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Objective: To study the protective effect of moxibustion for tripterygium-induced premature ovarian failure (POF) and its underlying mechanisms in rats., Methods: Forty-five female SD rats were randomly divided into normal control, POF model and moxibustion groups ( n ＝15/group). The POF model was induced by intragastric administration of Triptolide (40 mg/kg), once daily for 6 weeks. From the 4 th week after modeling, moxibustion was given at "Guanyuan" (CV 4) and bilateral "Sanyinjiao" (SP 6) for 10 min, once daily for 3 weeks. Pathological changes of ovary tissues were determined by hematoxylin-eosin (HE) staining. The serum estradiol (E 2 ), luteinizing hormone (LH), follicle-stimulating hormone (FSH), interleukin-6 (IL-6) and interleukin-1 β (IL-1 β) contents were measured by enzyme-linked immunosorbent assay (ELISA). The expression levels of phosphatidyl inositol 3- kinase (PI 3 K), protein kinase B (Akt) and mammalian target of rapamycin (mTOR) proteins of the ovarian tissue were detected by Western blot., Results: After modeling, HE staining showed that the numbers of ovarian follicles and follicular granulocytes and corpora luteum layers were decreased, and the number of corpora atretica was increased in the model group. The content of serum E 2 was markedly decreased and those of serum LH, FSH, IL-6 and IL-1 β were markedly increased in the model group ( P <0.01), and the expression levels of ovarian p-PI 3 K, p-Akt and p-mTOR were markedly increased after modeling relevant to the control group ( P <0.01). Following moxibustion, the pathological damage of ovarian tissue was improved, the contents of serum LH, FSH, IL-6, IL-1 β, and the levels of p-PI 3 K, p-Akt and p-mTOR proteins in the ovarian tissue were significantly decreased ( P <0.05, P <0.01), and the content of serum E 2 was markedly increased ( P <0.05) in comparison with the model group., Conclusion: Moxibustion can improve POF in POF rats, which may be related to its actions in inhibiting PI 3 K/Akt/mTOR signaling, down-regulating serum IL-6, IL-1 β, and regulating serum hormones.

To improve a fast and high-quality isolation method for culturing the primary cardiomyocyte and fibroblast in vitro, the neonatal Wistar rats were decapitated accordingly and left ventricles were isolated under the sterile condition. The ventricles were chopped and digested in the enzyme solution containing 0.5 mg/mL type II collagenase. During this process, the digesting time, frequency and stirring speed, centrifuging frequency and speed were strictly controlled. The cardiomyocytes were separated from the cardiac fibroblast by using the Percoll density gradient centrifugation. The cell viability was tested by staining with 0.2% trypan blue. The purity of cardiomyocytes and fibroblasts were determined by immunoflourescent staining with anti-cTnI, anti-Vimentin and anti-α-SMA antibodies. The results indicated that with this protocol, the viability and purity of cardiomyocytes were 92% and 95%. The automobile pulse of the adhered cardiomyocyte was visible. For fibroblasts, the cell viability and purity were 96% and 94%. Our results demonstrate that this advanced isolation method is reproducible, and can simultaneously produce high-quality primary cardiomyocytes and fibroblasts for the future study.