Anti-Colon Cancer Activity And Mechanism Action Of Total Diterpenoids From Scutellaria Barbata D.Don

Colorectal Cancer(CRC)is one of the most common malignancies worldwide,with the third highest incidence of cancer worldwide and the second highest mortality of cancerrelated mortality worldwide.In recent years,the treatment methods for CRC are constantly updated,and the combination therapy of targeted drugs is the main treatment method for CRC patients.Despite the development of drug therapy,the treatment of CRC has been difficult to break through,the prognosis is still poor,the median survival period is only 25-30 months.The pathogenesis of CRC is complicated.Previous studies showed that Wnt,PI3K/AKT,Hedgehog(HH),Erb B,Notch,NF-κB and MAPK can promote the carcinogenesis of CRC,while BMP,AMPK and Hippo can inhibit the development of CRC.Existing clinical reports have found that the HH signaling pathway plays an important role in the formation of CRC,which is involved in the dedifferentiation of the primary metastatic site of CRC and plays an important role in the adenoma-carcinoma pathway.In addition,HH signaling pathway has been shown to be involved in cell proliferation,migration,invasion and cell cycle of various tumor cells.Therefore,targeting HH pathway may be an important breakthrough point for the treatment and Prevention of CRC.Previous studies have shown that diterpenoid alkaloid monomers of Scutellaria barbata D.Don have good anti-tumor activity,and the anti-proliferation activity of Scutellaria barbata D.Don has good anti-colon cancer activity,but there is no complete study on the mechanism of anti-colon cancer.Because of the low content of diterpenoid alkaloid monomers,the enrichment of diterpenoids with similar structure and polarity(Bzl-D)can complement the study of its anti-tumor effect and mechanism.In this study,the dried whole grass of scutellaria barbata D.Don was percolated with85% ethanol,and the percolate was concentrated and extracted with petroleum ether,ethyl acetate,n-butanol and water,respectively,the diterpenoids were mainly concentrated in the petroleum ether layer and the ethyl acetate layer by TLC analysis.The anti-proliferation activity of the diterpenoids was screened by CCK8 method in vitro and the ethyl acetate part was confirmed as the follow-up part.Total diterpenoids(Bzl-D)were isolated from the ethyl acetate by Polyamide column chromatography with DCM,which accounted for 22.8% of the total mass of the ethyl acetate The chemical constituents of Bzl-D were analyzed and identified by UPLC-q TOF-MS,and 14 new croxane-type diterpenoids were identified.The effect of Bzl-D on the proliferation of HT29,HCT116 and SW480 cells was detected by CCK8 method,the results showed that Bzl-D had different effects on the viability of three kinds of colon cancer cells after 24 hours,especially on the proliferation of HT29 cells.The IC50 of Bzl-D was 22.4 μg/ml.Firstly,Western blotting was used to evaluate the regulatory effect of Bzl-D on HH pathway protein in HT29 cells,the expression of PTCH1,SMO,SHH and Gli1 proteins in HT29 cells were detected after treated with Bzl-D at different concentrations for 24 H.The results showed that compared with CTRL Group,the expression levels of GLI and SMO protein in HT29 cells treated with Bzl-D at 10 μg/ml were significantly decreased(p < 0.001),while the expression levels of PTCH1 and Shh were not significantly different(P > 0.05)The expression levels of GLI,SMO,SHH and PTCH1 protein were significantly decreased when the concentrations were increased to 20 and 40μg/ml(p < 0.001,p < 0.01,p < 0.05).These results suggest that Bzl-D can inhibit the proliferation of HT29 cells by regulating the expression level of major proteins of HH pathway.The target of HT29 was further studied by cell thermal migration assay(CETSA),the results showed that the expression level of Shh protein in Bzl-D Group was higher than that in CTRL group at 10 μg/ml(p < 0.001 at 40 ° C,42 ° C and 44 ° C,P < 0.01 at 46 ° C),suggesting that Bzl-D binds to SHH protein,and it has high thermal stability.Secondly,the effects of Bzl-D on migration and invasion of HT29 cells were evaluated by cell scratch test and Transwell Assay.The results showed that in the scratch test,compared with CTRL Group,the inhibition of Ht29 cell motility gradually appeared in the range of 5-20 μg/ml with the increase of Bzl-D concentration,compared with the CTRL Group,the Transwell Group showed a significant decrease in migration rate(p < 0.05,p < 0.01,p <0.001),and the Transwell group showed a significant decrease in migration rate(p < 0.05,p< 0.01,p < 0.001),in the same concentration range,the migration rate of HT29 cells was also significantly decreased in a dose-dependent manner(p < 0.001,p < 0.001,p < 0.001).At the same time,Transwell experiment also investigated the different invasiveness of BzlD to HT29 cells in the same range of concentration.The results showed that compared with CTRL group,Bzl-D decreased the invasive ability of HT29 cells in a concentrationdependent manner(p < 0.001),and Bzl-D inhibited the invasive ability of HT29 cells at different concentrations(p < 0.001).Annexin v-fitcpi staining,PI staining and Flow cytometry staining were used to evaluate the effect of Bzl-D on apoptosis and cell cycle of HT29 cells.The results showed that compared with CTRL group,Bzl-D induced G 2/M phase arrest in different degrees in the range of 5-20 μg/ml(p < 0.01,p < 0.001,p < 0.001).Finally,a colon cancer subcutaneous tumorigenic nude mouse model was constructed by subcutaneous injection of HT29 cells into nude mice,which were continuously intraperitoneally injected with Bzl-D(50 mg/kg)for 16 days,and mouse tumor volume and body weight were measured every two days,and mice were sacrificed on Day 16,the weight of tumor was measured and the pathological changes of tumor tissue were observed by HE staining.The results showed that Bzl-D(50mg/kg)could inhibit the growth of HT29 cell xenograft tumor in mice by 68%(p < 0.001),the tumors in the Bzl-D(50 mg/kg)group had more necrotic areas than those in the control group,which was basically consistent with the results of anti-tumor effect in vitro.Taken together,this study suggests that Bzl-D can target SHH to inhibit the expression of HH pathway-related proteins in vitro,thereby inhibiting the proliferative effects of colon cancer HT29 cells and affecting the migration and invasion ability of tumor cells,it can also induce apoptosis of tumor cells and arrest G 2/M phase cells,and significantly inhibit tumor growth in vivo.These results provide scientific basis for the rational development and utilization of Scutellaria barbata and the discovery of new anti-tumor drugs with obvious effects and clear components for CRC.