The Pharmacodynamic Study Of Double-drug Compatibility Of Astragalus And Salvia Miltiorrhiza On Renal Damage In Spontaneously Hypertensive Rats And The Pharmacology On AMPK Pathway

Objective: To observe of the pharmacodynamics of double-drug compatibility of Astragalus and Salvia miltiorrhiza(traditional Chinese invigorating Qi and promoting blood circulation herbs) on delaying the pathological process of hypertensive renal damage and the renal protective effects. And to explore the preliminary pharmacological mechanism of the double-drug compatibility through the study of renal morphology and the regulation on the mRNA and protein expression of AMPK alpha, CaMKK and Sestrin.Methods: 45 16-week-old spontaneous hypertension rats(SHR) were included in the experimental group, and 9 age-matched WKY rats were used as control group(W group).According to the random number table method, SHR were divided into five groups with 9rats per group: Astragalus group(H group), Salvia miltiorrhiza group(D group),double-drug compatibility of Astragalus and Salvia miltiorrhiza group(HD group),valsartan group(X group) and SHR model group(M group).(2) After 8-week-feeding, rats were given 2ml corresponding drugs intragastrically each day for 28 days in total.(3)Blood pressure was measured by tail artery pressure method.(4) After 28-day intragastric administration, we detected the level of circulating kidney injury markers, such as serum cysteine proteinase inhibitor C(Cystain C), creatinine(Cr) and blood urea nitrogen(BUN),and urinary Cystain C and urinary microalbumin(mALB) and N-acetyl beta D Glucosaminidase(NAG). Renal blood flow, rat kidney size and the speed of blood flow were observed by ultrasound Doppler.(5)The rats were sacrificed, the kidneys were collected, the morphology of renal tissue was observed by hematoxylin and eosin(HE)staining and TEM(Transmission electron microscopy).(6)The change of mRNA level of AMPK alpha, Sestrin, CaMKK in kidney was detected by real time fluorescence quantitative PCR and the change of protein level of AMPK alpha and Sestrin, CaMKK inthe kidney was detected by Western Blotting.Results:(1) After 4-week drug administration, the blood pressure of rats in double-drug compatibility of Astragalus and Salvia miltiorrhiza group and valsartan group was significantly lower than that in SHR model group, while the blood pressure of rats in astragalus group and Salvia miltiorrhiza group was decreased slightly, but there was no significant difference. Compared with SHR model group, m ALB and NAG of the medication group were significantly decreased(P<0.01), among which Salvia miltiorrhiza group showed the best efficacy. Serum Cystain C, Cr, BUN and urinary Cystain C, mALB,NAG demonstrated no significant difference among groups(P>0.05).(2) Compared with SHR model group, renal blood flow index(RI) in both left and right kidney of the medication group was significantly lower(P<0.05), and the effect of double-drug compatibility of Astragalus and Salvia miltiorrhiza group was the most obvious.(3) The results of real-time fluorescence quantitative PCR assay showed, compared with the SHR model group, the mRNA expression of AMPK? and Sestrin was significantly up-regulated(P<0.05), while the mRNA expression of CaMKK was significantly down-regulated(P<0.01) in all medication group, and the double-drug compatibility of Astragalus and Salvia miltiorrhiza group was the most effective drug. Western Blotting assay showed that the protein expression of AMPK alpha and Sestrin was significantly higher than that of SHR model group, while CaMKK protein expression was significantly inhibited in each medication group, and the effect of the double-drug compatibility of Astragalus and Salvia miltiorrhiza group was the most obvious.Conclusion:(1) Double-drug compatibility of Astragalus and Salvia miltiorrhiza demonstrated beneficial effects on the renal damage of SHR significantly, with better efficacy than Astragalus or Salvia miltiorrhiza alone.(2) The pharmacological mechanism of double-drug compatibility of Astragalus and Salvia miltiorrhiza was related to the up-regulation of mRNA and protein expression of renal AMPK alpha and Sestrin and down-regulation of CaMKK, i.e. activation of the AMPK pathway in SHR.