Adrenomedullin (ADM) exerts anti-oxidant, anti-inflammatory and anti-apoptotic effects in Leydig cells. However, the role and mechanism of ADM in the pyroptosis of Leydig cells are poorly understood. This study first showed the protective effects of ADM on the pyroptosis and biological functions of Leydig cells exposed to lipopolysaccharide (LPS) by promoting autophagy. Primary rat Leydig cells were treated with various concentrations of LPS and ADM, together with or without N-acetyl-L-cysteine (NAC) or 3-methyladenine (3-MA). Cell proliferation was detected through CCK-8 and BrdU incorporation assays, and ROS level was measured with the DCFDA assay. Real-time PCR, western blot, immunofluorescence, transmission electron microscopy, TUNEL and flow cytometry were performed to examine ADM’s effect on the pyroptosis, autophagy and steroidogenic enzymes of Leydig cells and AMPK/mTOR signalling. Like NAC, ADM dose-dependently reduced LPS-induced cytotoxicity and ROS overproduction. ADM also dose-dependently ameliorated LPS-induced pyroptosis by reversing the increased expression of NLRP3, ASC, caspase-1, IL-1β, IL-18, GSDMD, caspase-3, caspase-7, TUNEL-positive and PI and active caspase-1 double-stained positive rate, DNA fragmentation and LDH concentration, which could be rescued via co-incubation with 3-MA. ADM dose-dependently increased autophagy in LPS-induced Leydig cells, as confirmed by the increased expression of LC3-I/II, Beclin-1 and ATG-5; decreased expression of p62 and autophagosomes formation; and increased LC3-II/LC3-I ratio. However, co-treatment with 3-MA evidently decreased autophagy. Furthermore, ADM dose-dependently rescued the expression of steroidogenic enzymes, including StAR, P450scc, 3β-HSD and CYP17, and testosterone production in LPS-induced Leydig cells. Like rapamycin, ADM dose-dependently enhanced AMPK phosphorylation but reduced mTOR phosphorylation in LPS-induced Leydig cells, which could be rescued via co-incubation with 3-MA. In addition, pyroptosis was further decreased, and autophagy was further promoted in LPS-induced Leydig cells upon co-treatment with ADM and rapamycin. ADM may protect the steroidogenic functions of Leydig cells against pyroptosis by activating autophagy via the ROS–AMPK–mTOR axis.

This study aimed to explore the possible benefits of adrenomedullin (ADM) in preventing oxidative stress and inflammation by using an in vitro primary culture model of rat Leydig cells exposed to lipopolysaccharide (LPS). Cell proliferation was detected through CCK-8 and BrdU incorporation assays. ROS were determined with a DCFDA kit, and cytokine concentrations were measured with ELISA assay kits. Protein production was examined by immunohistochemical staining and Western blot, and gene expression was observed through RT-qPCR. Results revealed that ADM significantly reduced LPS-induced cytotoxicity, and pretreatment with ADM significantly suppressed ROS overproduction and decreased 4-HNE and 8-OHdG expression levels and concentrations. ADM pretreatment also significantly attenuated the overactivation of enzymatic antioxidants, namely, superoxide dismutase, catalase, thioredoxin reductase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase. ADM supplementation reversed the significantly increased gene expression levels and concentrations of TNF-α, IL-1β, TGF-β1, MCP-1 and MIF. ADM pretreatment significantly inhibited the gene expression and protein production of TLR-2 and 4. Furthermore, ADM pretreatment markedly reduced the phosphorylation of JNK, ERK 1/2 and p38, phosphorylation and degradation of IκBα and nuclear translocation of p65. Our findings demonstrated that ADM protects Leydig cells from LPS-induced oxidative stress and inflammation, which might be associated with MAPK/NF-κB signalling pathways.

