Matrine alleviates cisplatin‐induced acute kidney injury by inhibiting mitochondrial dysfunction and inflammation via SIRT3 / OPA1 pathway

Figures

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  FIGURE 1. Matrine inhibits cisplatin‐induced cell injury in HK2 cells. Cells were incubated with cisplatin (Cis, 10 μM) in the present or absent of matrine (MAT, 400 μΜ). (A–C) Cell viability was measured by CCK8 assay. (D and E) For apoptosis assay, the cells were stained with Annexin V‐FITC/PI cell apoptosis detection kit and analysed by flow cytometry. (F and G) Western blotting and quantification analysis of apoptosis‐related BAX and Bcl‐2 protein levels. The data are presented as the means ± SEM (n = 3). Bar graphs represent means ± SEM *p < 0.05 and **p < 0.01 versus control (CON); ## p < 0.01 versus cisplatin (Cis)

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  FIGURE 2. Matrine treatment improves mitochondrial function in cisplatin‐treated HK2 cells. (A and B) Mitochondrial ROS (mtROS) was determined by MitoSOX Red. (C and D) The mitochondrial membrane potential (Δψm) was detected by TMRM staining. (E) Representative immunofluorescence of mitochondrial morphology in HK2 cells, loaded with Mito‐tracker‐green (100 nM). (F) The ATP content in cells was detected by commercial ATP bioluminescence assay kit. (G and H) Western blotting and quantification analysis of mitochondria biogenesis‐related proteins (TFAM) and mitochondrial electron transport chain complex related proteins (NDUFS4 and ATP5a). Data represent 3 independent experiments, and are presented as the means ± SEM. **p < 0.01 versus CON group; # p < 0.05 and ## p < 0.01 versus Cis group

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  FIGURE 3. Matrine increases SIRT3 expression and its deacetylation activity. (A) Representative immunofluorescence of SIRT3 in HK2 cells with anti‐SIRT3 (red) and DAPI (blue). (B and C) The expression of SIRT3 was determined by Western blotting. (D) Cell lysates were immunoprecipitated (IP) with an anti‐SIRT3 or an anti‐OPA1 antibody and then immunoblotted (IB) with OPA1 and SIRT3 antibodies. Anti‐IgG antibody as a negative control. (E and F) Acetylation of OPA1 in HK2 cells with MAT treatment. The data are presented as the means ± SEM (n = 3). **p < 0.01 compared with CON group; # p < 0.05 and ## p < 0.01 compared with Cis group

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  FIGURE 4. Silencing of SIRT3 partially abolishes the protective effects of MAT on mitochondrial dysfunction and oxidative injury induced by cisplatin in HK2 cells. Cells were transfected with control siRNA (si‐NC) or SIRT3 siRNA (si‐SIRT3) for 6 h and treated with cisplatin in the presence or absence of MAT for 48 h. (A) The expression of SIRT3 was determined by Western blotting. (B) Acetylation of OPA1 in HK2 cells. (C) Representative immunofluorescence of mitochondrial morphology in HK2 cells. The mtROS level (D) and the ATP content (E) in HK2 cells. (F) The MDA content and CAT activity in cells were measured by chemical kit. The data are presented as the means ± SEM (n = 3). **p < 0.01 compared with CON group; # p < 0.05 and ## p < 0.01 compared with Cis group; & p < 0.05 and && p < 0.01 compared with Cis + MAT group

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  FIGURE 5. Matrine‐alleviated inflammation and cell injury in cisplatin‐treated HK2 cells was dependent on SIRT3. (A–C) The mRNA levels of IL‐1β, TNF‐α and IL‐6 in SIRT3‐knockdown HK2 cells. The cell viability (D) and apoptosis (E) were determined. (F) The expression of BAX and Bcl‐2 was assessed by Western blotting. The data are presented as the means ± SEM (n = 3). **p < 0.01 compared with CON group; ## p < 0.01 compared with Cis group; & p < 0.05 and && p < 0.01 compared with Cis + MAT group

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  FIGURE 6. Matrine treatment alleviates nephrotoxicity and apoptosis induced by cisplatin in mice. (A) The body weight, (B) the BUN level and (C) the serum CREA at 4 days after cisplatin injection. (D) Morphological changes of kidney tissues from different group were measured by haematoxylin and eosin and PAS staining. Arrows indicate acute tubular necrosis; pound signs indicate vacuolization. (E and F) The apoptosis in kidney tissues from mice was measured by TdT‐mediated dUTP nick‐end labelling (TUNEL) assay, and the TUNEL‐positive apoptotic cells were quantified by Image‐J software. (G) Western blotting and quantification analysis of apoptosis‐related BAX and Bcl‐2 protein levels. All data are quantified from 6 mice/group and shown as the means ± SEM. **p < 0.01 versus normal control (NC); ## p < 0.01 versus Cis

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  FIGURE 7. Matrine treatment activates SIRT3/OPA1 axis and rescues kidney mitochondrial function in cisplatin‐induced AKI mice. (A) Representative immunohistochemical staining of SIRT3 in mouse kidney sections among different groups. (B and C) SIRT3 expression and acetylation of OPA1 in mouse kidney sections were detected by Western blotting (n = 3). (D) mtROS was determined by MitoSOX Red staining in mouse kidney sections. (E–G) The mRNA levels of TFAM, NDUFS4 and ATP5a. (H) The ATP level in kidney tissues from mice. (I) Schematic diagram of mechanism that MAT prevented cisplatin‐induced cell injury and renal failure. The data are presented as the means ± SEM. All data are quantified from 4 mice/group and shown as the means ± SEM. **p < 0.01 versus the NC; # p < 0.05 and ## p < 0.01 versus Cis