Transcriptomic and proteomic features of a mouse model of sperm DNA damage induced by benzo(a)pyrene.

This study replicated a mouse model of sperm DNA damage induced by benzo(a)pyrene (BaP), and the transcriptomic and proteomic features of the model were examined to clarify the pathways related to BaP-induced damage to sperm DNA. Male mice in the BaP group were subjected to BaP at a dosage of 100 mg/kg/d or an equivalent quantity of saline solution in the control group for 60 days. Subsequently, the DNA fragmentation index (DFI) in sperm was assessed using a sperm chromatin structure assay (SCSA). RNA-seq and data-independent acquisition (DIA) were used to identify the mRNA and protein expression patterns in the testis. The sperm DFI significantly increased in the BaP group. Compared to the control group, the BaP group exhibited differential expression of 240 genes (referred to as DEGs) and 616 proteins (referred to as DEPs). These molecules included Aldh1a1, Cyb5r3, Fads1, Oxsm, Rcn3, and Prss45. Pathways in cancer, the PI3K-Akt signaling pathway, metabolic pathways, and the MAPK signaling pathway were the primary areas where these genes showed enrichment. BaP can damage the DNA of sperm and affect metabolism, the PI3K-Akt pathway, and pathways associated with cancer signaling.

Repair mechanism of Yishen Tongluo formula on mouse sperm DNA fragmentation caused by polystyrene microplastics.

CONTEXT: Plastics can break down into millions of microplastic (MPs, < 5 mm) particles in the soil and ocean. These MPs can then affect the function of the reproductive system. There is currently no effective solution to this problem aside from traditional Chinese medicine. We have previously used Yishen Tongluo formula (YSTL) to treat sperm DNA damage caused by some toxic substances. OBJECTIVE: To investigate the mechanism underlying the repair of mouse sperm DNA fragmentation caused by polystyrene microplastics by YSTL. MATERIALS AND METHODS: An animal model of polystyrene microplastic (PS-MP)-induced sperm DNA damage was replicated by gavage of SPF ICR (CD1) mice PS-MPs at 1 mg/d and treated with YSTL at 11.89, 23.78 and 47.56 g/kg, respectively, for 60 days. The Sperm DNA fragmentation index (DFI) of each group was detected and compared. The target genes of YSTL identified by transcriptomic and proteomic analyses were validated by qRT-PCR and western blotting. RESULTS: The DFI of the PS group (20.66%) was significantly higher than that of the control group (4.23%). The medium and high doses of the YSTL group (12.8% and 11.31%) exhibited a significant repairing effect. The most enriched pathway was PI3K/Akt. TBL1X, SPARC, hnRNP0, Map7D1, Eps8 and Mrpl27 were screened and SPARC was validated. DISCUSSION AND CONCLUSIONS: The precise mechanism by which YSTL inhibits PD-MPs DNA damage may be associated with the PI3K/Akt pathway and SPARC. It provides a new direction for using traditional Chinese medicine to prevent and repair reproductive system injury caused by MPs.

Transcriptomics and proteomics analysis to explore the mechanism of Yishen Tongluo formula repairing sperm DNA damage in rats.

The sperm DNA fragmentation index (DFI) is an objective indicator of male fertility. Currently, effective treatments for high sperm DFI are limited and traditional Chinese medicine (TCM) has certain advantages in this aspect. Yishen Tongluo formula (YSTL), a TCM formula, has been found to reduce DFI in patients. To better understand the mechanisms underlying its activity, we used transcriptomics and proteomics to analyse the potential target gene YSTL repairing tripterygium glycosides (TGs)-mediated sperm DNA damage in rats, followed by validation analyses using RT-qPCR and western blotting, which showed that relative to the control group, DFI was markedly elevated in the TGs group, but markedly lower in the YSTL group relative to the TGs group. KEGG pathway analysis of 119 differentially expressed genes and 158 DEPs identified using trend analysis revealed that they were enriched for apoptosis and base excision repair at the transcriptomic level and for microRNAs in cancer and complement and coagulation cascades at the proteomic level. Ttr and Pnpla2 were identified as potential target genes for YSTL. Our data show that YSTL can protect rat sperm DNA from TGs-induced damage, which may be related to apoptosis, DNA repair and other pathways, and the possible target genes are Ttr and Pnpla2.

