OBJECTIVE To investigate the mechanism of betaine(BET) in ameliorating oligoasthenozoospermia(OLI) through regulating the methylation of Bcl-2, Pi3k, and Akt series genes.
METHODS Sixty male Sprague-Dawley rats were randomly divided into blank control group, model groupTripterygium wilfordii glycosides(TWGs) 60 mg·kg–1·d–1, positive control group(L-carnitine, 300 mg·kg–1·d–1), low-, medium- and high-dose BET treatment groups(200, 400, 800 mg·kg–1·d–1). Testicular histopathology was examined by HE staining. Serum testosterone(T), luteinizing hormone(LH), and follicle-stimulating hormone(FSH) levels were measured using ELISA. Immunofluorescence staining was employed to analyze the expression of Caspase-3, BCL-2, PI3K, and AKT in testicular tissue. Western blotting was employed to determine the expression of BCL-2 and PI3K in testicular tissue. Methylation capture sequencing was performed to detect methylation levels of Bcl-2, Pi3k, and Akt series genes. Meanwhile, pi-RNA sequencing was used to assess the regulatory effects of BET on target genes. GO functional and KEGG pathway enrichment analyses were performed on potential target genes.
RESULTS Compared with the blank control group, the model group exhibited significant pathological changes in the testicular seminiferous tubules, including massive exfoliation of spermatogenic cells(P<0.001), disorganized arrangement, and tubular dilation; hormonal analysis showed decreased levels of T and FSH(P<0.01) and increased LH levels(P<0.01); immunofluorescence staining revealed that the expression of Caspase-3 was significantly up-regulated(P<0.001), while the expression of BCL-2, PI3K, and AKT was significantly reduced(P<0.001); Western blotting results indicated that BCL-2 and PI3K expression was significantly decreased in testicular tissue(P<0.01 or P<0.001). Compared with the model group, the medium- and high-dose BET treatment groups demonstrated notable improvements: the number of spermatogenic cells in the seminiferous tubules significantly increased(P<0.001), and cellular morphology improved; T and FSH levels rose(P<0.01), while LH levels decreased(P<0.01); immunofluorescence staining revealed that the expression of Caspase-3 was significantly reduced(P<0.001), while the expression of BCL-2, PI3K, and AKT was markedly up-regulated (P<0.001). Western blotting results showed that, compared with the model group, BCL-2 and PI3K expression in testicular tissue was significantly increased in the high- and low-dose BET groups(P<0.01 or P<0.001). Methylation capture sequencing revealed that BET significantly enhanced the methylation levels of target genes in the Bcl-2, Pi3k, and Akt families. pi-RNA sequencing further indicated that BET regulated the methylation of these target genes via the pi-RNA/PIWI complex. GO and KEGG pathway analysis suggested that this mechanism was associated with the PI3K-AKT signaling pathway.
CONCLUSION BET exerts therapeutic effects on the TWGs-induced OLI rat model. The underlying mechanism may involve BET promoting methylation of target genes(Bcl-2, Pi3k, and Akt) by supplying methyl groups, as well as activating the BCL-2-PI3K-AKT signaling pathway to up-regulate the expression of associated proteins.
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