Effect Of Berberine On Melanoma In Vivo Animal Experiments

Objective:To evaluate the role of berberine in the development of human melanoma A375 cell nude mice transplantation tumor and its related molecular mechanism.Methods: We created the human melanoma A375 cell model of nude mice transplantation based on our previous in vitro experiments.Then we randomly divided20 mice into 4 groups: control group,low-dose berberine group(10mg/kg),medium-dose berberine group(20mg/kg)and high-dose berberine group(25 mg/kg).When we finish our experiment,we measured the volume and weight of tumor,observed the effect of berberine on pathological changes of transplanted tumor in nude mice with the help of HE staining,determined the amount of Ki67 expression by immunohistochemical method to feflect on the ability of proliferation of tumor cells and understood the apoptosis about tumor cells checked by TUNEL essay.Finally,we detected the expression of m RNA and proteins about apoptosis-related factors,such as Bcl-2、Bax and Caspase-3,which is in the way of fluorescence quantitative PCR and Western blotting essay and then we used fluorescence quantitative PCR to detect mi R-582-5p 、 mi R-188-5p expression.Analysis of data was performed using the program of spss 23.0.Results: Measurements of tumor volume and weight at the end of the experiment showed that with increasing concentrations of berberine,the transplanted tumors decreased both in size and weight.The inhibitory effect of high dose of berberine was the most significant in the three experimental groups,whose tumor control rate is 49.43%,70.11%,72.34% in turn(P<0.01).After He staining,it revealed a large number of tumor cells with obvious atypia and visible mitotic figures in the control group,whereas,tumor cell necrosis was observed actually in the medium and high dose groups,and whose necrosis range was wider than in the lower dosage group,while vessel destruction was observed in the high dosage group after berberine intervention.Immunohistochemistry showed that there was a large number of brownish yellow positive cells before berberine was added in,and the number of Ki67 positive cells gradually decreased with the increase of berberine concentration.(P < 0.01)We can find that the number of apoptotic cells increased gradually in the low,medium and high dosage groups with the help of TUNEL essay.(P < 0.01)The following factors involved in apoptosis were measured by quantitative fluorescent PCR.Bax,caspase-3 m RNA expression levels increased and were statistically significant(P < 0.01)in the medium and high doses of berberine,but not in the low dose of berberine,whereas bcl-2 expression levels decreased and were significantly decreased with increasing concentrations of berberine(P < 0.01),also in the low dose of berberine.Results from Western blotting experiments show that the protein expression of Bax was increased gradually with increasing concentrations of berberine compared with the control group(P < 0.01 for medium and high doses),the protein expression levels of Bcl-2 was not statistically significant for low,medium and high doses,and the protein expression levels of Caspase-3 was enhanced(all P <0.01).There was no significant changes in the expression of mir-582-5p and mir-188-5p were observed in the berberine low dose group compared with the control group,,while the expression of the above two mi RNAs was upregulated in the medium and high dose groups(P < 0.05).Conclusion: Berberine has such function which involved inhibiting the growth of human melanoma A375 nude mice transplantation tumor and regulating the expression of apoptosis-related proteins in order to promote apoptosis,and mi R-582-5p 、mi R-188-5p may participate in the above way.But we need make a thorough investigation of the specific mechanism in subsequent experiments.