Transcriptome and genome sequencing investigating the molecular characteristics of patients with varicocele infertility.

The prevalence of varicoceles in male infertility is increasing; however, the exact mechanism is unknown, and no direct studies of varicose spermatic veins have been conducted. Three patients with varicocele infertility were included to explore the possible factors that cause varicocele infertility, and varicose and nearby normal veins were harvested by varicocelectomy. RNA sequencing was performed on six vascular samples, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the screened differential expressed genes which were validated by quantitative reverse transcription-polymerase chain reaction. The genomes of the patients were analysed using next-generation sequencing to screen for genetic factors behind varicocele infertility. 1171 genes were upregulated and 2772 were downregulated in varicose spermatic veins compared with those in normal veins. These genes were significantly enriched in the alcohol consumption pathway. HIST1H4C, HIST1H4F, HIST1H4K, TM9SF1, and TMEFF1 were significantly differentially expressed. The genomic results identified patients with mutations in CFTR, NANOS1, SRCAP, GATA4, GCM2, TUBB1, ALDH7A1, ANTXR1, and MAP3K1. In conclusion, our results indicated that Alcohol consumption may be a cause of varicoceles. Mutations in certain genes, such as CFTR, may be a cause of male infertility due to varicoceles.

Methylation status of TK1 correlated with immune infiltrates in prostate cancer.

TK1 is overexpressed in numerous cancers and is associated with to a poor prognosis. However, the relationship between methylation status of TK1 and Immune Infiltrates in Prostate Cancer (PCa) is unknown. The goal of this study was to use comprehensive bioinformatic analyses to elucidate the involvement relationship between methylation status of TK1 and Immune Infiltrates in PCa. TK1 mRNA expression and methylation data in PCa were investigated via GEPIA, TIMER, and UALCAN coupled with MEXPRESS data resources. We employed the LinkedOmics data resource to determine the signaling cascades linked to TK1 expression. Single-cell analysis was performed using the CancerSEA data resource. GeneMANIA and CancerSEA were used to analyze the correlation between TK1 and TK1 coexpressed genes. In addition, TIMER and TISIDB were adopted to assess tumor-invading immune cells and immunomodulators. CTD was utilized to detect the drugs acting on TK1. This study found that TK1 was overexpressed in PCa, and its contents were linked to tumor stage and prognosis. Genes co-expressed with TK1 were enriched in cascades involved in the ribosome, cell cycle, oxidative phosphorylation, DNA replication, oocyte meiosis, and the proteasome. The expression of TK1 along with its methylation status was found to be linked to tumor-invading immune cells, as well as PCa immunomodulators. We also examined the prospect of employing TK1 as a possible target for PCa therapy. This work provides the clinical value of TK1 hypermethylation in PCa and highlights new insights into its novel immunomodulatory functions.

[Effects of Yishen Tongluo Prescription on the PI3K-Akt-MTOR pathway and the protein and mRNA expressions of CatSper-1 and HSPA2 in the testis tissue of oligoasthenospermia rats].

