The Mechanism Of XueShiSanjia On Angiogenesis Of Hepatic Fibrosis By Regulating MiR29b-3p/VEGFA Based On Luobing Theory Of TCM

Objective:To reveal the molecular mechanism that XSSJS acts through miR29b-3p/VEGFA axis to inhibit hepatic fibrosis angiogenesis,the effect of XSSJS on the expression of hepatic fibrosis angiogenesis-related factors,namely VEGFA,PDGF,CD31,CD34,and P-Smad2/3 was tested through in vitro and in vitro experiments.Methodology: PART Ⅰ TCM theory studyAssociating Luobing theory in Traditional Chinese Medicine with hepatic fibrosis as to open up a new frontier in considering such an issue.PART Ⅱ In vitro experimentsa)The experimental groups include the blank group,Model group,miR-29b-3p group,and miR-29b-3p NC group.Except for the blank group,all the other groups utilize TGF-β1 to induce the vitalization of HSCs-T6.Subsequently,miR-29b-3p mimic was transfected in vitro,which serves as a means to amplify the expression of the endogenous gene of miR-29b-3p.Besides,the empty vectors of the mimics(miR-29b-3p mimics NC)was set as negative.CCK8 and BRDU were employed to test the cell vitality and proliferation after the transfection.RT-PCR was used for testing the expression of miR-29b-3p,VEGFA,PDGF,TGF-β,Smad2,Smad3 m RNA.Western Blot was used for testing the expression of VEGFA,PDGF,TGF-β,Smad2/3,P-Smad2/3.The immunofluorescence was used for testing the expression of CD31,VEGFA,PDGF,TGF-β,Smad2/3 and P-Smad2/3.b)Prepare the XSSJS-based serum,after the TGF-β1 intervention,the vitalized HSCs-T6 is subject to the transfection of miR-29b-3P mimics,followed by the intervention of XSSJS.The associated groups are as follows: Blank group,model group,XSSJS+miR-29b-3p group and XSSJS+miR-29b-3p NC group.CC8 measures the proliferation rate,RT-PCR for the m RNA expression of miR-29b-3p,VEGFA,PDGF,TGF-β,Smad2,Smad3.Western-Blot for the protein expression of VEGFA、PDGF、 TGF-β、Smad2/3、P-Smad2/3.Immunofluorescence was used to test the expression of CD31,VEGFA,PDGF,TGF-β1,Smad2/3,P-Smad2/3.c)In luciferase reporter gene test,according to the micro RNAs database(Target Scan,HMDD,miRTar Base),we first filtered out the potential targets for miR-29b-3P,which include VEGFA,TGF-β,SMAD,PDGF.In conjunction with the analysis on the expression trend of those targets,we finally identified VEGFA as the most optimal target gene for hepatic fibrosis angiogenesis.Therefore,we cloned the 3’UTR of the target gene featuring the binding sites for miR-29b-3P,and transferred the clone into the vector of the p GL3-basic Luciferase,as to verify and predict the binding sites of target gene VEGFA.PART Ⅲ In vivo experimentsa)The molding progress and group-based intervention: the serum immune method was used to build the negative contrast for the overexpression glandrelated virus(miR-29b-3p AAV)and miR-29b-3p AAV NC.From the 10 th week of the molding process,miR-29b-3p AAV was transfected in vivo for the Wister rats,and the following groups were considered: blank group,model group,miR-29b-3p AAV group,miR-29b-3p AAV NC group,XSSJS+miR-29b-3p AAV group,and XSSJS+miR-29b-3p AAV NC group.b)The bio-indicator test: The subject animal is dispatched via injection after 24 days of transfection,subsequent to which the hepatic coefficients were calculated,serum was collected for the test of HA,LA,FNA-α and IL6.HE and Masson paint are used for the visual observation of the pathological change in the hepatic tissues.RT-PCR is used for examining the expression of miR-29b-3p,VEGFA,PDGF,TGF-β1,Smad2,and Smad3 m RNA.The MVD were calculated via microscope.Result: PART Ⅰ In vitro experimentTest 1a)The transfection of miR-29b-3p yields statistical high significance(p<0.001)by RT-PCR test.According to the CCK8 test,compared to the blank group,the model and miR-29b-3p NC groups exhibit an increased proliferation rate between 24 h and 48h(p<0.05).However,in the miR-29b-3p group,the high proliferation has been reversed.In addition,The BRDU test results have the same trend change of the CCK8.b)The RT_PCR result shows: Compared to the blank group,the model group has an increased expression(p<0.05)in VEGFA,PDGF,TGF-β,Smad2 and Smad3 m RNA,but a decrease in miR-29b-3p m RNA(p<0.05).Compared to the model group,the miR-29b-3p group possesses a decrease in the m RNA expression of VEGFA,Smad2 and Smad3(p<0.05).c)Western Blot test shows: The model group’s significance(p<0.05)is indicated by the strengthening of the expression in VEGFA,PDGF,TGF-β and P-Smad2/3 in comparison of the blank group.While the miR-29b-3p group displays,in a reversed manner,the weakening of the expression in VEGFA,PDGF,TGF-β(p<0.05).d)Immuno Florescence result shows: The protein expression of CD31,VEGFA,PDGF,TGF-β from the model group has increased(p<0.05)in comparison with the blank group.The protein expression of VEGFA,PDGF、TGF-β、Smad2/3 from the miR-29b-3p group has decreased(p<0.05).Test 2a)serum for 24 h,the proliferation rate has decreased evidentlyin the model group(p<0.05).The XSSJS+miR-29b-3p group presents a significant reduction(p<0.05)in the expression level of VEGFA,PDGF,TGF-β,Smad2 and Smad3 m RNA compared to the model group.b)RT-PCR test result shows: The m RNA expression of VEGFA,PDGF,TGF-β,Smad2 