Andrographolide Induces Nephrotoxicity Through Inhibiting SIRT3-dependent Signaling Pathways

Background: Andrographolide(Andrographolide,Andro)is one of the main bioactive components of andrographis paniculata,which owns a diterpene lactone structure.It has a variety of pharmacological activities,such as anti-inflammatory,antibacterial,anti-tumor,anti-virus,immunomodulatory and hepatoprotective effects.Clinically,it is mainly used for the treatment of inflammation-related diseases such as upper respiratory tract infection,pneumonia,acute tonsillitis and so on.With the continuous expansion of the scope of application of andrographolide,the adverse reactions of andrographolide are gradually increasing,in which urinary system damage is the most common,and even acute renal injury may occur.However,so far,there are few studies on the specific mechanism of renal injury induced by andrographolide,which is worthy of in-depth discussion.Aims: To observe whether andrographolide can induce renal injury in NRK-52 E cells and mice in vitro,and further explore the molecular mechanism,ours preliminary experimental basis can provide for clinical rational and safe drug use.Methods:(1)CCK-8 assay was used to detect the effect of andrographolide on the proliferation of NRK-52 E cells.Hoechst staining and flow cytometry were used to detect the effect of different concentrations of andrographolide on the apoptosis rate of NRK-52 E cells.The effects of different concentrations of andrographolide on the expression of apoptosis-related proteins in NRK-52 E cells were detected by Western Blot method.(2)Western Blot was used to detect the effect of andrographolide at different concentrations on the expression of SIRT3 protein in NRK-52 E cells.The SIRT3 protease activity was detected by kit.After the overexpression of SIRT3,CCK-8 assay was used to detect the effect of andrographolide on the proliferation of NRK-52 E cells,Hoechst staining and flow cytometry were used to detect the effect of andrographolide on the apoptosis rate of NRK-52 E cells.Western Blot assay was used to detect the effect of andrographolide on the expression of apoptosis-related proteins in NRK-52 E cells.(3)Western Blot method was used to detect the effect of andrographolide at different concentrations on the expression of AMPK-related proteins in NRK-52 E cells.(4)KM mice were given different doses of andrographolide(0,750,1500 mg/kg/d)for 8 days,the body weight of mice was weighed and recorded every day.The levels of blood urea nitrogen(BUN)and serum creatinine(CR)were detected by related kits,and the renal tissue was detected by HE staining.The protein expression levels of caspase3 and SIRT3 in renal tissue were detected by Western Blot.Results:(1)CCK-8 assay showed that andrographolide could inhibit the proliferation of NRK-52 E cells in a time-and dose-dependent manner,Hoechst staining and flow cytometry showed that with the increased concentration of andrographolide,the apoptosis rate of NRK-52 E cells was increased;Western Blot assay showed that the protein express of caspase 3 was inhibited by andrographolide in a time-and dose-dependent manner.(2)Compared with the group without treatment,with the increased concentration of andrographolide,the protein expression of SIRT3 was decreased,and the protease activity of SIRT3 was decreased.After overexpression of SIRT3,CCK-8 assay showed that the survival rate of NRK-52 E cells in Andro+SIRT3group was significantly higher than that in Andro group.The results showed that pretreatment with specific overexpression of SIRT3 in NRK-52 E cells could significantly alleviate the inhibition of cell proliferation induced by andrographolide.Hoechst staining and flow cytometry showed that the rate of apoptosis in Andro+SIRT3 group were significantly lower than those in Andro group.Western Blot assay showed that the protein expression of Caspase 3(cleaved/pro)in Andro+SIRT3 group was significantly lower than that in Andro group,indicating that pretreatment with NRK-52 E cell specific overexpression SIRT3 could significantly alleviate andrographolide-induced proliferation inhibition and apoptosis.(3)Western Blot assay showed that after NRK-52 E cells were treated with different concentrations of andrographolide for 24 h,the expression of LKB1,AMPK,P70S6 K and S6 was basically unchanged,but the phosphorylation level of LKB1,AMPK,P70S6 K and S6 changed,p-LKB1 and p-AMPK were significantly up-regulated,p-P70S6 K and p-S6 were significantly down-regulated.(4)After intragastric administration of andrographolide at different doses for 8 days,the body weight of mice had no significant change,but with the increase of andrographolide dose,the levels of BUN and CR in blood increased,and glomerular hypertrophy was appeared in renal tissue by HE staining.Western Blot assay showed that the expression of apoptosis-related protein cleaved/pro-caspase-3 increased with the increase of andrographolide dose,and the protein expression level of SIRT3 decreased.Conclusion:(1)Andrographolide inhibits the proliferation and induces apoptosis of renal NRK-52 E cells.(2)Andrographolide regulates the proliferation and apoptosis of NRK-52 E cells by inhibiting SIRT3.(3)SIRT3 may not affect the downstream LKB1-AMPK signal pathway to regulate the proliferation and apoptosis of NRK-52 E cells.(4)Andrographolide induced renal injury in KM mice and affected the protein expression of SIRT3.