A 779

Animal experiment:

A bilateral OVX operation is use to induce postmenopausal osteoporosis in female Wistar albino rats. The animals are generally anesthetized using single IP injection of ketamine + xylazine combination (80 mg/kg and 5 mg/kg; respectively). After ligation, the ovaries are excised from a longitudinal incision made on the dorsolateral body region. Sham rats have the same procedure without the ligation and excision. The risk of postoperative infection is eliminated by applying fusidic acid topical antibiotic cream twice weekly for 4 weeks. Following the sham and OVX operations, animals are divided into eight groups (n = 8/group) including, Sham, Sham + Ang(1-7), Sham + A-779, Sham + Ang(1-7)+A-779, OVX, OVX + Ang(1-7), OVX + A-779 and OVX + Ang(1-7)+A-779. Eight weeks following sham and OVX operations, osmotic pumps are implanted subcutaneously to deliver Ang(1-7) (200 ng/kg/min), A-779 (400 ng/kg/min) or a mixture of Ang(1-7) and A-779 (200 ng/kg/min and 400 ng/kg/min; respectively) for 6 consecutive weeks. Body weights of all animals are monitored and recorded at the beginning and every week throughout the study. Animals' general health is maintained during periods of treatments. At the end of treatments periods, animals are placed fasting in metabolic cages for 16 hr and urine samples are collected and frozen at −70°C. Then, the blood samples are collected by cardiac puncture under ketamine + xylazine anesthesia and serum samples are obtain by centrifugation at 4000 RPM to. Animals are then sacrificed by decapitation and the uterine tissues and femoral bones are removed and cleaned. Uterine tissues are cleaned from fats and the wet weights are determined directly and expressed as g/100 g body weight. In each rat, both femurs are removed and cleaned from soft tissues. The left femoral bone samples are frozen at −70°C until analyzed, while the right femoral bones are kept in 10% formalin for mico-CT and minerals analysis.

References:

[1]. Abuohashish HM, et al. Angiotensin (1-7) ameliorates the structural and biochemical alterations of ovariectomy-induced osteoporosis in rats via activation of ACE-2/Mas receptor axis. Sci Rep. 2017 May 23;7(1):2293.
[2]. Abuohashish HM, et al. ACE-2/Ang1-7/Mas cascade mediates ACE inhibitor, captopril, protective effects in estrogen-deficient osteoporotic rats. Biomed Pharmacother. 2017 Aug;92:58-68.
[3]. Yan WF, et al. Effects and related mechanism of angiotensin-(1-7) on Toll-like receptor 4-mediated oxidative stress in human umbilical vein endothelial cells. Zhonghua Xin Xue Guan Bing Za Zhi. 2017 Mar 24;45(3):223-229.