The Improvement Effects Of The Sika Deer Antler Proteins Combination With Cisplatin On PC-3M Cells And Alleviation Cisplatin Nephrotoxicity

In the present study,we explored that Sika deer antler proteins may be an ideal agent for the combination therapy of cisplatin possesses the synergy and attenuation effect in cancer.Sika deer antler proteins and Sika deer antler polysaccharide were extracted by ultrasound device,and the contents and purities were determined byBradford method and phenol sulfuric acid method respectively.The purity of Sika deer antler proteins was 86.35%,and the purity of Sika deer antler polysaccharide was 82.45%for the pharmacological experiment.MTT method was used to screen the antitumor activity of Sika deer antler proteins and Sika deer antler polysaccharide on human hepatoma cells?HepG2?,human lung cancer cells?NCI-H460?and human prostate cancer?PC-3M?,and the cytotoxicity of Sika deer antler proteins and Sika deer antler polysaccharide on human embryonic kidney cells?HEK 293?was determined.The mechanisms of anticancer activity of Sika deer antler proteins on PC-3M cells were studied by plate cloning assay,flow cytometry and Western blotting.The MTT results showed Sika deer antler proteins(200-800?g·mL^-1)could suppress proliferation capacity and induce apoptosis on PC-3M cells?P<0.01?,and no significant inhibitory effect on HepG2 and NCI-H460.In addition,there were no toxic effect on HEK 293 cells.Sika deer antler polysaccharide(0-800?g·mL^-1)did not inhibit the three kinds of cancer cells,and no toxic effect on HEK 293 cells.Compared with control group,the colony formation of the PC-3M cells significantly was reduced by Sika deer antler proteins alone and in combination with cisplatin.The flow cytometry results showed that Sika deer antler proteins alone and in combination with cisplatin could induce the apoptosis of PC-3M cells,and the apoptotic rates were 72.79%and 99.6%,respectively.The Western blotting results showed that Sika deer antler proteins alone and in combination with cisplatin could significantly increase the expression of cleaved-caspase-3 and the ratio of Bax/Bcl-2?P<0.01?.Sika deer antler proteins exerts an anti-tumor effect by inhibiting cell proliferation and inducing apoptosis of PC-3M cells.To establish the cytotoxicity model of cisplatin on HEK 293 cells and the nephrotoxicity model of cisplatin on mice,and investigated the effect of Sika deer antler proteins against cisplatin-induced nephrotoxicity.The cytotoxicity assay in vitro,the levels of Super oxide dismutase?SOD?,Glutathione?GSH?,Lactate dehydrogenase?LDH?,Malondialdehyde?MDA?,tumour necrosis factor alpha?TNF-??,and Interleukin-6?IL-6?,and expression of caspase-3,Bax,and Bcl-2 were measured.And we observed the renal cell apoptosis by TUNEL staining.The cytotoxicity assay results showed that Sika deer antler proteins improved antioxidant defense,attenuated apoptosis by decreasing expression levels of cleaved-caspase-3and Bax,increasing Bcl-2 expression,and inhibiting apoptosis positive cells in HEK293 cells.In addition,Sika deer antler proteins treatment inhibited markedly the levels of TNF-?and IL-6.Sika deer antler proteins can relieve cisplatin-induced nephrotoxicity by improving antioxidase activity,suppressing inflammation,and inhibiting apoptosis in vitro.The nephrotoxicity test in vivo is shown that the levels og renal function,oxidative stress,and inflammatory factors,and expression of apoptosis-related proteins were measured in vivo.Renal tissues were stained with TUNEL and H&E to observe renal cell apoptosis and pathological changes.The nephrotoxicity test results showed that Sika deer antler proteins also ameliorated cisplatin-induced histopathologic changes,and BUN and SCr?P<0.05?.Western blotting analysis showed Sika deer antler proteins decreased clearly expression levels of cleaved-caspase-3 and Bax,and increased the expression level of Bcl-2?P<0.01?.Additionally,Sika deer antler proteins markedly regulated oxidative stress markers and inflammatory cytokines?P<0.05?.TUNEL staining showed decreased apoptosis after Sika deer antler proteins treatment?P<0.01?.Sika deer antler proteins can relieve cisplatin-induced nephrotoxicity by improving antioxidase activity,suppressing inflammation,and inhibiting apoptosis in vivo.Biological safety of Sika deer antler proteins was evaluated by acute oral toxicity test,bone marrow micronucleus assay and sperm aberration experiment.The biological safety test results showed that no mortalities or adverse effects was observed in Kunming mice following acute oral gavage with Sika deer antler proteins at a dose of 8.0 g/kg body weight during the acute toxicity test.Furthermore,the negative results obtained in bone marrow micronucleus test and sperm malformation assay demonstrated that Sika deer antler proteins had no obvious toxicity to cause mutagenicity of chromosome and genital cells.