| BackgroungRenal interstitial fibrosis(RIF)is thought to be a common pathway in the progression of chronic kidney disease(CKD)to end stage renal disease(ESRD),and epithelial-mesenchymal transition(epithelial-mesenchymal transition(EMT)plays a key role in RIF.Gut flora disorders are also considered to be a key factor in promoting CKD progression in current studies,but the link between gut flora and renal interstitial fibrosis is not yet clear.As ESRD has poor prognosis in clinical practice,it can only be maintained by hemodialysis or kidney transplantation,which brings great pain to patients;and according to related materials,there are about 130 million CKD patients in China,among which about 1/3 CKD patients develop to end-stage renal disease,which also brings great medical burden to the health care system.Therefore,how to delay the progression of CKD to ESRD is the concern of many scientists.Currently,it is believed that by inhibiting RIF and regulating intestinal flora is an effective therapeutic strategy to protect the kidney.And Chinese medicine has unique advantages in improving interstitial renal fibrosis and regulating intestinal flora.ObjectivePreliminary investigation of the mechanism of action of silymarin in ameliorating interstitial renal fibrosis based on intestinal flora.MethodsForty-six male C57 mice were selected and divided into normal and model groups after 1 week of acclimatization feeding.The model group was induced with 0.25%adenine feed for a period of 10 weeks.At the end of modeling,three mice were randomly selected from the normal group and the model group,and the success of the model was verified by renal pathological examination combined with serum indexes.The mice in the model group were divided into 4 groups:model group,0.4%,0.8%and 1.6%silymarin group,8 mice in each group.The silymarin feed intervention cycle was 8 weeks.The general conditions of mice such as body weight,food intake and water intake were observed during the intervention period,and blood and kidneys of rats were collected for testing after 8 weeks.Serum creatinine,urea nitrogen and 24h urine protein were detected by kits;renal histopathological changes were detected by HE and Masson staining;16S rRNA sequencing was used to detect the difference of intestinal flora in each group of mice and correlation analysis was made by correlation with clinical indexes.The expression of TGF-β1 and p-Smad2 in the kidney tissues of mice was observed by protein immunoblotting and immunohistochemical staining.Results①The mice in the normal group had moist and shiny hair,normal activity and slowly increasing body weight;the mice in the adenine model group had dull hair,dull color,reduced body weight and reduced activity;the mice in the 0.4%silymarin group had improved their hair,activity,body weight,feeding and other general conditions compared with the model group.②The results of biochemical indexes showed that the serum creatinine and urea nitrogen were significantly increased in the model group compared with the normal group;the 0.4%silymarin group could significantly reduce the serum creatinine and urea nitrogen(P<0.05).③Histopathological sections of the kidney showed that the kidney tissue of the normal group was intact,with no significant expansion or atrophy of the glomerulus and tubules,and the interstitium was intact without inflammatory cell infiltration and fibrosis;compared with the normal group,the kidney tissue of the model group showed significant tubular expansion,large inflammatory cell infiltration in the interstitium,and significant fibrosis;compared with the model group,the kidney tissue of the 0.4%silymarin group showed significantly lower levels of interstitial inflammation Compared with the model group,the level of interstitial inflammation and the degree of fibrosis in the kidney of 0.4%silymarin group were significantly reduced.④ The results of microbial 16S rRNA amplicon sequencing showed that the relative abundance of Ruminococcaceae,Anaerotruncus,and Anaeroplasma was significantly higher in RIF mice compared with the normal group,and showed positive correlation with creatinine and urea nitrogen values in serum;on the contrary,the relative abundance of Akkermansia was significantly lower in RIF mice and showed negative correlation with creatinine and urea nitrogen values in serum.The 0.4%silymarin intervention significantly decreased the relative abundance of Ruminococcaceae,Anaerotruncus and Anaeroplasma and significantly increased the relative abundance of Akkermansia in RIF mice,and changed the correlation between the flora and clinical indexes.⑤ Western blot showed that compared with the normal group,the expression of E-cadherin and anti-inflammatory cytokine IL-10 in the kidney of the model group decreased significantly,and the expression of N-cadherin,α-Smooth muscle protein(α-SMA),proinflammatory cytokine IL-1β,TNF-α was significantly increased.Immunohistochemistry and Western blotting showed TGF-β1 and p-Smad2 increased significantly.Compared with the model group,the expression of E-cadherin and anti-inflammatory cytokine IL-10 in the kidney of 0.4%silybin group increased significantly,and the expression of N-cadherin,α-SMA,IL-1β,TNF-α decreased significantly.Immunohistochemistry and Western blotting showed the expression on TGF-β1 and p-Smad2 decreased significantly(P<0.05).ConclusionSilybin changed the structure of intestinal flora in renal interstitial fibrosis model mice.The mechanism of improving renal interstitial fibrosis may be to regulate the relative abundance of some intestinal flora,reduce the toxin into the blood and reduce the secretion concentration of inflammatory factors,so as to regulate TGF-β1/Smad2 signaling pathway delays renal tubular epithelial cell mesenchymal transformation and improves renal interstitial fibrosis. |
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