基于DRD4/NOX4通路探讨左归丸改善肾阴虚型甲亢大鼠氧化应激的作用机制OA

Objective:To decipher the mechanism by which Zuoguiwan(ZGW)treat hyperthyroidism in rats with kidney-Yin deficiency based on the dopamine receptor D4(DRD4)/nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 4(NOX4)signaling pathway.Methods:The rat model of kidney-Yin deficiency was induced by unilateral intramuscular injection of dexamethasone(0.35 mg·kg-1).After successful modeling,the rats were randomized into model,methimazole(positive control,5 mg·kg-1),low-,medium-,and high-dose(1.85,3.70,7.40 g·kg-1,respectively)ZGW,and normal control groups.After 21 days of continuous gavage,the behavioral indexes and body weight changes of rats were evaluated.The pathological changes of the renal tissue were observed by hematoxylin-eosin staining.The serum levels of thyroid hormones[triiodothyronine(T3),thyroxine(T4),thyroid-stimulating hormone(TSH)],renal function indexes[serum creatine(Scr)and blood urea nitrogen(BUN)],energy metabolism markers[cyclic adenosine monophosphate(cAMP)and cyclic guanosine monophosphate(cGMP)],and oxidative stress-related factors[superoxide dismutase(SOD),malondialdehyde(MDA),and NADPH)]were measured by enzyme-linked immunosorbent assay(ELISA).Western blot was employed to analyze the expression of DRD4,NOX4,mitochondrial respiratory chain complex proteins[NADH:ubiquinone oxidoreductase subunit S4(NDUFS4)and cytochrome C oxidase subunit 4(COX4)],and inflammation-related protein[tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6),p38 mitogen-activated protein kinase(MAPK)]pathway in the renal tissue.Results:Compared with the normal group,the model group showed mental malaise,body weight decreases(P<0.01),inflammatory cell infiltration in the renal tissue,a few residual parotid glands in the thyroid,elevations in serum levels of T3,T4,Scr,BUN,cAMP,cAMP/cGMP,MDA,and NADPH(P<0.01),down-regulation in protein levels of TSH,SOD,and DRD4(P<0.05,P<0.01),and up-regulation in expression of NOX4,p-p38 MAPK/p38 MAPK,and inflammatory factors(P<0.01).Compared with the model group,ZGW increased the body weight(P<0.05,P<0.01),reduced the infiltration of renal interstitial inflammatory cells,restored the thyroid structure and follicle size,lowered the serum levels of T3,T4,Scr,BUN,cAMP,cAMP/cGMP,MDA and NADPH(P<0.05,P<0.01),up-regulated the expression of TSH,SOD and DRD4(P<0.05,P<0.01),and down-regulated the expression of NOX4,p-p38 MAPK/p38 MAPK,and inflammatory factors(P<0.05,P<0.01).Moreover,high-dose ZGW outperformed methimazole(P<0.05).Conclusion:By activating DRD4,ZGW can inhibit the expression of NOX4 mediated by the p38 MAPK pathway,reduce oxidative stress and inflammatory response,thereby ameliorating the pathological state of hyperthyroidism due to kidney-Yin deficiency.This study provides new molecular mechanism support for the clinical application of ZGW.