Molecular Mechanism Of Andrographolide Induced Renal Inflammation,Fibrosis And Aging

BackgroundAndrographolide is one of the main effective active ingredients extracted from Andrographis of traditional Chinese herbal medicine.It is commonly used clinically for the treatment of upper respiratory tract infections,dysentery and other infectious diseases.Because of its special efficacy in bacterial and viral upper respiratory tract infections and dysentery,it is known as a natural antibiotic.However,with the wide application of these traditional Chinese medicine injections in clinical practice,the adverse reactions they show are more and more,common adverse reactions are rash,dizziness,gastrointestinal reactions,more serious kidney damage,severe anaphylactic shock,acute renal failure,etc.,among which the adverse reactions of kidney damage have caused widespread concern in the domestic medical community.Andrographolide toxicity has become an important obstacle restricting their clinical application.The existing research mostly focuses on the pharmacodynamics and clinical research of andrographolide and its derivatives,and there are not many studies on its nephrotoxicity,renal inflammatory response and fibrosis and molecular mechanism.Therefore,it is necessary to conduct in-depth research on this,in order to provide an important basis for the better development and application of andrographolide and its derivatives.AimsTo study whether Andrographolide indeces renal inflammation,fibrosis and aging in vitro renal NRK-52 E cells,and further explore its mechanism of action.To study the induced nephrotoxicity of Andrographolide in vivo and its effect on SIRT3-dependent signaling pathway in c57 mice.Methods(1)Real-time PCR was used to detect the effects of Andrographolide at different concentrations on inflammation,fibrosis and cell aging of NRK-52 E cells.The effects of Andrographolide at different concentrations on fibrosis of NRK-52 E cells were detected by Western blot.The effects of Andrographolide at different concentrations on the aging of NRK-52 E cells were detected using a p16 immunofluorescence kit.The effect of Andrographolide at different concentrations on reactive oxygen species in NRK-52 E cells was detected by DCFH-DA fluorescence probe method reactive oxygen species detection kit.γ-H2 AX immunofluorescence kit was used to detect the effect of Andrographolide at different concentrations on DNA damage of NRK-52 E cells.Adopt β-Detection of Andrographolide related to aging in NRK-52 E cells using galactosidase activity detection kit β-Activity of galactosidase.(2)Real-time PCR was used to detect the effect of Andrographolide on the m RNA expression of SIRT3 in NRK-52 E cells at different concentrations and at different times.Western blot method was used to detect the effect of Andrographolide at different concentrations and time on the protein expression of SIRT3 in NRK-52 E cells.The effects of Andrographolide at different concentrations on the activity of SIRT3 in NRK-52 E cells were detected with the kit.(3)Real-time PCR assay was used to detect the m RNA expression level of inflammation related factors in NRK-52E-Vector and NRK-52E-SIRT3 cells induced by Andrographolide.The effects of Andrographolide on the fibrosis of NRK-52E-Vector and NRK-52E-SIRT3 cells were detected by Real-time PCR and Western Blot methods.Real-time PCR assay was used to detect the m RNA expression level of Andrographolide on NRK-52E-Vector and NRK-52E-SIRT3 cell aging related factor p16.The effects of Andrographolide on the aging of NRK-52E-Vector and NRK-52E-SIRT3 cells were detected using a p16 immunofluorescence kit.The effect of Andrographolide on reactive oxygen species in NRK-52E-Vector and NRK-52E-SIRT3 cells was detected by using a DCFH-DA fluorescence probe assay for reactive oxygen species detection kit.γ-H2 AX immunofluorescence kit was used to detect the effect of Andrographolide on DNA damage in NRK-52E-Vector and NRK-52E-SIRT3 cells.Adopt β-Detection of Andrographolide related to aging of NRK-52E-Vector and NRK-52E-SIRT3 cells using galactosidase activity detection kit β-Activity of galactosidase.The effect of Andrographolide on the interaction between SIRT3 and p53 was detected by immunoprecipitation and Western Blot assay.(4)The effects of Andrographolide on serum creatinine(CR),urea nitrogen(BUN)and other biochemical indexes in mice kidney were detected by the kit.H&E staining and Masson staining were used to detect the pathological changes of kidney tissue in mice.The protein expression level of SIRT3 in mice was detected by Real-time PCR assay.Using a kit to detect the expression of inflammatory factors in mouse serum.Real-time PCR and Western Blot methods were used to detect the expression of fibrosis-related genes in mice induced by Andrographolide.Real-time PCR assay was used to detect the m RNA expression level of Andrographolide on aging related factor p16 in mice.ResultsIt can be shown by Real-time PCR assay,Western Blot assay and immunofluorescence assay that Andrographolide can induce inflammatory response,fibrosis and senescence in NRK-52 E cells.It can be concluded by kit,Real-time PCR assay and Western Blot that Andrographolide can inhibit the activity of SIRT3,and it can be shows that Andrographolide can inhibit SIRT3 activity by regulating SIRT3 and thus affect the inflammation,fibrosis and senescence of NRK-52 E cells by using Real-time PCR assay,Western Blot,kits and immunofluorescence assay after overexpression of SIRT3 in stable transgenic cell lines by immunoprecipitation and Western Blot assay.It can be concluded that there is an interaction between SIRT3 and p53,and Andrographolide can affect the interaction between SIRT3 and p53.When c57BL/16 J mice were injected with different concentrations(250 mg/kg and 500 mg/kg)of Andrographolide,the serum creatinine and urea nitrogen levels in mice were increased.The results of H&E staining and inflammatory factor kit proved that Andrographolide could damage kidney tissues and produce inflammatory responses in mice.Masson staining,Real-time PCR assay and Western Blot assay could prove that Andrographolide could induce kidney fibrosis in mice.Real-time PCR assay and Western Blot assay proved that andrographolide could inhibit the activity of SIRT3 in mice and induce renal senescence.Conclusions(1)Andrographolide induces inflammation,fibrosis,and senescence in renal NRK-52 E cells.(2)Andrographolide induces inflammation,fibrosis and senescence in NRK-52 E cells by regulating the SIRT3/p53 pathway.(3)Andrographolide induces inflammation,fibrosis and senescence in the kidney in mice.