and Smad3 m RNA are significance increase in the model group(p<0.05).Compared to the model group,the XSSJS+miR-29b-3p group present a decrease in the m RNA expression of VEGFA、PDGF、TGF-β、Smad2 Smad3.Compared with XSSJS+ miR-29b-3p NC group,the m RNA expression levels of VEGFA,PDGF,TGF-β,Smad2 and Smad3 in XSSJS+ miR-29b-3p group were significantly decreased.c)Western Blot test shows: The protein expression of VEGFA,TGF-β,P-Smad2/3 significantly rise in the Model group.While VEGFA、TGF-β、P-Smad2/3 declined from XSSJS+miR-29b-3p group compared with model and XSSJS+miR-29b-3p NC group(P<0.05).d)Immunofluorescence test result shows: The protein expression of CD31,VEGFA,TGF-β,Smad2/3,P-Smad2/3 significantly increased from the model group,and decreased from the XSSJS+miR-29b-3p(P<0.05).While,compared with XSSJS+miR-29b-3p NC group those bio-markers indicates no significance.e)Luciferase gene test: After co-transfection of the luciferase reporter plasmid with miR-29b-3p,exogenous miR-29b-3p significantly reduced the luciferase activity of the wild-type VEGFA 3’UTR,but had no effect on the luciferase activity of the mutant VEGFA 3’UTR.These results indicate that Mir-29B-3p has a direct targeted regulatory relationship with VEGFA.PART Ⅱ In vivo experimenta)The HE and MASSON show: Result after the over-expression of miR-29b-3p,compared to the blank group,the model group led to the visual observation of the denaturation of hepatic cells largely in bubble forms,the cell nucleus are centralized and the cytoplasm swollen.The hepatic cells around the central vein formed regional necrosis.Accretion of collagenous fibre cells are spotted,accompanied by medium level of fibrosis.Compared to the model group,the hepatic lobules from the miR-29b-3p group display a clear boundary.Hepatic cells and sinusoids are observed in radiate patterns elongating along the central vein,the sinusoids present no noticeable signs of dilation and compression as well inflammation,cellular infiltration is conspicuous.Result from over-expressing miR-29b-3p while the XSSJS in use,the XSSJS+miR-29b-3p group has relatively insignificant accretion of phorocytosis,a part of veins were connected to each other,forming bridges,surrounding the portal area are a small number of inflamed cells in focal infiltration.The MASSON positive staining area of XSSJS + Mir-29b-3p group was significantly decreased compared with the model group and Mir-29B-3p NC group,which was statistically significant.b)Elisa result shows: Compared with the blank group,LN,HA,TNA-α and IL-6 values have all risen in model group.after the over-expression of miR-29b-3p,all of the previous values have significantly dropped except for the HA.Overexpressing miR-29b-3p along with the intervention of XSSJS led to a decrease in LN,TNA-α,compared with XSSJS+miR-29b-3p NC,and the result is statistically significant.c)RT-PCR result shows: The expression values of VEGFA,TGF-β,PDGF,Smad2 and Smad3 m RNA from the model group have all increased compared to the blank group,especially that the VEGFA value has grown for 37 times and miR-29b-3p m RNA has significantly dropped.Over-expressing miR-29b-3p along with the intervention of XSSJS led to a decrease in all values.The decreased value of VEGFA,PDGF m RNA indicates a significant over-expression compared with miR-29b-3p.Compared with XSSJS+miR-29b-3p NC group,all values were reduced in XSSJS+miR-29b-3p group.d)Western-Blot result shows: the values of VEGFA、TGF-β、PDGF、P-Smad2/3 have significantly increased from the model group.After the overexpression of miR-29b-3p AVV with the intervention of XSSJS,VEGFA,TGF-β,P-Smad2/3 have decreased(statistically significant)compared with model and XSSJS+miR-29b-3p NC groups.e)Immunofluorescence test result shows: Compared to the blank group the values of CD31,CD34,PDGF,TGF-β,P-Smad2/3,VEGFA for the model group have increased(p<0.05).Compared to the model group,all the protein expression,except for PDGF,have decreased.the protein expression from the XSSJS+miR-29b-3p group,except for PDGF,have all decreased compared to the model group(p<0.05).While,there is no statistical significance between XSSJS+miR-29b-3p NC and XSSJS+miR-29b-3p group.f)MVD test: The hepatic vascular density(MVD)level of HF rats in model control group and miR-29b-3p NC group was significantly increased.Compared with the model group and XSSJS+ Mir-29b-3p NC group,MVD in XSSJS+ Mir-29B-3p group was significantly decreased.Conclusion:a)Based on the in vitro experiment,the overexpression of miR-29b-3p is capable of significantly reducing the expression of VEGFA,and also curb the expression of other factors related to hepatic fibrosis.According to luciferase reporter gene test,VEGFA is the downstream target gene of miR-29b-3p has been confirmed.Moreover,we acquired preliminary evidences showing that the hepatic fibrosis related angiogenesis could be inhibited via the adjustment of miR-29b-3p/VEGFA axis using XSSJS.b)The vivo experiment for Wister rats further demonstrated that XSSJS could curb the hepatic fibrosis related angiogenesis through regulating the miR-29b-3p/VEGFA axis,as well as reflected by the apparent improvement in the pathological symptoms.