| Objective:To study the anti-hepatic fibrosis effect and explore the mechanism of Qiwei Qinggan Powder in vivo and in vitro.Methods:（1）50 male Wistar rats were divided into 5groups with 10 rats per group:blank group,hepatic fibrosis model group and Qiwei Qinggan Powder low,middle and high dose group[135,270,405 mg/（kg·d）].Except blank group,each group was treated intragastric administration with 50%CCl4-peanut oil solution twice a week.Rats in the drug group were treated intragastric administration with Qiwei Qinggan Powder at the corresponding dose once a day.After 10 weeks,the contents of ALT,AST and ALP were measured with serum after anesthesia and centrifugation.The liver tissue was taken,the gross shape was observed,weighed,and the liver index was calculated.The liver tissue was taken,the content of HYP was measured;the liver tissue was taken,fixed,embedded,sectioned and stained with HE and Masson to observe the histopathological changes of the liver.Theα-SMA in the liver tissue was detected by immunohistochemistry.By using transcriptome sequencing technology,combined with bioinformatics analysis,a large number of literatures were searched to screen and enrich the signal pathway related to hepatic fibrosis.Extraction of RNA from liver tissue,detection ofα-SMA,CollagenⅠ,JAK2 and STAT3 m RNA content by q-PCR.Extraction of protein from liver tissue,detection ofα-SMA,CollagenⅠ,JAK2,p-JAK2,STAT3 and p-STAT3 expression level by Western Blot.（2）16 Wistar rats were divided into two groups:blank group and Qiwei Qinggan Powder group[1350 mg/（kg·d）].The rats in the treatment group were treated intragastric administration with Qiwei Qinggan Powder once a day.After 7 days,anesthesia,blood collection and centrifugation were performed.The serum in the same group was combined,inactivated at 56℃and filtered by filter membrane.HSC was divided into blank group,low（10%）,middle（15%）and high（20%）dose group.The content ofα-SMA,CollagenⅠ,JAK2,STAT3 m RNA were detected by q-PCR.The expression ofα-SMA,CollagenⅠ,JAK2,STAT3,p-JAK2,p-STAT3 were detected by Western Blot.MTT method was used to detect the effect of different concentration of drug serum and different administration time[24h,48h,72h]on cell proliferation,and to determine the inhibition effect and the best effect time of Qiwei Qinggan Powder on cell proliferation.Examine the apoptosis ratio of HSC-T6 with Flow Cytometry.Results:（1）The liver of the blank group was bright red,soft and smooth;the liver of the model group was dark red or even yellow,very rough;the liver of the high,medium and low dose group was dark red,slightly rough.Compared with the blank group,the liver index of the model group was significantly increased（P<0.01）,the serum level of ALT,AST,ALP,HYP were significantly increased（P<0.01）;compared with the model group,the liver index of the low dose group was decreased（P<0.05）,the serum level of ALT,AST,HYP of each dose group were decreased（P<0.01）,ALP content of the high dose group was significantly decreased（P<0.01）.HE and Masson staining showed that the structure of the liver lobules in the blank group was complete,the hepatocytes were arranged orderly,the plasma was even,and there was no degeneration,necrosis,inflammatory cell infiltration,fibrous tissue proliferation;the structure of the liver lobules in the model group was damaged,the arrangement of the liver plates was disordered,a large number of inflammatory cells infiltration,balloon like changes of liver cells,fibrous tissue proliferation,and some samples even showed bridging connection and pseudolobules.In each dose group of Qiwei Qinggan Powder,the fibroproliferation,inflammatory cell infiltration,cell degeneration and necrosis decreased.From the Masson staining results,compared with the blank group,the collagen content in the model group increased significantly（P<0.01）;compared with the model group,the collagen content in each dose group decreased significantly（P<0.01）.The results of immunohistochemistry showed that compared with the blank group,the positive expression area ofα-SMA in the model group increased greatly,while that in each dose group of Qiwei Qinggan Powder decreased.The results of q-PCR showed that compared with the blank group,the m RNA levels ofα-SMA,CollagenⅠin the model group increased significantly（P<0.05）.Compared with the model group,the m RNA levels ofα-SMA,CollagenⅠin the each dose group of Qiwei Qinggan Powder decreased significantly（P<0.05）.Western Blot results showed that compared with the blank group,α-SMA and CollagenⅠprotein in the model group increased significantly（P<0.01,P<0.05）.Compared with the model group,α-SMA and CollagenⅠprotein in each dose group of Qiwei Qinggan Powder decreased significantly（P<0.01）.（2）The JAK2/STAT3 signal pathway was obtained after transcriptome sequencing and literature search.（3）The results of q-PCR in vivo showed that compared with the blank group,the levels of JAK2 and STAT3 m RNA in the model group increased significantly（P<0.05）;compared with the model group,the levels of STAT3 m RNA in the high dose group of Qiwei Qinggan Powder decreased significantly（P<0.01）;the levels of JAK2 m RNA in the low dose group decreased significantly（P<0.05）.Western Blot results showed that compared with the blank group,JAK2,p-JAK2,STAT3,p-STAT3 protein in the model group increased significantly（P<0.01,P<0.01,P<0.01,P<0.05）;compared with the model group,JAK2,p-JAK2,STAT3 protein in each dose group decreased significantly（P<0.01）;p-STAT3protein in the high and middle dose groups decreased significantly（P<0.01,P<0.05）.The results of q-PCR showed that compared with the control group,the m RNA expression ofα-SMA,JAK2 and STAT3 in each dose of serum group decreased significantly（P<0.01）,and CollagenⅠm RNA in each dose of serum group decreased significantly（P<0.01,P<0.05,P<0.01）.Western Blot results showed that compared with the control group,the expression ofα-SMA,CollagenⅠ,JAK2,p-JAK2,STAT3 and p-STAT3 protein in the low dose serum group decreased significantly（P<0.01）;the expression ofα-SMA,CollagenⅠ,JAK2,p-JAK2,STAT3 and p-STAT3 protein in the middle dose serum group decreased significantly（P<0.01）;the expression ofα-SMA,CollagenⅠ,p-JAK2 and p-STAT3 protein in the high dose serum group decreased significantly（P<0.01）.The results of MTT showed that at 24 h,the OD value of each dose group of drug serum decreased significantly（P<0.01）,the inhibition rate of proliferation was 42.95%,50.89%,44.93%;at 48 h,the OD value of middle and high dose groups of drug serum decreased significantly（P<0.01）,the inhibition rate of proliferation was 32.03%,33.55%;at 72 h,the OD value of each dose group of drug serum decreased significantly（P<0.01）,the inhibition rate of proliferation was 21.36%,33.26%,33%.However,there was no significant difference in the inhibition rate of cell proliferation among the drug containing serum groups at 24 h,48 h and 72 h（P>0.05）,so follow-up experiments were carried out at 24 h after administration.The results of flow cytometry showed that compared with the control group,the apoptotic rate of the low and high dose serum group was significantly higher（P<0.01,P<0.05）.Conclusion:Mongolian medicine Qiwei Qinggan Powder has the effect of treating hepatic fibrosis and can inhibit the activation,proliferation and apoptosis of HSC.Meanwhile,in the process of anti hepatic fibrosis,Qiwei Qinggan Powder can reduce the expression of JAK2,p-JAK2,STAT3 and p-STAT3 in JAK2/STAT3 signal pathway.Therefore,we speculate that Qiwei Qinggan Powder may affect HSC through JAK2/STAT3 signaling pathway,so as to play an anti hepatic fibrosis role. |
