| Objective: To observe the effect of syndrome differentiation of traditional Chinese medicine based on spleen on disease-free survival(DFS)in patients with stage III gastric cancer after radical surgery;to study the effect of urinary benzoic acid,ursolic acid,on apoptosis of gastric cancer cells in vitro Molecular mechanism to further explore the role of gastrointestinal Anyang in inhibiting gastric cancer.Methods: The multi-center non-randomized concurrent control study was used to include the stage III cases of gastric cancer after radical resection in the Chinese medicine + chemotherapy group based on spleen-based syndrome differentiation and the chemotherapy group in the same period without taking the traditional Chinese medicine.Kaplan-The Meier method and the Cox proportional hazard model were used to analyze the factors affecting the disease-free survival(DFS)after radical gastrectomy for single-factor and multi-factor analysis,and the stratified analysis was based on clinical pathological staging.In vitro gastrointestinal urinary active ingredient ursolic acid was used to intervene human gastric cancer cell lines MKN45 and SGC7901,and CCK8 method was used to detect the inhibitory effect of ursolic acid on the proliferation of human gastric cancer cells MKN45 and SGC7901.IC50 values of each group were calculated.Flow cytometry was performed.The cell cycle and apoptosis of gastric cancer cells MKN45 and SGC7901 were observed by Hoechst staining,and the apoptosis-related proteins of human gastric cancer cell line MKN45,Bax,Bcl-2,Bcl-x and Caspase3 were detected by Western Blot assay.The expression changes and explored the mechanism of ursolic acid-induced apoptosis in gastric cancer cells.Results:(1)A total of 555 patients with stage III gastric cancer after radical resection were enrolled in the study,including 253 patients in the Chinese medicine + chemotherapy group and 302 patients in the chemotherapy alone group.Cox multivariate analysis showed that Lauren classification(P = 0.011),clinical pathological stage(P = 0.000)and Chinese medicine treatment(P = 0.000)were independent prognostic factors for DFS after radical gastrectomy.According to the stratified stratification of clinical pathological stage,193 cases of stage IIIB subgroup,including 88 cases of traditional Chinese medicine + chemotherapy group and 105 cases of chemotherapy alone,were analyzed by Cox multivariate analysis.The results showed that Chinese medicine(P=0.027)and Lauren The typing(P=0.014)was an independent influencing factor of DFS after radical resection of stage IIIB.The median DFS of the Chinese medicine + chemotherapy group was not reached,and the median DFS of the chemotherapy group was 31.2 months(P=0.008).The disease-free survival rates of the chemotherapy group at 1,3,and 5 years were 88%,64%,and 56%,respectively.The disease-free survival rates of the chemotherapy group were 78%,45%,and 36%,respectively.The difference was statistically significant.In 108 cases of IIIC subgroup,including 40 cases of traditional Chinese medicine + chemotherapy group and 68 cases of chemotherapy alone,Cox multivariate analysis showed that Chinese medicine(P=0.042)was an independent influencing factor of DFS in patients with stage IIIC of radical gastrectomy.The median DFS of the Chinese medicine + chemotherapy group was 59.1 months,and the median DFS of the chemotherapy group was 21.7 months(P=0.007).The disease-free survival rate of the Chinese medicine + chemotherapy group was 84% at 1,3,and 5 years,respectively.61%,43%,the disease-free survival rate of the chemotherapy group was 75%,30%,and 19%(P=0.013),respectively,and the difference was statistically significant.(2)CCK8 results showed that after ursolic acid intervention MKN45 and SGC7901,the proliferation inhibition rate of the two cells increased with the increase of drug concentration and prolonged action time.Flow cytometry showed that after ursolic acid intervention in cell line MKN45 and SGC7901 24 H,the percentage of apoptosis was increased in the high concentration group(at IC50)compared with the blank control group(0μM)(P<0.05).Statistically significant,MKN45 cell apoptosis mainly occurs in the late stage,and is arrested in the G2 phase.SGC7901 apoptosis mainly occurs in the early stage and is blocked in the S phase.Hochest staining showed that after ursolic acid intervention in MKN45 cell line 24 H,the chromatin of the cells was concentrated,and apoptotic bodies appeared,showing obvious apoptosis characteristics.Western-blot experiments showed increased expression of proapoptotic proteins caspase3 and Bax,and down-regulation of anti-apoptotic proteins Bcl-x and Bcl-2.Conclusion:(1)The spleen-based syndrome differentiation treatment is an independent protective factor affecting DFS in patients with stage III gastric cancer after radical surgery.It can prolong the disease-free survival of patients with stage IIIA,IIIB and IIIC after radical gastrectomy.(2)Ursolic acid inhibited the proliferation of gastric cancer cells MKN45 and SGC7901 in a dose-dependent manner,blocking cell cycle in G2 and S phase respectively;apoptosis of gastric cancer cells MKN45 and SGC7901 was induced in late and early stages,... |
