| Objective:Observe the anti-tumor effect of the method of JianPi HuaTan in TCM through in vivo and in vitro studies and explore its regulation mechanism of EMT in lung cancer,providing experimental evidence for the JianPi HuaTan method and JianPi HuaJi recipe.Methods:(1)In vitro study:We prepared the medicated serum of JianPi HuaJi recipe,MTT assay and cell scratch experiment were used to observe the effect of medicated serum on the proliferation and migration of lung cancer A549 cells.The effect of medicated serum on the regulation of EMT markers E-cad,N-cad,Snail,Slug and Twistl mRNA and protein expression in A549 cells were detected by qRT-PCR and Western Blot experiment.(2)In vivo study:BALB/c-nu mouse lung cancer xenograft model was established by subcutaneous injection of lung cancer A549 cells,mice were sacrificed after 14 days of intervention in groups after successful model building,and the tumor volume of each group was compared.The effect of medicated serum on the regulation of EMT markers E-cad,N-cad,Snail,Slug and Twistl mRNA and protein expression in tumor tissues were detected by qRT-PCR and Western Blot experiment.Results:(1)In vitro study:The results of the MTT assay showed that compared with the control group,the medicated serum of JianPi HuaJi recipe with a concentration of 10%-25%can effectively inhibited the proliferation of lung cancer A549 cells(P<0.01),and the inhibitory effect was time-concentration dependent.The concentration of the medicated serum reaches the optimal concentration at 20%;the results of the cell scratch assay showed that the cell migration rate of the medicated serum group was significantly lower than that of the control group(P<0.01);The results of qRT-PCR experiments showed that compared with the control group,the expression of E-cad mRNA in the medicated serum group was significantly up-regulated,and the expression of N-cad,Snail,Slug and Twistl mRNA was significantly down-regulated(P<0.01);Western blot experiment results showed that the expression of E-cad protein in the medicated serum group was significantly up-regulated,and the expression of N-cad,Snail,Slug,and Twistl protein was significantly down-regulated;the results of qRT-PCR experiment and Western blot experiment were basically the same,which proved that the medicated serum of JianPi HuaJi recipe has a certain regulatory effect on EMT-related markers of lung cancer.(2)In vivo study:After 21 days of subcutaneous injection of A549 cells in mice,all mice subcutaneously transplanted tumors reached 100-150mm3,suggesting that the tumor-bearing mouse model was successfully constructed.After the completion of the model building,mice in the TCM group were gavage with JianPi HuaJi recipe,the mice in the control group were treated with sterile ddH2O by gavage,and the mice in the cisplatin group were treated with intraperitoneal injection of 0.2mg/ml cisplatin solution.During the drug intervention,there was no death of mice in each group,and no significant differences in diet,drinking,and daily activities were seen.After 14 days of drug intervention,the mice were sacrificed,and the tumor volume of each group was compared.The results showed that compared with the model group,the tumor volume of the JianPi HuaJi recipe group and the cisplatin group were significantly reduced,and the results were statistically different(P<0.05);The results of qRT-PCR experiments showed that compared with the model group,the expression of E-cad mRNA in the JianPi HuaJi recipe group and the cisplatin group was significantly up-regulated(P<0.01),and the mRNA expressions of N-cad,Snail,Slug and Twistl were all significant down-regulation(P<0.05);Western Blot experiment results showed that compared with the model group,the E-cad protein expression levels of the JianPi HuaJi recipe group and the cisplatin group were significantly up-regulated(P<0.01),while N-cad,Snail,Slug and Twistl protein expression levels were significantly down-regulated(P<0.05);the results of qRT-PCR and Western blot experiment were basically the same,indicating that JianPi HuaJi recipe has a regulatory effect on EMT-related markers and inhibited the growth of subcutaneous transplanted tumors in nude mice by regulating EMT of lung cancer.Conclusion:The JianPi HuaJi recipe established according to the method of JianPi HuaTan can effectively inhibited the proliferation and migration of lung cancer A549 cells,and significantly inhibited the growth of lung cancer subcutaneously transplanted tumors in BALB/c-nu mice.The anti-tumor mechanism may be up-regulated E-cad,down-regulate the expression of N-cad,Snail,Slug and Twistl,and then reverse the epithelialmesenchymal transition. |
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