Study On The Treatment Of Experimental Zebrafish Liver Fibrosis With New PPAR Dual Agonists F3SM And Danning Tablets

Objectives:To establish a model of zebrafish liver fibrosis induced by diethylnitrosamine,and to evaluate whether the model was successfully established according to pathological findings.Then detect liver index,body weight and aspartate aminotransferase,to observe the effect of diethylnitrosamine on zebrafish.After the model was stabilized,treat zebrafish with the new anti-fibrotic drugs F3SM,GFT505,OCA,HUJIAOJIAN,and DANNINGPIAN.After the treatment,evaluate and analyze the liver tissue histopathological sections,liver index and body weight,to determine whether each group of drugs has a therapeutic effect on liver fibrosis,and to explore the molecular mechanism of drug exerting therapeutic effects by detecting changes in liver fibrosis-related gene expression such as tgfbr2b,smad2.Methods:The 6-month-old adult zebrafish was soaked in diethylnitrosamine culture water with a dissolved concentration of 150 mg/L for 6 weeks to construct a liver fibrosis model.After the model was established and stabilized,it was randomly divided into F3SM group,GFT505 group,OCA group,HUJIAOJIAN group,DANNINGPIAN group,negative control group and normal control group,with 20 zebrafish in each group.Each group of zebrafish was treated with drug dose:F3SM group:25ug/d;GFT505 group:25ug/d;OCA group:25ug/d;HUJIAOJIAN group:40ug/d;DANNINGPIAN group:90ug/d;negative control group and the normal control group had a normal diet.After 3 weeks of treatment,dissect the liver tissue for liver index,and pathological sections.RT-qPCR was used to detect the changes of transcription levels of liver fibrosis-related genes such as tgfbr2b,smad2.Results:After 6 weeks of exposure with diethylnitrosamine,liver tissue pathological HE staining showed liver tissue disorder,hepatocyte degeneration and necrosis,fibrous nodules,and the Picric acid Sirius red staining showed collagen fiber deposition;liver index and body weight decreased significantly;there was no significant change in aspartate aminotransferase.HE staining of each drug treatment group showed that the degree of fibrotic lesions was reduced,and the range was reduced.The Picric acid Sirius red staining showed that the collagen fiber deposition changed significantly compared with the negative control group.In terms of liver index and body weight,the model group was significantly different from the normal control group;the liver index of GFT505 group and DANNINGPIAN group was slightly higher than that of the negative control group(P<0.05),which was statistically significant.The liver index and body weight of the other treatment groups were higher than those of the negative control group,but it was not statistically significant.RT-qPCR results:F3SM group:After the treatment with F3SM,the transcription level of tgfbr2b gene was down-regulated(P<0.01),with statistical significance.The transcription level ofsmad2 gene was down-regulated(P<0.05),which was statistically significant.GFT505 group:After the treatment with GFT505,the transcription level of tgfbr2b gene was down-regulated(P<0.05),which was statistically significant.The transcription level of smad2 gene was significantly down-regulated(P<0.01),which was statistically significant.OCA group:After the treatment with OCA,the transcription level of tgfbr2b gene was significantly down-regulated(P<0.01),with significant difference.The transcription level of smad2 gene was down-regulated(P<0.001),which was statistically significant.HUJIAOJIAN group:After the treatment with HUJIAOJIAN,the transcription level of tgfbr2b gene was significantly down-regulated(P<0.01),with statistical significance.But the transcription level of smad2 gene was not significantly changed.DANNINGPIAN group:After the treatment with DANNINGPIAN,the transcription level of tgfbr2b gene was significantly down-regulated(P<0.01),with statistical significance.But the transcription level of smad2 gene was not significantly changed.Conclusions:Six-month-old zebrafish adult fish were immersed in diethyl nitrosamine at a concentration of 150 mg/L.After 6 weeks of exposure,the zebrafish liver fibrosis model was successfully established.The model is stable and reproducible.After 3weeks of the treatment,the pathological changes showed that the degree of liver fibrosis was improved.Each group of drugs had a certain degree of therapeutic effect on diethylnitrosamine-induced liver fibrosis zebrafish.By detecting the mRNA level of tgfbr2b and smad2 gene,it can be inferred that each group of drugs can play a role by down-regulating liver fibrosis-related genes and TGF?-Smad signaling pathway.