acids(SCFA),proinflammatory cytokines IL-6,IL-1?,IL-18,TNF-? in liver tissue and AMPK signaling pathway in liver tissue of different dietary groups.And to investigate the effect of changes in dietary factors on NAFLD and the related mechanism of NAFLD.Methods42 male SD rats aged 6 weeks and body mass in the range of 140-180 g were randomly divided into two dietary groups: control group(n = 12)and model group(n= 30)after one week adaptive feeding.The control group was fed with the common diet,and the model group was fed with high fat diet.After 8 weeks,6 rats were randomly killed in each group,and the establishment of NAFLD rat model was determined according to the biochemical and pathological results of rat liver.Then the model diet group was randomly divided into four dietary patterns: high fat diet group,high carbohydrate diet group,high protein diet group and common diet group,6 rats in each group,high fat diet group continued to give high fat diet,the rest each group was replaced with dietary feed,that is,high carbohydrate diet group given high carbohydrate feed,high protein diet group given high protein feed,the common diet group given ordinary feed.Rats were sacrificed at 12 week.Total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),high density lipids(LDL-C),and high density lipids(HDL-C)were measured by biochemical method.IL-6,IL-1?,IL-18 and TNF-? were detected by enzyme-linked immunosorbent assay(ELISA).The serum short-chain fatty acids(AMPK)and phosphorylated adenylate activated protein kinase(p-AMPK?)in liver tissue were measured by immunohistochemistry(IHC).Real-time quantitative PCR(Real Time)was used to detect the expression of adenylated protein kinase(ACC)and HMGR mRNA transcription levels in liver tissue.Results1.Results of NAFLD rat model building The levels of TC,TG and LDL-C in the liver tissue homogenate of the model group were significantly higher than those in the control group,and HDL-C decreased significantly.Liver histomorphology and HE staining showed that the lipid deposition of hepatocytes in model group increased.The results showed that the 8-week high fat diet could cause hepatocellular steatosis,that is,high-fat diet successfully induced the establishment of NAFLD rat model.2.Results of dietary factors intervene1)Liver histomorphology and pathology results By observing the morphological findings of rat liver tissue,the liver tissue of the control group was dark red,the liver was soft and the liver margin was sharp,and there was no obvious abnormal change.the liver tissue volume of the common diet group was slightly larger,the color was darker than that of the normal liver,the liver surface coarse grainy,edge slightly narrowed,the liver texture is still soft;the liver volume of the rats in the high fat diet group,the high protein diet group and the high carbohydrate diet group was significantly larger,the liver tissue showed pale and the liver margin was blunt,and the nodular and focal changes were seen on the surface.HE staining showed that the structure of the hepatic lobule was clear and complete,the cytoplasm was uniform and the nucleus was seen in the central part of the control group.The hepatic lobule structure and partial hepatocyte edema were seen in the common diet group,and mild fatty steatosis;The liver lobular structures of high-fat diet group,high-carbohydrate diet group and high-protein diet group were unclear,the border and the structure was not obvious,cytoplasm loose,liver structure disorder,hepatocyte swelling was obvious,the balloon-like changes in the cytoplasm can be seen in varying sizes of lipid droplets,were medium-severe steatosis.2)Results of liver biochemical markers of liver tissue Compared with the control group,the levels of TG,TC and LDL-C in the liver homogenate of high-fat diet group,high-carbohydrate diet group,high-protein diet group and common diet group were increased and the HDL-C level was decreased(P<0.05).Compared with the common diet group,the levels of TC,TG and LDL-C in the liver tissue homogenate of high-fat diet group,high-carbohydrate diet group and high-protein diet group were increased and the level of HDL-C was decreased(P<0.05).3)The level of SCFA in serum Compared with the control group,the levels of acetate and total SCFAs in the serum of the high-fat diet group,the high-carbohydrate diet group,the high-protein diet group and the common diet group were significantly higher,the difference was statistically significant(P <0.05);the propionate and butyrate levels were not significantly different.Compared with the common diet group,the levels of acetate and total SCFAs in serum of the high-fat diet group,the high-carbohydrate diet group and the high-protein diet group were significantly higher,the difference was statistically significant(P <0.05);the propionate and butyrate levels were not significantly different.4)The concentration of IL-6,IL-1?,IL-18,TNF-? in liver tissue Compared with the control group,the levels of IL-6,IL-1?,IL-18 and TNF-? in the high-fat diet group,the high-carbohydrate diet group,the high-protein diet group and the common diet group were higher,the difference was statistically significant(P<0.05).Compared with the common diet group,The levels of IL-6,IL-1?,IL-18 and TNF-? in liver tissue homogenate of the high-fat diet group,the high-carbohydrate diet group and the high-protein diet group were higher,the difference was statistically significant(P<0.05)5)Expression of AMPK-?2 and p-AMPK? in liver tissue Compared with the control group,the expression levels of AMPK-?2 and p-AMPK? in the liver tissues of the high-fat diet group,the high-carbohydrate diet group,the high-protein diet group and the common diet group were lower,the difference was statistically significant(P <0.05).Compared with the common diet group,the expression levels of AMPK-?2 and p-AMPK? in liver tissue of the high-fat diet group,the high-carbohydrate diet group and the high-protein diet group were lower,the difference was statistically significant(P <0.05).6)Transcription levels of ACC and HMGR mRNA in liver tissue Compared with the control group,the transcription levels of ACC and HMGR mRNA in the liver tissue homogenate of the high-fat diet group,the high-carbohydrate diet group,the high-protein diet group and the common diet group were higher,the difference was statistically significant(P <0.05).Compared with the common diet group,the transcription levels of ACC and HMGR mRNA in liver tissue homogenate of the high-fat diet group,the high-carbohydrate diet group and the high-protein diet group were significantly higher,the difference was statistically significant(P <0.05).Conclusion1.The NAFLD rat model was successfully established after 8-weeks high fat diet,indicating that long-term high-fat diet can lead to lipid deposition in the liver and induce hepatic steatosis.2.The levels of serum acetate and total SCFAs were increased in high fat diet group,high carbohydrate diet group and high protein diet group,while the levels of propionate and butyrate were not significantly different,suggesting that SCFA may have an effect on the pathogenesis of NAFLD,in which acetate may play a role in the pathogenesis of NAFLD.3.The levels of IL-6,IL-1?,IL-18 and TNF-? in the liver tissue homogenate of the high-fat diet group,the high-carbohydrate diet group and the high-protein diet group were significantly increased,indicating that IL-6,IL-1?,IL-18,TNF-?promote the development of chronic inflammatory reaction of NAFLD.4.The expression levels of AMPK-?2 and p-AMPK? in the high-fat diet group,the high-carbohydrate diet group and the high-protein diet group were decreased,while the transcription levels of ACC and HMGR mRNA in the liver homogenate of high-fat diet group,the high-carbohydrate diet group and the high-protein diet group were increased,suggesting that AMPK activity and the transcription levels of ACC and HMGR could affect the lipid regulation process of liver cells,thus affecting the development of NAFLD.5.Changes in dietary patterns such as high-fat,high carbohydrate and high protein and other dietary risk factors can aggravate the development of NAFLD inflammatory response process,indicating that high fat,high carbohydrate and high protein and other irrational diet are risk factors of NAFLD.