| Objective:Modern studies have shown that the total flavonoids of Spatholobi Caulis has anti-tumor pharmacological effects,inhibits the growth of cervical cancer HeLa cells and has high safety.In this study,whether TGF-β1/Notch signaling pathway is associated with the occurrence and development of cervical carcinoma was studied at cellular level and animal level,and at the same time we explored the molecular mechanism of flavonoids of Spatholobi Caulis through TGF-β 1/Notch signaling pathway in the intervention of cervical carcinoma.It provides scientific theories and experimental basis for the research and development of Zhuang medicine Zhuang Medicine Spatholobi Caulis in the treatment of cervical carcinomaMethods:1.The Spatholobi Caulis crushed,70%ethanol water bath reflux extraction,obtained crude extract,weighed,and Separation of macroporous resins by column chromatography.Elute with different concentrations of ethanol,(0%,30%,70%)collect 30%,70%ethanol eluate,dry and weigh.Rutin was used as a standard sample,UV spectrophotometer method was used to detect the crude extract of Spatholobi Caulis and Spatholobi Caulis 30%,70%ethanol eluate flavonoids2.In vivo experiments,nude mice transplanted tumor models were established by subcutaneous injection,divided into 7 groups for administration,observe the survival condition and tumor growth of nude mice;The mRNA expressions of Notchl,TGF-β1,Heyl and Snail were detected by PCR in nude mice;Determination of interleukin-6(IL-6)and tumor necrosis factor(TNF-α)in serum of nude mice by enzyme-linked immunosorbent assay(ELISA);Determination of superoxide dismutase(SOD)content in tumor tissues of nude mice by hydroxylamine method and malondialdehyde(MDA)content in thiobarbituric acid method;Hematoxylin and eosin(HE)staining was used to observe the histopathological changes of each tumor3.In vitro experiments using human cervical carcinoma HeLa cells as the research object,using CCK-8 method to observe the inhibitory effect of total flavonoids of Spatholobi Caulis on HeLa cells and the time-effect relationship;The expression of Notch 1,TGF-β 1,Hey 1 and Snail mRNA in HeLa cells was detected by RT-PCR.The expression of Notchl,TGF-β 1,Heyl and Snail protein in HeLa cells was detected by Western blot;Using inverted microscope to observe cell growth and morphological changesResults:1.Extraction,purification and content determination of total flavonoids from Spatholobi Caulis:the content of flavonoids in 30%ethanol extract of Spatholobi Caulis was 61.88%,the content of flavones in the extract of 70%ethanol was 43.13%,the content of flavonoids in Spatholobi Caulis extract was 19.90%by UV spectrophotometry2.The anti-cancer activity of total flavonoids from Spatholobi Caulis in vivo and its mechanism study showed that:thirty-five nude mice were inoculated with human HeLa cell suspension and the rate of tumor formation was 100%after one week.Compared with the model group,the DAPT group,the positive group,and the Spatholobi Caulis high-low-dose group all inhibited the growth of tumor tissue(P<0.01 or P<0.05).The tumor inhibition rates of the positive group,the DAPT group,and the high,medium,and low doses of Spatholobi Caulis were 54.93%,42.26%,40.10%,25.44%,and 20.83%,respectively.Compared with the model group,Spatholobi Caulis total flavonoids high,medium dose group and positive group both can decrease the expression of IL-6 in serum of tumor-bearing nude mice(P<0.05),and those in the DAPT group decreased significantly IL-6 expression levels(P<0.01).Compared with the model group,the high dose group and the positive group could decrease the level of TNF-α in the tumor-bearing nude mice(P<0.05 or P<0.01),and DAPT group significantly reduced the expression of TNF-α in the tumor-bearing nude mice serum(P<0.01).Compared with the model group,the high-dose total flavonoids of Spatholobi Caulis could significantly reduce the content of MDA in the nude mice(P<0.01),and both the DAPT group and the positive group could significantly reduce the content of MDA in the tumor tissue(P<0.01).Compared with the model group,the high and medium dose of total flavonoids of Spatholobi Caulis could significantly increase the activity of SOD in tumor tissue of nude mice(P<0.01 or P<0.05),DAPT group and positive group could significantly increase the tumor tissue dynamics of SOD(P<0.01).PCR results showed that compared with the model group,the total flavonoids of Spatholobi Caulis increased the expression of NotchlmRNA,TGF-β1mRNA,Hey1mRNA and SnailmRNA(P<0.01 or P<0.05)in tumor-bearing nude mice.The results of HE staining showed that the tumor cells in the model group were numerous and closely arranged.Compared with the model group,the cancer cells in the positive group,the DAPT group,and the Spatholobi Caulis group were reduced and the necrosis was more serious.3.The anti-cancer activity of total flavonoids from Spatholobi Caulis in vitro and its mechanism study showed that:Compared with the normal group,the positive group could significantly inhibit the proliferation of HeLa cells at a concentration of 5 ×10-4 g·L-1(P<0.01),and the total flavonoids of Spatholobi Caulis at 0.025～0.8g·L-1 significantly inhibited HeLa cell proliferation(P<0.01),and inhibition of a certain dose-effect relationship.The results of PCR showed that compared with the normal group,the positive group and the total flavonoids of Spatholobi Caulis could significantly increase the expression of Notch 1mRNA,TGF-β 1mRNA,HeylmRNA and SnailmRNA in HeLa cells(P<0.01 or P<0.05).The results of western blot showed that:compared with the normal group the expression of Notch 1,TGF-β 1,Heyl and Snail protein in the positive group and the total flavonoids of Spatholobi Caulis both increased significantly(P<0.01 or P<0.05).The cells were observed under an inverted microscope,compared with the normal group,the positive group,the DAPT group,and the total flavonoids of Spatholobi Caulis flavonoids acted on the HeLa cells,and the cell morphology was pyknosis,the gap became larger,and the apoptotic appearance appearedConclusion:1.The total flavonoids of Spatholobi Caulis have inhibitory effects on human cervical carcinoma HeLa cells in vitro and in vivo2.The inhibitory effect of total flavonoids from Spatholobi Caulis on human cervical carcinoma HeLa cells is related to the intervention of TGF-β1/Notch signaling pathway.The total flavonoids of Spatholobus suberectus can inhibit tumor by upregulating TGF-β1,regulating Notch signaling pathway,up-regulating Heyl and Snail genes.At the same time the total flavonoids of Spatholobi Caulis can remove free radicals in the tumor tissue,inhibit the secretion of IL-6 and TNF-α in nude mice serum,exert anti-oxidation and anti-inflammatory effects,and it can promote the apoptosis of tumor cells to improve the pathological morphology of tumor tissues in nude mice and further inhibit the development of tumors. |
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