Anemoside B4 Attenuates Kidney Injury By Inhibiting ROS/MAPKs Signaling Pathway

Part one Anemoside B4 attenuates cisplatin-induced acute kidney injuryObjective: Cisplatin is a highly effective chemotherapeutic agent commonly used in the treatment of a wide variety of malignancies.However,its clinical usage is severely limited due to its serious side effects,especially nephrotoxicity.Anemoside B4,the most abundant saponin in the roots of Pulsatilla chinensis(Bunge),has anti-inflammation activity in vitro.However,the role of anemoside B4 in cisplatin-induced nephrotoxicity is unclear.In the present study,in vitro model of cisplatin-induced apoptosis in renal cell and in vivo model of acute kidney injury in mice were established to investigate the protective effects and molecular mechanism of Anemoside B4 on cisplatin-induced nephrotoxicity.Method: The protective effect of anemoside B4 on cisplatin-induced Balb/c mice acute renal failure was evaluated by the test of body weight,blood biochemical criterion and HE staining.The effects of anemoside B4 on HEK 293 T cell viability was measured by CCK8,and the cell morphology was observed with microscope.Apoptosis associated proteins such as p53,Bcl-2,Bax,Cleaved-caspase 3,MAPK family protein levels of ERK,JNK,p38,and inflammation-associated proteins including p65,COX-2,IL-1β were measured by western blot to elucidate the underlying mechanism.Furthermore,the intracellular antioxidant capacity including superoxide dismutase(SOD),glutathione(GSH),catalase(CAT)were estimated using chemical kits,then the intracellular reactive oxygen species(ROS)were analyzed by flow cytometry.Results: Compared with animals in normal group,percentage of weight growth was reduced by a single dose of cisplatin(20 mg/kg)in mice(P < 0.001),while the ratio of kidney weight was increased(P < 0.05).At the same time,the levels of serum creatinine(P < 0.05)and blood urea nitrogen(P < 0.01)were dramatically increased,and obvious inflammatory infiltration and renal tubular swelling were observed in kidney tissue.Thedecrease of mice weight and the increase of kidney index(P < 0.05)were inhibited by pretreated with 100 mg/kg anemoside B4(bid).The increased levels of serum Crea and BUN(P < 0.05)could also be inhibited,as well as the renal swelling and inflammatory infiltration.The viability of HEK-293 T cells was significantly reduced by 20 μM cisplatin(P < 0.001),and the cell morphology was damaged,compared with that in the vehicle group.While,the toxicity of cisplatin was significantly improved by pretreatment with 12μM anemoside B4(P < 0.001).In vivo and vitro models of cisplatin-induced nephropathy,apoptotic proteins Bax,p53,Cleaved-caspase 3 were significantly increased(P < 0.05),Bcl-2 was suppressed(P <0.05),and inflammation-related proteins COX-2,IL-1β,RAGE,S100A9 were also increased(P < 0.05).These altered expression of apoptosis proteins and inflammation-related proteins were significantly improved by 12 μM anemoside B4(P <0.05).In addition,the expression levels of MAPK family proteins,such as p-JNK(P <0.01),p-p38(P < 0.05),p-ERK(P < 0.05),and that of a NF-κB signal pathway protein,p-p65(P < 0.05),were up-regulated by induction of cisplatin.Accordingly,the increasing ROS levels(P < 0.01),and decreasing of SOD,CAT activities(P < 0.01)and GSH content(P < 0.05).While the increased intracellular expression of p-JNK,p-p38,p-ERK,p-p65(P< 0.05)and ROS release(P < 0.05)were significantly improved by treatment of anemoside B4,simultaneously the antioxidant capacity were restored(P < 0.05).The results suggested that anemoside B4 could inhibit cisplatin-induced acute kidney injury.Part two Anemoside B4 attenuates streptozocin-induced diabetic nephropathyObjective: Diabetes is a metabolic diseases characterized by high blood glucose.At present,there are more than 100 million diabetic patients in China,which has been one of diseases that seriously threaten people’s health.Diabetic nephropathy is one of the most common microvascular complications of type 1 and type 2 diabetes.Renal damage is reported in 40-50% of patients with type 1 diabetes,and about 30% in patients with type 2diabetes.The pathogenesis of diabetic nephropathy is