| Objective:To observe whether the Xueshi Sanjia powder based on the theory of "Zhu Ke Jiao" can play the role of anti liver fibrosis by regulating HSC autophagy and inhibiting hepatic sinusoidal capillarization,so as to reveal the scientific connotation of the theory of "host guest interaction" and provide some scientific basis for the theory of clinical treatment of liver fibrosis with traditional Chinese medicine.Method 1:Xueshi Sanjia powder group(high,medium and low)and Biejiajian Pill group,with 10 rats in each group.Except for the blank control group,the other groups of rats were established by intraperitoneal injection of pig serum according to the method reported in the literature.After the completion of modeling,each group was given corresponding drugs for treatment,lasting for 4 weeks.At the end of the treatment,the samples were taken for relevant detection.Method 2:in vitro experiment:(1)preparation of medicated serum:30 male Wistar rats were divided into five groups after adaptive feeding for one week,including normal group,Xueshi Sanjia powder(high,medium and low)group and Biejiajian Pill group,with 6 rats in each group.(2)cell model:hepatic stellate cells were treated with TGFβ1 for 0,12,24 and 48 hours to induce the activation of hepatic stellate cells to replicate the cell model of hepatic fibrosis.The cells were divided into six groups:normal group,model group,Xueshi Sanjia powder(high)group,modified Sanjia powder(middle)group,Xueshi Sanjia powder(low)group,Biejiajian Pill group(3)medicated serum intervention:after the completion of the model,the corresponding medicated serum was used to intervene the cells.After the intervention,cells were collected or fixed for follow-up study.Result:1.Xueshi Sanjia powder can improve the pathological damage of liver fibrosis rats(1)The effect on the general condition of ratsAfter modeling,except for the blank control group,the other groups of rats gradually showed mental depression,slow response,slow behavior,poor appetite,less smooth fur,and some of them even had tail fur shedding and slow growth of body weight.After treatment,the rats in the blank control group had rapid weight growth,fat body,quick reaction and no abnormal performance.In the model group,the rats continued to show poor overall state,dry and yellow fur,hyperemia of eyeballs,swelling of limbs,abdominal distension and stiffness,purple and dark tongue,sparse stool,et;In the high-dose group,the gloss of skin and hair increased significantly,the weight increased significantly,the mental state improved significantly,the activity increased significantly,the phenomenon of clustering reduced,the diet increased,and the stool returned to normal.After treatment,the mental state of the rats in the Xueshi Sanjia powder(medium and low)dose groups was improved,the fur was glossy and smooth,but the color was still dark,the weight increased,the diet increased,but the stool was sparse and the abdomen was stiff;after treatment,the overall condition of the rats in the Biejiajian Pill group was improved to a certain extent.(2)The effect on liver injuryThe liver injury of the treatment groups could be improved in varying degrees,and the serum ALT,AST,HA and LDH of the Xueshi Sanjia powder(high)dose group were significantly decreased(P<0.01);compared with the Biejiajian Pill group,the indexes of the Xueshi Sanjia powder treatment groups had no significant difference There was significant difference(P>0.05).(3)The effect on pathological morphologyExtensive fibrosis and hepatocyte degeneration and necrosis appeared in the model group;part of the veins in the Xueshi Sanjia powder(high)dose group were connected to form a bridge,and no obvious other abnormality was found;the local hepatic sinuses in the Xueshi Sanjia powder(medium)dose group were slightly dilated,congestion,and local hemorrhage A small amount of inflammatory cells infiltration in the portal area;diffuse fatty degeneration of hepatocyte2.Xueshi Sanjia powder can regulate the autophagy of HSCs and play the role of anti liver fibrosis(1)The effect on autophagy index expression of HSCs in vivoCompared with the blank control group,the protein expressions of beclin-1,p62,LC3 Ⅰ/Ⅱ and LC3 in the model group were significantly different(P<0.05/P<0.01);compared with the model group,the treatment groups could improve the expression of autophagy protein of HSCs in different degrees,and the protein expressions of beclin1,p62,LC3 Ⅰ/Ⅱ and LC3 in the high-dose group were significantly different(P<0.05)/Compared with Biejiajian Pill group,Western blot showed that there was no significant difference in the protein expression of beclin-1,p62 and LC3 Ⅰ/Ⅱ between the two groups(P>0.05),but there was significant difference in p62 and LC3 between the two groups(P<0.05).(2)The effect on the expression of activation index of HSCs in vivo modelThe expression of TIMP-1 was significantly different(P<0.01);compared with Biejiajian Pill group,Q-PCR results showed that the expression of MMP-1 was significantly decreased in middle dose group(P<0.01),and TIMP-1 was significantly increased in low dose group(P<0.01)There was no significant difference in TIMP-1 and MMP-1 among the three groups(P>0.05).(3)The effect on the activity of HSCs in vitro modelThe morphology of HSC in the normal group had little change compared with that before the intervention,and the cells grew well,proliferated steadily,and the size was uniform;in the model group,the cells proliferated rapidly,and the cell morphology showed star like changes,showing a significant activation state;in the three groups of Xueshi Sanjia powder(high,medium,and low),the cells grew slowly,and Biejiajian Pill also showed a certain degree of cell proliferation inhibition.(4)The effect on the expression of autophagy related proteins of HSCs in vitroIn each treatment group was improved in varying degrees,and the expressions of beclin-1,p62,LC3 Ⅰ/Ⅱ and LC3 in the Xueshi Sanjia powder(high)dose group were significantly different(P<0.01/P<0.05).(5)The effect of HSCs activation protein expression in vitrothe protein expressions of TIMP-1,MMP-1,COL-1 and LN in the Xueshi Sanjia powder(high)dose group were significantly different(P<0.01/P<0.05).3.Xueshi Sanjia powder can inhibit hepatic sinusoidal capillarization(1)Influence on LSECs apertureCompared with the blank control group,the number of fenestra of Lsecs in the model group decreased or even disappeared,the number of fenestra of lsecs increased,and the basement membrane formed(P<0.01);compared with the model group,the number of fenestra of lsecs in each treatment group decreased in varying degrees,and the fenestra of lsecs in the high-dose group of Jiajian Sanjia powder was significantly lower Compared with the Biejiajian Pill group,the window of lsecs in the modified Sanjia powder(low)dose group decreased significantly(P<0.05).(2)The effect on the markers of hepatic sinusoidal capillarizati on(3)The effect on the composition of basement membraneThe expression of coii-1 and LN mRNA in each treatment group was improved in varying degrees,in which the expression of coll-1 mRNA in the Xueshi Sanjia powder(high)dose group was significantly decreased(P<0.01),and the expression of LN mRNA was significantly increased(P<0.01)Compared with Biejiajian Pill group,there were significant differences in coii-1 mRNA and LN mRNA in low-dose group(P>0.05).Conclusion:1.Xueshi Sanjia powder can improve the general situation and pathological damage degree of liver fibrosis rats.2.Xueshi Sanjia powder can regulate the autophagy of HSCs and inhibit liver fibrosis.3.Xueshi Sanjia powder can inhibit the capillarization of hepatic sinuses.4.Regulating HSCs autophagy and reversing hepatic sinusoidal capillarization may be the molecular mechanism of Xueshi Sanjia powder’s anti hepatic fibrosis effect.5.Part of the scientific basis for the treatment of liver fibrosis based on the theory of"host guest interaction" may be related to HSCs autophagy and hepatic sinusoidal capillarization... |
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