| Objective:to observe the influence of Jiajiansanjiasan on the HIF-1α/VEG F A/VEGFR-2 and PDGFB/PDGFR/βsignal pathway of pathological angiogenesis of rats with immuno-hepatic fibrosis, todiscuss the effect and mechanism of Jiajiansanjiasan, finally to uncover partly the scientific connotation of’Zhukejiao(Pathogen and the right-Qi integrated to induce disease)’Methods:60 SPF Wister male rats were chose to be divided into normal control group 20(normal group), model control group20 (model group), positive control group 10(Biejiapian group) and Jiajiansanjiasan group 10(Sanjiasan group). Porcine serum was adopted and injected intraperitoneally for all rats except the normal control group to set up immuno-hepatic fibrosis rat model. Intraperitoneal injection was lasted for 12 weeks, and then six rats of the normal group and six rats of the model group were executed randomly. Liver tissue was obtained. The pathological morphology of liver was observed under light microscope after dyed by HE and MASSON to estimate the model. The liver tissue and blood sample of the model group were kept and marked as model 1 group; the corresponding indexes were detected and compared with other group in further experiment. After the model setting up was succeeded, intragastric administration of Jiajiansanjiasan was given to the Sanjiasan group, intragastric administration of Fufang Biejia Ruangan Tabletwas given to the Biejiapian group, while intragastric administration of normal saline was given to normal and model group (marked as model 2 groups). All rats were treated for 60 days before executed. Liver tissue of rat was obtained. The pathological morphology of liver was observed under light microscope after dyed by HE and MASSON. Ultrastructure of hepatic cells and sinusoidal endothelial cells was observed under electron microscope. Microvessel density was observed after marked by CD34. Protein level of HIF-1α, VEGFA/VEGFR-2 and PDGFB/PDGFR-β was detected by wester-blot method. mRNA level of HIF-1α, VEGFA/VEGFR-2 and PDGFB/PDGFR-βwas detected by RT-PCR method. Blood sample was drawn from the rat heart, hepatic function indexes were detected by fully automatic biochemical analyser, indexes of hepatic fibrosis were detected by ELISA method.Results:1. Hepatic pathology observation of the model ratsBy the ends of modeling, the hepatic tissue of the model 1 group had appeared fibrous bands derived from the portal area and central vein which enclosed the hepatic lobule. There was trend of pseudolobuli formation or had formed pseudolobuli. By then ends of be treated 60 days, the hepatic tissue of the model 2 group had appeared obvious fibrous tissue around the portal area and central vein, there was obvious pseudolobuli and fibrous contaction compared with model 1 group.2. The indexes of hepatic function and fibrosisThe AST/ALT/GLB level of the model 2 group was higher than the normal group and the model 1 group(P<0.01 or P<0.05), the ASL level of the sanjiasan group was significant lower than the model 2 group(P<0.01) but had no significant difference with the normal group(P>0.05), the GLB level was higher than the normal group(P<0.05); the AST level of the biejiapian group was significant lower than the model 2 group(P<0.01) and had no significant difference with the normal group and sanjiasan group(P>0.05).The HA/LN/CⅣ/PCⅢ level of the model 2 group was significantly higher than the other four groups which includes model 1 group(P<0.01 or P<0.05); thePCⅢ/LA level of the sanjiasan group was higher than the normal group(P<0.01 or P<0.05), PCⅢ/LA/LN level was lower than the model 2 group(P<0.01 or P< 0.05);thePCⅢ/LA level of the biejiapian group was higher than the normal group(P <0.01 or P<0.05), the PCIII/LA/LN level was lower than the model 2 group(P <0.01 or P<0.05), but had no significant difference with the sanjiasan group(P> 0.05).3. Microvessel density (MVD)The MVD levels of the groups which were modeled were significantly higher than normal group(P<0.01 or P<0.05);The MVD level of the model 2 group was significantly higher than Sanjiasan group and Biejiapian groups(P<0.01),but there was no significant difference comparing with the model 1 group(P>0.05). The MVD level of the Sanjiasan group was significantly lower than the model 2 group(P<0.01), but there was no significant difference comparing with Biejiapian group(P>0.05).4. The HIF-α-VEGFA/VEGFR-2 and PDGFB/PDGFR-β signal pathwayThe VEGFA level of the model 1 group was higher than the normal group(P< 0.05), all indexes of the model 2 group were higher than the normal group(P< 0.01 or P<0.05), the VEGFA and PDGFR- β level of the sanjiasan group was higher than the normal group(P<0.05), HIF-1α level was lower than the normal group(P<0.01), the HIF-1 α/VEGFR-2/PDGFB level was lower than the mordel 2 group(P<0.05), the HIF-1 α/VEGFR-2 level was lower than the biejiapian groupHIF-1 α/VEGFR-2; the VEGFA/VEGFR-2/PDGFR-β level of the biejiapian group was higher than the control group(P<0.05), the PDFGB level of the model 2 group was lower than the model 2 group(P<0.05), there was no significantdifference between the sanjiasan group(P>0.05)。The HIF-1 α/VEGFA/VEGFR-2/PDGFB/PDGFR-β mRNA of the model 2 group was higher than the normal group(P<0.01 or P<0.05), the HIF-1 a /VEGFR-2/PDGFB mRNA of the sanjiasan group was lower than the model 2 group(P<0.01), there was no significant difference between the normal and biejiapian group(P>0.05).Conclusion:1. The pathomorphism observation, hepatic function and fibrosis indexes have shown that intraperitoneal injection of porcine serum can set up immuno-hepatic fibrosis rat model successfully. The best model appeared 60 days after modeling. Not only had the pathological morphology shown the liver is under fibrosis condition, but also the hepatic fibrosis indexes had appeared obvious abnormity in the serum level.2. the serum AST, HA, LN and PCIII level of the sanjiasan group was lower than the model 2 group, which has shown that Jiajiansanjiasan can improve the hepatic function and fibrosis severity of rats with immuno-hepatic fibrosis, it has function to anti-hepatic fibrosis..3. The MVD of the sanjiasan group was lower than the MVD of the model 2 group, which has shown Jiajiansanjiasan can prevent pathological angiogenesis effectively of rats with immuno-hepatic fibrosis; it may be the mechanism of its anti-hepatic fibrosis function.4.the HIF-1α/VEGFR-2/PDGFBprotein and mRNA of sanjiasan group was lower than the model 2 group, which has shown Jiajiansanjiasan can prevent pathological angiogenesis of rats with immuno-hepatic fibrosis, the mechanism may be it can promote the micro-circulation and release anaerobic condition to affect the expression of HIF-1α/VEGFR-2/PDGFBprotein and mRNA, finally affect the HIF-1α/VEGFA/VEGFR-2 and PDGFB/PDGFR-β signal pathway.
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