OBJECTIVE: To study the effect of Yishen Tongluo Prescription (YTP) on the testis tissue of the male rats with oligoasthenospermia (OAS) and its action mechanism based on the PI3K-AKT-mTOR pathway. METHODS: We randomly divided 48 SPF male SD rats into 8 groups: normal control, OAS model control, L-carnitine (LC), high-dose YTP, low-dose YTP, Omipalisib inhibitor (OI), OI + high-dose YTP, and OI + low-dose YTP, with 6 rats in each group. We established a model of OAS in the latter 7 groups by intragastric administration of tripterygium wilfordii polyside, followed by intervention with corresponding drugs. After treatment, we obtained semen parameters from the rats, observed pathological changes in the testis tissue by HE staining, and determined the expressions of the PI3K-AKT-mTOR signaling pathway-related proteins and mRNA by Western blot and real-time fluorescence quantitative PCR (qRT-PCR). RESULTS: Sperm concentration and total sperm motility were significantly improved in the LC and YTP groups compared with those in the OAS model control group (P < 0.001). HE staining showed irregularly arranged spermatogenic cells and narrowed lumina and widened gaps of seminiferous tubules in the OAS model controls, as well as similar pathological changes in the LC, YTP and OI + YTP groups. Significant up-regulation was observed in the protein expressions of p-Akt, CatSper-1 and HSPA2 in the LC group (P < 0.05), those of p-Akt, mTOR, catsper-1 and HSP2 in the low-dose YTP group (P < 0.05) and that of PI3K in the high-dose YTP group (P < 0.05) compared with those in the model controls. There were no statistically significant differences in the expressions of PI3K, mTOR and catsper-1 between the OI and model control groups (P > 0.05), nor in those of PI3K, p-Akt, mTOR, CatSper-1 and HSPA2 between the OI + YTP and the former two groups (P > 0.05). The mRNA expressions of PI3K, mTOR, CatSper-1 and HSPA2 were remarkably higher in the LC and YTP groups than in the model control (P< 0.05), with those of catsper-1 and PI3K even more significantly up-regulated in the high-dose than in the low-dose YTP group (P< 0.001; P< 0.05). Statistically significant differences were not observed in the mRNA expressions of PI3K, mTOR, CatSper-1 and HSPA2 between the model control and OI groups (P > 0.05), nor in those of PI3K, mTOR, catsper-1 and HSPA2 between the model control and OI + YTP groups (P > 0.05). CONCLUSION: Yishen Tongluo Prescription can improve sperm quality and pathological changes of the testis tissue in rats with Tripterygium glycoside-induced OAS, which might be attributed to its ability of up-regulating the expressions of the PI3K Akt mTOR pathway-related proteins and mRNA in the testis tissue.

[Clinical and mechanism research on functional erectile dysfunction treated with moxibustion and qiangshen shugan qiwei decoction].

OBJECTIVE: To observe the clinical therapeutic effect on functional erectile dysfunction (FED) of kidney deficiency and liver stagnation treated by Baixiao moxibustion and qiangshen shugan qiwei decoction. METHODS: A total of 120 patients with FED were randomized into an observation group (60 cases, 2 cases dropped off) and a control group (60 cases, 4 cases dropped off). In the control group, the patients were treated with oral qiangshen shugan qiwei decoction (Radix Rehmanniae Praeparata, Rhizoma Dioscoreae, Rhizoma Curculiginis, Herba Epimedii, etc.), one dose daily. In the observation group, on the base of the treatment as the control group, Baixiao moxibustion was exerted along the distribution of governor vessel on the back, 30 min each time, once a week. The treatment duration was 4 weeks in the two groups. Before and after treatment, the scores of international index of erectile function-5 (IIEF5), erectile hardness scale (EHS), erectile dysfunction inventory of treatment satisfaction (EDITS) and TCM symptoms were observed in the patients of the two groups. Before and after treatment, the levels of serum sex hormones (testosterone [T], luteinizing hormone [LH], estradiol [E2], prolactin [PRL]) and the expressions of relevant proteins (matrix metalloproteinase-3 [MMP-3], matrix metalloproteinase-9 [MMP-9] and vascular endothelial growth factor [VEGF]) were determined in the patients of the two groups separately. The penile vascular function (peak systolic velocity [PSV], end-diastolic velocity [EDV], resistance index [RI]) was compared before and after treatment. Besides, the clinical therapeutic effect was evaluated. RESULTS: After treatment, the scores of IIEF5, EHS and EDITS were all increased as compared with the values before treatment (P<0.05), and the scores of TCM symptoms were reduced as compared with the values before treatment in the patients of the two groups (P<0.05). After treatment, the scores of IIEF5, EHS and EDITS in the observation group were higher than those in the control group (P<0.05), and the score of TCM symptoms was lower than the control group (P<0.05). After treatment, the levels of T and LH in serum of the two groups and the expressions of serum MMP-3 and MMP-9 in the observation group were all increased as compared with those before treatment (P<0.05), the levels of E2 and PRL in the observation group and VEGF expressions in the two groups were all reduced as compared with those before treatment (P<0.05). The levels of serum T and LH and the expressions of MMP-3 and MMP-9 in the observation groups were all higher than those in the control group (P<0.05), and the levels of E2 and PRL and VEGF expression were lower than the control group (P<0.05). After treatment, PSV was increased and EDV was reduced as compared with those before treatment in the patients of the two groups (P<0.05), RI in the observation group was reduced as compared with that before treatment (P<0.05). After treatment, PSV in the observation group was higher and EDV and RI were lower than the control group (P<0.05). The total effective rate was 87.9% (51/58) in the observation group, higher than 76.8% (43/56) in the control group (P<0.05). CONCLUSION: The combined therapy of Baixiao moxibustion and qiangshen shugan qiwei decoction effectively increases erectile function, erectile hardness and treatment satisfaction in the patients with functional erectile dysfunction of kidney deficiency and liver stagnation, as well as improves penile vascular function. The effect mechanism of this therapy may be related to the regulation of serum sex hormones and relevant proteins.

