Study On The Inhibitory Effect And Molecular Mechanism Of Four Kinds Of Phenylethanoid Glycosides Deprived From Cistanche Deserticola On Malignant T Cells

Objective To study the killing and proliferation-inhibiting effects of a mixtures of four phenylethanoid glycoside(echinacoside,acteoside,cistanoside A and 2-Acetylacteoside,hereinafter referred to as CPhGs)deprived from Cistanche deserticola on malignant T cells,as well as the underlying molecular mechanism.Methods 1.Based on network pharmacologic analysis,the cell signaling pathways affected by the bioactive components of Cistanche deserticola for inhibiting T cell lymphoma were predicted.2.The killing and proliferation-inhibiting effects of CPhGs on malignant T cell lines Jurkat,HH,Myla and Hut78 were observed under microscope and detected by flow-cytometry and CCK8 assay.3.In above-mentioned cell lines,the CPhGs induced apoptosis was detected by flow-cytometry,and the expression and activation of apoptotic factors were detected by real-time quantitative polymerase chain reaction(qRT-PCR)and Western Blot.4.The induction of pyroptosis of malignant T cells by CPhGs.In Jurkat and Hut78 cell lines,the activation of GSDMD,the key protein of pyroptosis,and the release of LDH was detected.The rescue effects on cell death induced by CPhGs was further observed by silencing GSDMD with siRNA,as well as by using Caspase-1 and 4 inhibitor VX765,confirming that CPhGs lead to pyroptosis in malignant T cells.5.Effect of CPhGs on NLRP3 inflammasome-IL-1β cell proliferation signaling.The influences of CPhGs on NLRP3 expression and IL-1βrelease were examined by Western Blot.The inhibitory effect of CPhGs on cell proliferation was confirmed by forced expression of NLRP3 protein in malignant T-cells under CPhGs treatment.6.A xenograft model of T cell lymphoma(TCL)in nude mice was established with Hut78 cell line.CPhGs were injected intraperitoneally to observe the weight and tumor size changes in 18 days and the tumor inhibition rates were calculated to confirm the effect of CPhGs on malignant T cells in vivo.The expressions of some key proteins in the signaling pathway were also detected by immunohistochemistry.Results 1.According to the network pharmacology screening analysis of traditional Chinese medicine database(TCMSP),the results showed that the inhibitory effect of Cistanche deserticola on T cell lymphoma may be related to PI3 K / AKT,Caspase,P53,PTEN,BAX,Bcl2 and other apoptotic factors.2.CPhGs killed malignant T cells and inhibited their proliferation in a time and dose dependent manner.3.CPhGs induced apoptosis in a concentration-dependent manner.CPhGs influenced SIRT2/P53 and PI3K/AKT signaling axes,and activated both intrinsic and extrinsic apoptotic signaling pathways.4.CPhGs induced pyroptosis by activating caspase-1-GSDMD canonical pyroptosis pathway,caspase-4/ 5-GSDMD no-canonical pyroptosis pathway and caspase3-GSDME pyroptosis bypass.CPhGs increased the release of pyroptosis factor LDH in the cell supernatant in a dose-dependent manner.After silencing GSDMD gene by siRNA and treating with caspase-1and 4 inhibitor VX-765,the killing and proliferation-inhibiting effectd induced by CPhGs could be partially rescued.VX765 also restored the expression of pyroptosis-related proteins and reduced LDH release.The results indicate that in addition to apoptosis,pyroptosis also plays a role in the killing of malignant T cells by CPhGs.5.It is worth mentioning that we found CPhGs inhibited NLRP3 protein expression and force expression of NLRP3 restored cell proliferation,suggesting that CPhGs exerted an inhibitory effect on tumor cell proliferation and metastasis by inhibiting NLRP3 signaling.Therefore,CPhGs lead to the separation of activating pyroptosis signaling and inhibiting of NLRP3 inflammatory cell proliferation signaling.6.The xenograft in nude mice showed that CPhGs could significantly inhibit the growth of TCL.HE staining of tumor tissue showed that CPhGs could increase the necrosis of tumor cells.Immunohistochemistry showed that CPhGs increased p53 expression and decreased NLRP3 expression in tumor tissues.Conclusion Our study shows that CPhGs can trigger malignant T cell killing and proliferation-inhibting.CPhGs induce apoptosis by regulating SIRT2/P53,PI3K/AKT signaling pathways and activating intrinsic/extrinsic apoptotic pathways.On the other hand,CPhGs induced pyroptosis by activating caspase-1-GSDMD canonical,caspase-4/5-GSDMD non-canonical pyroptotic pathways and caspase3-GSDME pyroptotic bypass.In addition,our study found for the first time that CPhGs can block the NLRP3-IL 1β signaling axis and conseqiently inhibit the proliferation of malignant T cells while activating the canonical pathway of pyroptosis.In conclusion,our results confirmed the anti-tumor effect of a mixture of 4 kinds of phenylethanoid glycosides and revealed the underlying molecular mechanisms,which provided an experimental basis for the new strategy of Cistanche deserticola or phenylethanoid glycosides in the treatment of T cell malignant tumors.