Background: The Chinese herbal compound Heshouwuyin has been shown to downregulate the apoptotic rate of testicular tissue cells in Wistar naturally aging rats, and this effect might be related to the mitochondrial pathway [15]. Apoptotic protease activating factor-1 (Apaf-1) is a major component of the apoptotic complex, which is a key element of the mitochondrial endogenous apoptotic pathway [13]. To further clarify the mechanism of Heshouwuyin in the mitochondrial apoptotic pathway, this study used Apaf-1 as a target to explore the mechanism by which Heshouwuyin inhibits the Apaf-1 pathway of spermatogenic cell apoptosis. Methods: In this study, an aging model of rat spermatogenic cells was established using free radical oxidative damage. Flow cytometry was used to detect the apoptosis rate of germ cells and the inhibitory effect of Heshouwuyin. Apaf-1 was specifically knocked down by siRNA interference technology, and mitochondrial membrane potential was measured. qRT-PCR, Western blotting and immunofluorescence analyses were used to detect the expression of the key genes Cyt c, Caspase-9 and Caspase-3 in the mitochondrial apoptotic pathway of spermatogenic cells.Results: Heshouwuyin reduced the mRNA and protein expression levels of Cyt c, Caspase-9 and Caspase-3 in senescent spermatogenic cells. In these cells, the mRNA and protein expression levels of Cyt c did not change significantly after specific knockdown of Apaf-1, and the mRNA and protein expression levels of Caspase-9 and Caspase-3 decreased significantly. This finding indicated that knockdown of Apaf-1 could decrease the mRNA and protein expression levels of the downstream pro-apoptotic genes Caspase-9 and Caspase-3. Although Cyt c was an upstream gene of Apaf-1, knockdown of Apaf-1 had no significant effect on Cyt c expression. Conclusion:The inhibition of spermatogenic cell apoptosis by Heshouwuyin was closely related to the Cyt c/Apaf-1/Caspase-9/Caspase-3 pathway. The inhibition of apoptosis by Heshouwuyin not only involved the Apaf-1 pathway, but other signaling pathways.

Context: The Chinese herbal formula Heshouwu decoction (Heshouwuyin) has protective effects on testicular function in aging male rats, but the mechanism is unknown. Objective: This study investigated whether Heshouwuyin affects the testicular function of aging rats by regulating the insulin/IGF signalling pathway. Materials and methods: Sixteen-month-old male Wistar rats in the Heshouwuyin group and the naturalaging group were orally administered Heshouwuyin granules (0.056 g/kg) or equivalent normal saline for 60 d. The testicular tissue of 12-month-old male Wistar rats was removed as a young control group (n ¼ 10). The testicular tissue and spermatogenic cells were studied. Results: The immunofluorescence results revealed that the insulin receptor (INSR)-(0.056 ± 0.00548), insulin receptor substrate 1(IRS1)-(0.251 ± 0.031), IRS2 (0.230 ± 0.019)-and insulin-like growth factor 1 (IGF1)positive cell rate (0.33 ± 0.04) in the aging group was higher than that in the young control group (0.116 ± 0.011, 0.401 ± 0.0256, 0.427 ± 0.031, 0.56 ± 0.031; p < 0.01), and the IGF-binding protein 3 (IGFBP3)positive cell rate (0.42 ± 0.024) was lower than that (0.06 ± 0.027) in the young group (p < 0.01). The intervention of Heshouwuyin reversed the above phenomena. The qPCR and immunoblot results were consistent with those of the immunofluorescence. The same results were obtained in spermatogenic cells. Conclusions: Our research shows that Heshouwuyin can regulate the insulin/IGF signalling pathway to improve testicular function, and provides an experimental basis for further clinical use.