[Yishen Tongluo Recipe combined with minimally invasive surgery for the treatment of varicocele-associated asthenospermia].

OBJECTIVE: To observe the clinical effect of Yishen Tongluo Recipe (YTR) combined with minimally invasive surgery (MIS) in the treatment of varicocele-associated asthenospermia (of the kidney-deficiency and collateral-obstruction type). METHODS: We randomly divided 116 patients with varicocele-associated asthenospermia and up to the inclusion criteria into three groups and treated them by YTR combined with MIS (low spermatic vein ligation at the external inguinal ring orifice under the microscope) (the YTR+MIS group, n = 39), YTR alone (the YTR group, n = 38), or MIS alone (the MIS group, n = 39). At 12 weeks after treatment, we compared the total effectiveness rate, the percentage of progressively motile sperm (PMS), sperm straight line velocity (VSL), sperm DNA fragmentation index (DFI) and symptoms among the three groups of patients. RESULTS: After treatment, the total effectiveness rate was significantly higher in the YTR+MIS than in the YTR and the MIS group (89.74% vs 63.16% and 64.10%, P < 0.05), and so were the percentage of PMS (31.67 ± 3.93 vs 24.48 ± 3.15 and 25.57 ± 3.37, P < 0.05) and VSL (ï¼»30.19 ± 5.32ï¼½ vs ï¼»27.19 ± 5.48ï¼½ and ï¼»27.55 ± 5.24ï¼½ µm/s, P < 0.05), but the DFI was remarkably lower in the former than in the latter two groups (11.25 ± 8.27 vs 15.87 ± 5.45 and 15.26 ± 4.36, P < 0.05). The symptoms were improved more significantly in the YTR+MIS than in the YTR and MIS groups (P < 0.05). CONCLUSIONS: Yishen Tongluo Recipe combined with minimally invasive surgery, as an effective and safe strategy, can significantly improve sperm quality and sperm DNA integrity in patients with varicocele-associated asthenospermia.

[Protective effect of Yishen Tongluo Recipe against benzo(a)pyrene-induced sperm DNA methylation changes in male rats].

OBJECTIVE: To explore the protective effect of Yishen Tongluo Recipe (YTR) against aberrant sperm DNA methylation in male rats exposed to benzo(a)pyrene (BaP). METHODS: Thirty male SD rats of the SPF grade were randomly divided into three groups of equal number: solvent control, BaP exposure and YTR intervention. The animals of the solvent control group were injected intraperitoneally with 0.5% DMSO while those of the other two groups with BaP at 0.1 mg/kg/d, all for 60 days, and at 31 days of BaP exposure, those of the YTR group were treated intragastrically with YTR for 30 days. Then, the left epididymides were harvested from all the rats and sperm suspensions collected and centrifuged for extraction of sperm DNA. The methylated DNA immunoprecipitation sequencing (MeDIP-seq) technique was used to detect the whole-genome DNA methylation in different groups. RESULTS: Exposure to BaP induced the up-regulation of 828 genes encoding mRNA in the sperm DNA, while YTR intervention produced a significant protective effect on the transforming growth factor ß3 (TGF-ß3), cystic fibrosis transmembrane conductance regulator (CFTR) and recombination activating gene 1 (RAG1), and down-regulated the expressions of 3 227 genes. BaP exposure also caused the up-regulation of 783 genes encoding lncRNA in the sperm DNA, and YTR treatment exhibited an evident protective effect on 62 of the up-regulated genes, induced the down-regulation of 3 378 genes, and showed a protective effect on 56 of the down-regulated genes. CONCLUSIONS: YTR has a protective effect against aberrant sperm DNA methylation in male rats exposed to BaP, which may be associated with lncRNA.