Long noncoding RNA, RNA component of mitochondrial RNA processing endoribonuclease (RMRP) plays an important role in cancer development and is closely correlated with prognosis in cancer patients. However, whether RMRP affects prognosis in patients with hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to investigate the expression level of RMRP in HCC and its correlation with prognosis in patients with HCC and explain the effects and associated mechanisms by conducting an in vitro study. The high expression level of RMRP was correlated with poor prognosis in patients with HCC. Using in vitro analysis, RMRP knockdown suppressed HCC cell proliferation, invasion, and migration (P < .05). miRNA‐206 overexpression had similar effects in HCC cell lines (Bel‐7402 and Huh‐7). Using Western blot analysis and cellular immunofluorescence detection, RMRP downregulation significantly suppressed TACR1/Erk1/2 pathway, while miRNA‐206 was significantly upregulated (P < .05). RMRP downregulation inhibits HCC‐related biological activities by the regulation of miRNA‐206/TACR1.

BackgroundMDR1 gene encoding P-glycoprotein is an ATP-dependent drug efflux transporter and related to drug resistance of yolk sac carcinoma. Ultrasound microbubble-mediated delivery has been used as a novel and effective gene delivery method. We hypothesize that small interfering RNA (siRNA) targeting MDR1 gene (siMDR1) delivery with microbubble and ultrasound can down-regulate MDR1 expression and improve responsiveness to chemotherapeutic drugs for yolk sac carcinoma in vitro.MethodsRetroviral knockdown vector pSEB-siMDR1s containing specific siRNA sites targeting rat MDR1 coding region were constructed and sequence verified. The resultant pSEB-siMDR1 plasmids DNA were encapsulated with lipid microbubble and the DNA release were triggered by ultrasound when added to culture cells. GFP positive cells were counted by flow cytometry to determine transfection efficiency. Quantitative real-time PCR and western blot were performed to determine the mRNA and protein expression of MDR1. P-glycoprotein function and drug sensitivity were analyzed by Daunorubicin accumulation and MTT assays.ResultsTransfection efficiency of pSEB-siMDR1 DNA was significantly increased by ultrasound microbubble-mediated delivery in rat yolk sac carcinoma L2 (L2-RYC) cells. Ultrasound microbubble-mediated siMDR1s delivery effectively inhibited MDR1 expression at both mRNA and protein levels and decreased P-glycoprotein function. Silencing MDR1 led to decreased cell viability and IC50 of Vincristine and Dactinomycin.ConclusionsOur results demonstrated that ultrasound microbubble-mediated delivery of MDR1 siRNA was safe and effective in L2-RYC cells. MDR1 silencing led to decreased P-glycoprotein activity and drug resistance of L2-RYC cells, which may be explored as a novel approach of combined gene and chemotherapy for yolk sac carcinoma.

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Objective The characteristics of bladder calculi and their correlation with hypertension in Southern Hunan, China were analyzed retrospectively to guide the prevention and treatment of bladder calculi and hypertension. Materials and methods A retrospective cohort study of medical records was conducted in the First Affiliated Hospital of University of South China from December 2017 to December 2021. The subjects were patients over 14 years old who were diagnosed with bladder calculi by imaging examination and whose calculi were removed. Analysis was conducted on the patients’ basic characteristics and calculus composition, characteristics, and relationship with hypertension. Results A total of 352 cases of bladder calculi were enrolled. The age ranged from 15 years to 89 years with an average of (65.56 ± 11.94) years. Most of the respondents came from rural areas (61.4%). Calcium oxalate was the main component of the stones. Calcium oxalate and carbonate apatite were the most common mixed stones, and pure uric acid was the most common single stone. The pure uric acid stones were significantly larger than the pure calcium oxalate stones (P < 0.05). In addition, 26.1% of the patients with bladder calculi experienced hypertension. Binary logistic regression showed that hypertension was an independent risk factor for bladder calculi (B = 2.584, OR = 1.685, P < 0.05). Conclusion The management of bladder calculi in Southern Hunan can be guided by local demographic data. Hypertension may be one of the independent risk factors for bladder calculi.