| Objective:Cervical cancer is one of the common gynecological tumors. About 300 thousands women die of cervical cancer all over the world every year, and the death ratio is still the highest. Recently, on the basis of three traditional therapy methods,the study of immune therapy is increasingly becoming a focus. Tumor can escape from immunological surveillance through immunosuppressive substances caused by immunosuppressive substance secreted from tumor cells, and this is the immportant mechanism of the occurrence and development of tumors. Therefore, studying the down- regulating effects on tumor immunosuppression and immunosuppressive substance secretion and seeking for the effective drugs that can reverse these kind of immunosuppression are important parts of discovering anti-tumor drugs, ascertaining their immunological mechanisms and guiding their proper clinical application. But the previous anti-tumor studies of artesunate(ART) were always about up-regulating immunofunctions of patients, inducing apoptosis and differentiation of tumor cells, killing cancer cells directly or inhibiting tumor growth and etc, the relatively systemic studies had still reported fewer about the down-regulating effects of ART on tumor immunosuppression through decreasing the secretion of immunosuppressive substances. Accordingly, based on foregone studies, more reports were showed on the immunosuppressions from cultured Hela cells caused by substances of TGF-β1,IL-10 and PGE2. The studies still had not been reported about exosomes. Our study aimed to discover the novel anti-tumor mechanism of ART. It is expected to provide experimental basement and the theoretic evidences through analyze the effects of ART on the secreted immunosuppressive substances form Hela cells, to exploit especially characteristic anti-tumor traditional Chinese medicines that have our own property right,to guide the proper application for clinical uses.Methods:The suitable inoculated concentration and cultured time of Hela cells were detected by MTT.Recultured supernatant of Hela cells after treated with ART respectively for 48 hours were collected concentration which had no change on the proliferation of Hela cells has been determined, the supernatants were called ART-S. Followed by being washed completely to remove the traditional Chinese medicines, and recultured twice with fresh medium,the supernatants we collected were called ART-S1 and ART-S2. The corresponding supernatants of Hela cells treated without ART as control ,they were called control-S,control-S1 and control-S2. The concentrations of TGF-β1 and IL-10 were measured by quantitative ELISA ,and that of was detected by ultraviolet spectrophotometry for A278.Exosomes from the supernatants were isolated by gradient centrifuge methods,their were observed through SEM,and the concentrations of protein in exosomes were measured by ultraviolet spectrophotometry for A280 . All data were analyzed to discover the kinds and levels of down-regulating immunosuppressive substances secreted from Hela cells treated with ART.Results:1.The inoculated concentration determined by MTT was 2×105/mL, and the cultured time was 48h.2.The top concentration of ART non-poisonous was 3.13mg/mL. 3. For supernatants of Hela treated without ART, all of the four immunosuppressive substance were secreted steadily ,the concentration of TGF-β1 was highest to (989.226±34.836)ng/mL, the concentration of IL-10 was( 3.391±0.164) pg/mL , the concentration of PGE2 was (1.318±0.076 ) pg/mL, A280 of Exosomes was (0.178±0.027).4.For Hela cells treated with ART, the concentrations of TGF-β1,IL-10,PGE2 decreased greatly in ART-S(2.859±0.520)ng/mL(P < 0.001, only 0.29% of control-S)(,3.010±0.217)pg/mL(P<0.05, 88.76% of control-S),(1.147±0.931 )pg/mL(P <0.05, 58.43% of control-S); there were no significant difference of Exosomes in ART-S( 0.143±0.029, P>0.05 ) .Compared with ART-S1, the concentration of TGF-β1,PGE2,and Exosomes A280 in ART-S1 decreased (0.009±0.001)ng/m(lP <0.001, only was 0.00008% of control-S1),(1.132±0.041)pg/mL(P<0.05, 52.67% of control-S1 ) ,0.063±0.142 ( P<0.05 , only was 35.4% of control-S1 ) ; the concerntration of IL-10 increased (3.121±0.137) pg/mL(P<0.05). The concentration of TGF-β1 in ART-S2 increased highly(0.576±0.032) ng/mL, (P<0.001 , was 0.06% of control-S2), the concentration of IL-10 and PGE2 had no changes(3.207±0.082) pg/mL, (P>0.05),( 1.206±0.010) pg/mL( P>0.05). Exosomes in ART-S2 rised greatly(0.135±0.017, P<0.05), while compared with control-S2 decreated was 75.84%. The morphology of Exosomes from Hela cells and without ART under SEM had no difference from each other.Conclusions:1.Hela cells can secreted four detected immunosuppressive substance steadily.2. ART can down-regulated the concentrations of TGF-β1,IL-10,PGE2 and Exosomes secreted by Hela cells .3. TGF-β1 may be the dominant immunosuppressive mole -cules secreted by Hela cells.4. Down-regulating the secretion of immunosuppressive substances may be the novel anti-tumor mechanism of ART.
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