Effect Of Matrine Combined With Cisplatin On Tumor Microvessel Density And Expression Of COX-2 And MMP2 In Hepatocellular Carcinoma Transplanted Nude Mice

Objective: To investigate the inhibitory effect of matrine enhanced cisplatin on human hepatocellular carcinoma(HCC)cell line Huh7 xenograft in nude mice and its related molecular mechanism.Methods:(1)BALB / c nude mice were subcutaneously injected with human HCC cell line Huh7 to construct the xenotransplantation model.After tumor formation,24 nude mice were randomly divided into four groups(6 mice in each group): control group(physiological saline 10 mg / kg),matrine group(matrine 100 mg / kg),cisplatin group(cisplatin 2 mg / kg),and combination group(matrine 100 mg / kg + cisplatin 2 mg / kg).Each group was administered by intraperitoneal injection according to body weight,once every other day,seven days as a course of treatment,and two courses of treatment.(2)To observe the growth of tumor in each group,the nude mice were sacrificed the next day after the administration,the tumor was excised,and the size of each tumor tissue was measured and weighed,compare the changes of tumor volume and tumor mass,calculate the tumor suppression rate in each group.(3)Immunohistochemistry was used to detect the expression of CD34 antibody in tumor vascular endothelial cells to determine the number of microvessels,calculate and compare the size of microvessel density(MVD)in each group.Western blot and immunofluorescence were used to detect the expression of cyclooxygenase-2(COX-2)and matrix metalloproteinase 2(MMP2)in each group.The correlation between COX-2 and MMP2 expression and CD34-MVD was analyzed in nude mice.(4)SPSS 24.0 software was used to analyze the experimental data,all data were normal distribution test.Normal distribution data between the two groups were compared using two independent sample t test,multiple groups were compared using ANOVA analysis,correlation analysis between the two groups using Pearson correlation coefficient.non-normal distribution data were analyzed and compared by rank sum test.Spearman rank correlation coefficient was used for correlation analysis between the two groups.All statistical analyses were bilateral.All measurement data were expressed as mean±standard deviation((?) ± s),and the difference was statistically significant(P < 0.05).Results :(1)Compared with the control group,the combination group,cisplatin group and matrine group could reduce the tumor volume and weight,and the tumor inhibition rates were 44.67%±6.40%,57.17%±13.90% and 81.00%±18.60%,respectively,and the difference was statistically significant(P < 0.05).(2)The immunohistochemical results showed that the expression of CD34-MVD was the highest in the control group,and the expression of CD34-MVD was decreased in matrine group,cisplatin group and combination group,while the expression of CD34-MVD was the lowest in the combination group(P < 0.05).(3)Western blot analysis showed that compared with the control group,the expressions of COX-2 and MMP2 in matrine group,cisplatin group and combination group were significantly decreased(P < 0.05),and the expressions of COX-2 and MMP2 in combination group were lower than those in matrine group and cisplatin group(P < 0.05).(4)Immunofluorescence results showed that COX-2 was mainly located in the cytoplasm of Huh7 HCC cells,and MMP2 was mainly located in the cytoplasm and membrane.COX-2 and MMP2 were highly expressed in the control group,followed by cisplatin group and matrine group,with the lowest expression in the combined group(P < 0.05).(5)In all tumor tissues,CD34-MVD was positively correlated with the expressions of COX-2 and MMP2,and the correlation coefficients r were0.634 and 0.606,respectively,and the P values were all less than 0.05.The difference was statistically significant.At the same time,the expressions of MMP2 and COX-2 were also positively correlated with each other,r = 0.866,P < 0.001.Conclusion:(1)Matrine combined with cisplatin has synergistic antitumor effect on HCC cells.(2)The expression of COX-2 and MMP2 in HCC Huh7 tumor-bearing nude mice was positively correlated with the expression of CD34-MVD,suggesting that COX-2 and MMP2 may be important angiogenesis factors of HCC.(3)The molecular mechanism of matrine enhancing cisplatin inhibiting angiogenesis in HCC may be to reduce the expression levels of COX-2 and MMP2 to inhibit the growth and invasion of cancer cells.