complex,including hyperglycemia,inflammation and oxidative stress.Due to the protective effect of anemoside B4 on acute kidney injury,we established a vivo models of streptozocin-induced diabetic rats and a vitro model of high glucose-induced mesangial cells proliferation to investigate itstherapeutic effect on diabetic nephropathy and elucidate the underlying mechanism.Method: The protective effect of anemoside B4 on streptozotocin-induced SD rats diabetic nephropathy was evaluated by the test of body weight,blood glucose monitoring,blood biochemical criterion,immunofluorescence,ELISA assay and HE,PAS and Masson staining,furthermore calculated the incidence of cataract.The effects of anemoside B4 on SV40 MES13 cell viability was measured by CCK8 kits.The intracellular antioxidant capacity including superoxide dismutase(SOD),glutathione(GSH),catalase(CAT)were estimated using chemical kits,intracellular reactive oxygen species(ROS)was analyzed by flow cytometry,and the levels of MAPK family proteins including p-JNK,p-p38,and that of NF-κB signal pathway protein,p-p65 were measured by western blotting.Results: 7 days after a single injection of streptozotocin(55 mg/kg),rats with blood glucose ≧ 16.7 mmol/L were selected as the model animals for diabetes.These diabetic rats had a decrease in percentage of weight growth(P < 0.001),while the intake of water and food(P < 0.001),ratio of kidney and liver weight were significantly increased(P <0.01),the dramatic increase in levels of blood glucose(P < 0.001),and serum TG(P <0.05),TC(P < 0.05),BUN(P < 0.01)and ALT(P < 0.05)levels was observed.In the renal tissues of diabetic rats,PAS staining showed an increase in the areas of glomeruli(P< 0.01)and glycogen(P < 0.001)and the glomerular mesangium expanded,Masson staining showed an increase in tubulointerstitial fibrosis and collagen fibers(P < 0.05),ELISA assay indicated the contents of cytokines IL-1β(P < 0.05),IL-6(P < 0.001),and TNF-α(P < 0.001)were increased rapidly.In addition,66.67% of DM rats suffered from cataract,while podocytes had not significantly apoptosis.Compared with rats in the diabetic group,8 weeks after injection of anemoside B4(5 mg/kg,bid)could accelerate the increase of body weight(P < 0.05),while water intake(P < 0.05)and blood glucose(P <0.01),kidney index(P < 0.01),liver index(P < 0.05),and serum TG(P < 0.05),TC(P <0.001),BUN(P < 0.05),and cytokines IL-1β(P < 0.05),IL-6(P < 0.001),TNF-α(P <0.05)were significantly reduced.At the same time,Morphological observation of kidney could also showed the reduction of glycogen(P < 0.01)and collagen fibers(P < 0.05),and the incidence of cataract dropped to 31.25%.The proliferation of SV40 MES13 cells increased rapidly by the stimulation with high glucose(25 m M)for 24 h(P < 0.01),accompanied with a significantly increased release of ROS(P < 0.01).The high glucose-induced cell proliferation and release of ROS were effectively inhibited by thepretreatment with anemoside B4(P < 0.05),while the activities of CAT and levels of GSH were significantly increased(P < 0.05).In vivo and vitro hyperglycemia models,the expression of MAPK family proteins including p-JNK(P < 0.01),p-p38(P < 0.05),and NF-κB signal pathway protein p-p65(P < 0.05)were up-regulated.While anemoside B4 significantly inhibited expression of p-JNK,p-p38,p-p65 in SV40 MES13 cells and expression of p-p38,p-p65 in renal tissues(P < 0.05).In summary,these results suggested that anemoside B4 could also attenuates streptozocin-induced diabetic nephropathy.Conclusion: Our data indicate that anemoside B4 could significantly attenuate cisplatin-induced acute kidney injury and diabetic nephropathy.In vitro,it could also inhibit the apoptosis of renal cells and proliferation of mesangial cells.The mechanism of these therapeutic effects may be related to the inhibition of the expression of apoptosis-associated proteins,the release of inflammatory factors and activation of the MAPKs and NF-κB signaling pathway,while inhibiting oxidative stress and increasing the activity of antioxidant enzymes.Taken together,the strong antioxidant and anti-inflammatory effects of anemoside B4 may provide therapeutic potential for multiple kidney injury.