The Mechanism Of Yangjing Capsule In Regulating StAR To Promote Testosterone Synthesis Via LncRNA H19

【Objective】Late-onset hypogonadism(LOH)is a common disease in the male aging process,its core mechanism is the functional decline and reduced number of Leydig cells,presenting a series of clinical symptoms of relative lack of testosterone.Exogenous testosterone supplementation is the main treatment method of LOH,but there are still some controversies and limitations.Therefore,TCM intervention on LOH meets the clinical needs,and it has important clinical significance to explore its internal mechanism.Previous scholars have reported that Lnc RNA H19 could regulate the expression of steroidogenic acute regulatory protein(St AR)at the post-transcriptional level.Previous studies have also confirmed that Yangjing Capsule(YC)could enhance the activity of St AR promoter and then promote testosterone synthesis,but the upstream mechanism of St AR regulation was not elucidated.Based on the above background,we investigated whether YC could regulate St AR through Lnc RNA H19 in promoting testosterone synthesis.【Methods】1.Animal experiment: BALB/c old male LOH model mice and BALB/c young male mice were used as animal subjects and divided into control group,Yangjing Capsule suspension(YCS)treated group(low,medium and high dose)and young group with continuous gavage for 4 weeks.The tissue morphology of the mouse testis was visualized by HE staining.The localization and expression of St AR protein in mouse testis were observed by immunohistochemistry in paraffin sections.Serum testosterone and free testosterone concentrations were determined by ELISA.The m RNA and protein expressions of St AR,Lnc RNA H19 and Micro RNA Let7 b m RNA were detected by q RT-PCR and Western Blot,respectively.2.Cell experiment: Mouse Leydig tumour cell line(MLTC-1)was cultured as in vitro to logarithmic phase,treated with low(0.01 mg/ml),medium(0.1 mg/ml)and high(1 mg/ml)dose Yangjing Capsule water extract(YCWE)for 24 h.The testosterone concentration of the cell supernatant was determined by ELISA.The m RNA and protein expressions of St AR,Lnc RNA H19 and Micro RNA Let7 b m RNA in MLTC-1 cells were detected by q RT-PCR and Western Blot,respectively.A lentiviral stable strain with knockdown Lnc RNA H19 was constructed and subsequently treated with high dose YCWE for 24 h.The m RNA levels of Lnc RNA H19 and St AR,cell supernatant testosterone were detected by q RT-PCR and ELISA,respectively.【Results】1.Animal experiment: YCS significantly increased serum testosterone and free testosterone concentrations in LOH model mice,upregulated Lnc RNA H19,St AR m RNA and St AR protein in testicular tissue and downregulated the expression of Micro RNA Let7 b m RNA.YCS was able to increase the Leydig cell number in mice.2.Cell experiment: YCWE could increase the concentration of testosterone in cell supernatant,upregulate the expression of Lnc RNA H19,St AR m RNA and St AR protein in MLTC-1 cells,and downregulate the expression of Micro RNA let7 b m RNA.After the construction of lentivirus stable strain knockdown Lnc RNA H19,the levels of Lnc RNA H19,St AR m RNA and cell supernatant testosterone in MLTC-1 cells decreased significantly.After YCWE intervention,both Lnc RNA H19 and St AR m RNA expression were upregulated,and the testosterone concentration in the cell supernatant increased significantly.【Conclusion】1.Yangjing Capsule could promote testosterone synthesis,which might be related to regulating the transcription and translation of St AR in steroid hormone synthesis.2.Yangjing Capsule could regulate the transcription and translation of St AR by upregulating the expression of Lnc RNA H19 m RNA.3.Yangjing Capsule suspension could increase the number of Leydig cells and improve the function of Leydig cells.4.Yangjing Capsule water extract could reverse the inhibition of St AR transcription and testosterone synthesis by lentiviral stable strain with knockdown of Lnc RNA H19.5.Lnc RNA H19 might be one of the key genes for Yangjing Capsule to promote testosterone synthesis.Yangjing Capsule probably regulated the transcription of St AR by Lnc RNA H19 and then promoted testosterone synthesis,this study provided theoretical basis and data support for clinical application of Yangjing capsule in the treatment of LOH.