Study On Alleviation Of Gentamicin Nephrotoxicity By Sika Deer Antler Protein

Sika deer antler has a long history of medicine and is a commonly used medicine for treating kidney-yang deficiency.According to the Pharmacopoeia of People's Republic of China,sika deer antler is used to invigorate kidney yang,strengthen the bones and muscles,and replenish sperm and blood,etc.In the previous study,the water extract of sika deer antler has a certain improvement effect on the nephrotoxicity induced by platinum drugs,but the nephrotoxic mechanism and action pathway of the drug are different,and the protective effect of sika deer antler on GM nephrotoxicity has not been reported.GM is widely used and very effective aminoglycoside antibiotic.It usually used to treat gram-negative infection.However,it is limited in clinical application due to its nephrotoxicity.Therefore,finding natural adjuvant drugs that can prevent or reduce GM nephrotoxicity has become a research hotspot.With this background,the objective of this study is to assess the renoprotective efficacy of sika deer antler against GM nephrotoxicity and study the potential mechanisms of renal protection.Sika deer antler proteins?SDAPR?and sika deer antler polysaccharide?SDAPO?were extracted by ultrasound assisted solvent method of sika deer antler active ingredient,and the contents and purity were determined by Braford method and phenol sulfuric acid method respectively.The purity of SDAPR was 92.60%?polysaccharide content?5.00%?,and the purity of SDAPO was 91.30%for the pharmacological experiment.To study the effect of water-soluble components in velvet antler on GM-induced cytotoxicity of human embryonic kidney 293?HEK293?cell,a model of GM-induced HEK293 cytotoxicity in vitro was established.MTT assay was used to determine the cytotoxicity of GM,and SDAPR and SDAPO for HEK293 cell proliferation.And MMT assay was also used to screen for SDAPR and SDAPO to reduce the activity of GM-induced cell injury.Biochemical detection kits were used to detect the content of lactate dehydrogenase?LDH?in the supernatant of HEK293 cell culture medium,the contents of glutathione?GSH?and malondialdehyde?MDA?in HEK293 cell lysates,and the activity of superoxide dismutase?SOD?in cell lysates,respectively.At the same time,changes of cell morphology were observed by optical microscopy.The results showed that GM significantly reduced the viability of HEK293 cells in a dose-dependent manner,with an IC₅₀ of 3.00 mg·mL^-1?P<0.01?and SDAPR(0.50-4.50 mg·mL^-1)had significant cell proliferation capacity for HEK293 cells?P<0.01?and no cytotoxicity to HEK293 cells.Pretreatment with SDAPR significantly inhibited the decrease in HEK293 cell viability induced by GM,and relieved the GM-induced decrease in GSH and SOD as well as the increase in MDA and LDH?P<0.01?.SDAPR can attenuate GM-induced HEK293 cytotoxicity,and its mechanism may be related to promoting cell proliferation and reducing oxidative stress.To establish the cytotoxicity and apoptosis model of GM on HEK293 cells,and investigate the effect of SDAPR on GM-induced apoptosis of HEK293 cells.ELISA assay was used to determine the early acute kidney injury biomarkers,such as level of neutrophil gelatinase associated lipocalin?NGAL?,cystatin-C?Cys-C?and kidney injury molecule 1?KIM-1?.The mitochondrial membrane potential??m?MMP?in HEK293 cells was determined using the JC-10 mitochondrial membrane potential kit.The nuclear morphology,apoptosis rate and expression of apoptosis-related proteins Bcl-2 and Bax in HEK293 cells were detected by Hoechst33258 fluorescent staining,flow cytometry and Western blotting.Our study results showed that SDAPR reduced GM induced cytotoxicity caused by apoptosis as evidenced by increased levels of NGAL,Cys-C and KIM-1?P<0.05,P<0.01?,and mitochondrial membrane potential??m along with reduced nuclear debris and apoptosis rate?P<0.01?.Western blotting analysis showed SDAPR decreased expression level of Bax,and increased the expression level of Bcl-2?P<0.01?.Collectively,SDAPR protects HEK293 cells from GM-induced cytotoxicity,which may be related to SDAR antagonizing apoptosis.To investigate the effect of SDAPR on GM-induced nephrotoxicity in mice,establish a model of nephrotoxicity induced by GM-induced ICR in mice.After the experiment,the body mass and renal mass were recorded to calculate the renal index.The levels of blood urea nitrogen?BUN?and creatinine?Cr?in serum,the activity of catalase?CAT?and SOD,and the contents of MDA,GSH,tumor necrosis factor??TNF-??and interleukin-6?IL-6?in serum were detected by biochemical detection kits,respectively.The pathological changes in renal tissue were observed by H&E staining.The results showed that treatment with SDAPR?50,100 and 200 mg/kg?significantly inhibited the GM-induced increase in the levels of renal index,BUN,Cr,MDA,TNF-?and IL-6?P<0.05,P<0.01?,as well as the decrease in the levels of SOD,CAT and GSH?P<0.05,P<0.01?.The renal pathological changes induced by GM were improved.SDAPR can effectively ameliorate the GM-induced nephrotoxicity,which may be related to the inhibition of oxidative stress and inflammation.Combining to the results of in vitro and in vivo experiments,SDAPR can effectively alleviate GM-induced HEK293 cytotoxicity and protect HEK293 cells from GM-induced cell damage and apoptosis.The mechanism is related to promoting cell proliferation,inhibiting oxidative stress and antagonizing apoptosis.At the same time,SDAPR can also alleviate GM-induced kidney injury in mice,and its mechanism is related to inhibition of oxidative stress and inflammatory response.This study systematically studied the effect of SDAPR on the induction of drug-induced renal injury by aminoglycoside drug-GM,and provided a theoretical basis for the study of the mechanism of kidney-reinforcing action of traditional Chinese medicine-Sika deer antler.