Studies On The Isolation And Mechanism Of Antagonistic Gentamicin Nephrotoxic Protein From Sika Deer Antler

Deer antler is regarded as a kind of traditional precious animal medicine in China.It is warm in nature,sweet and salty,and it belongs to the kidney meridian and liver meridian.It has traditional functions such as strengthening kidney yang and replenishing essence.The protein component of deer antler is the most important component for its pharmacological activity.Modern pharmacological studies have found that Sika deer antler protein(SDAPR)has a mitigating effect on the nephrotoxicity induced by gentamicin(GM),but its active protein components have not been clear,and the mechanism of action has not been fully elucidated.Therefore,the purpose of this study is to explore the active protein components and their molecular mechanisms of Sika deer antler that have a protective effect on GM-induced acute kidney injury.The total protein of sika deer antler(SDAPR)was separated by Sephadex dextran gel filtration chromatography and the sika deer component proteins SDAP1 and SDAP2 were used as the research objects.The sika deer component protein components were detected by liquid chromatography tandem mass spectrometry combined with proteomics.Bioinformatics methods were used to screen out the material basis and molecular mechanism of protecting drug-induced nephrotoxicity.Through mass spectrometry detection and identification,there were 16 credible proteins in SDAP1.The molecular weight of the protein was mainly distributed between 15.6-71.4k Da and the isoelectric point was between 4.74-9.6 in SDAP1.There were 11 credible proteins in SDAP2.The molecular weight was mainly distributed between 15.6-68.8k Da,and the isoelectric point was between 4.74-9.6 in SDAP2.The common proteins of interest in SDAP1 and SDAP2 were analyzed by GO and KEGG,including sequence alignment,three-dimensional structure establishment and phylogenetic tree analysis.The results showed that SDAP1 and SDAP2 may reduce GM-induced nephrotoxicity by regulating the PI3K/AKT signaling pathway.The protective effect of deer antler component proteins on the damage of GM in human embryonic kidney 293(HEK293)and its molecular mechanism were studied.Using the in vitro toxicity model of GM-induced HEK293 cell injury,the effective doses of SDAPR,SDAP1 and SDAP2 on the survival rate of GM-induced cells were screened by the MTT method and RTCA real-time cell analysis technology.Biochemical kit analysis method were used to detect the changing level of oxidative stress markers in HEK293 cells.The DCFH-DA fluorescent probe method was used to detect the changes of reactive oxygen species(ROS)in HEK293 cells,and the ELISA method was used to detect the levels of inflammatory cytokines,tumor necrosis factor alpha(TNF-α)and interleukin 6(IL-6).The apoptosis level of HEK293 cells was detected by Annexin V/PI flow cytometry and Hoechst 33258 staining.Western Blot was used to determine the expression levels of Bax,Bcl-2,P53 and Cleaved-Caspase 3 in each group of HEK293 cells,and measured the expression levels of PI3K-Nrf2 pathway related proteins in each group of cells,including p-PI3K/PI3 K,p-AKT/AKT,Nrf2,HO-1,NQO1 and keap-1protein expression levels.The results showed that the administration of SDAPR,SDAP1 and SDAP2 significantly increased the survival rate of GM(3mg/m L)-induced HEK293 cells(P<0.01);significantly reduced LDH,MDA,TNF-α,IL-6 and ROS levels in GM-induced HEK293 cells(P<0.01),and significantly increased CAT and SOD activities and GSH levels in GM-induced HEK293 cells(P<0.01);reduced the degree of nuclei fragmentation caused by GM in Hoechst 33258 staining,and significantly reduced the apoptotic rate of HEK293 cells(P<0.01),reduced the increase of Bax,P53 and cleaved-caspase 3 protein expression in GM-induced HEK293 cells,increased the expression of Bcl-2 protein,and reduced the apoptosis of GM-induced HEK293 cells.Finally,the Western Blot method was used to improve the expression levels of p-PI3 K,p-AKT,HO-1 and NQO1 in GM-induced HEK293 cells(P<0.01),the inhibition of Nrf2 translocation in GM-induced HEK293 cells was improved(P<0.01),and the degradation of keap-1 in HEK293 cells was promoted(P<0.01).SDAPR,SDAP1 and SDAP2 pretreatment can reduce the GM-induced toxicity,oxidative damage,inflammation and apoptosis of HEK293 cells by regulating the PI3K-Nrf2 pathway.The improvement effect of SDAP1 and SDAP2 on GM-induced acute kidney injury in mice and its mechanism were studied.A GM-induced acute kidney injury model in mice was established,the body weight of the mice was recorded and the kidney index was calculated;serological methods were used to detect the levels of creatinine(Cr)and urea nitrogen(BUN)in the serum of mice,and the biochemical kits were used to detect MDA and GSH levels,SOD and CAT activities in mice kidney tissue;ELISA method detected TNF-α and IL-6 levels in mouse kidney tissue;H&E and Masson staining methods observed histopathological changes;Hoechst 33258 and TUNEL staining method was used to detect the apoptosis of kidney tissue;Western Blot method was used to determine the expression levels of Bax,Bcl-2 and Cleaved-Caspase 3 in mice kidneys,and the expression of PI3K-Nrf2 pathway related proteins in each group of kidney levels,including protein expression levels of p-PI3K/PI3 K,p-AKT/AKT,Nrf2,HO-1 and NQO1.The results showed that SDAP1 and SDAP2 pretreatment can significantly inhibit the increase in renal index and renal function indexes Cr and BUN in GM-induced mice(P<0.01),and enhanced the SOD,CAT activity and GSH levels in mouse kidney tissue(P<0.01),inhibited the levels of TNF-α and IL-6 in the kidney tissue of mice(P<0.01),improved the pathological changes of kidney tissue and renal cell apoptosis,reduced the increase of Bax and cleaved-caspase 3 protein expression in GM-induced mice kidney tissue,increased the expression of Bcl-2 protein,and reduced the apoptosis of mice kidney cells induced by GM.Finally,SDAP1 and SDAP2 reversed GM-induced reduction of p-PI3 K,p-AKT,Nrf2,HO-1,and NQO1 protein expression levels in mice kidney tissue(P<0.01).In conclusion,SDAP1 and SDAP2 may play a role in antagonizing GM-induced acute kidney injury in mice by regulating the levels of oxidative stress and apoptosis mediated by PI3K-Nrf2.Combined with the results of proteomics and pharmacological experiments in vitro and in vivo,it can be concluded that sika deer antler component proteins SDAP1 and SDAP2 can effectively regulate PI3K-Nrf2 pathway,reduce GM-induced oxidative damage,inflammatory response and apoptosis,and significantly improve GM-induced HEK293 cell injury and acute kidney injury in mice.It provided a theoretical basis for the clinical treatment of acute kidney injury caused by GM and the kidney-tonifying mechanism of traditional Chinese medicine sika deer antler.