Mouse hygiene status-A tale of two environments for mast cells and allergy.

Animal models, including those employing the use of house mice (Mus musculus), are crucial in elucidating mechanisms in human pathophysiology. However, it is evident that the impreciseness of using laboratory mice maintained in super-hygienic barrier facilities to mirror relevant aspects of human physiology and pathology exists, which is a major limitation in translating mouse findings to inferring human medicine. Interestingly, free-living wild mice are found to be substantially different from laboratory-bred, specific pathogen-free mice with respect to various immune system compartments. Wild mice have an immune system that better reflects human immunity. In this review article, we discuss recent experimental findings that address the so-called "wild immunology", which reveals the contrasting immune features between laboratory-raised mice and their wild companions as well as laboratory mice that have been exposed to a natural rodent habitat. A particular focus will be given to the development of pulmonary mast cells and its possible impact on the use of "naturalized" or "rewilded" laboratory mice as experimental asthma models.

Human placental extract suppresses mast cell activation and induces mast cell apoptosis.

BACKGROUND: Human placental extract (HPE) has been documented to facilitate the healing of certain disorders including allergy. However, the effects of HPE on the functionality of mast cells, a critical cell type in allergic diseases, have not been reported. METHODS: To investigate the effects of HPE on the regulation of allergy with respect to the biological functions of mast cells, the mast cell line C57 or HMC-1 cells were treated with HPE followed by the assessment of cell proliferation, apoptosis, activation, chemotaxis and phagocytosis. Mouse peritoneal mast cells were also investigated for their responses to induction of apoptosis by HPE in vivo. Furthermore, the effect of HPE on mast cell degranulation was confirmed using the passive cutaneous anaphylaxis (PCA) assay, an acute allergy model. RESULTS: HPE was capable of suppressing mast cell proliferation and inducing mast cell apoptosis. Mast cell degranulation in response to compound 48/80- or anti-DNP IgE and DNP-mediated activation was suppressed. In addition, treatment with HPE compromised the production of cytokines by mast cells and cell chemotaxis. These observations were consistent with the dampened passive cutaneous anaphylaxis (PCA) assay following treatment with HPE. CONCLUSION: This study revealed a suppressive effect of HPE on overall mast cell activities, suggesting a potential regulatory role of HPE on the alleviation of allergic diseases through mast cells.

Exercise-induced irisin improves follicular dysfunction by inhibiting IRE1α-TXNIP/ROS-NLRP3 pathway in PCOS.

BACKGROUND: Excessive production of androgen drives oxidative stress (OS) and inflammasome activation in ovarian granulosa cells (GCs). Therefore, the induced follicular developmental disorder is the major cause of infertility in women with polycystic ovary syndrome (PCOS). Exercise-induced upregulation of irisin is capable of regulating metabolism by reducing OS and inflammation. Exercise has been shown to alleviate a range of PCOS symptoms, including maintaining a normal menstrual cycle, in several clinical trials. METHODS: Female Sprague-Dawley (SD) rats and primary ovarian cells were treated with two different androgens, dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), to simulate a hyperandrogenic environment, followed by eight weeks of exercise training and irisin intervention. The levels of reactive oxygen species (ROS), tissue inflammation and fibrosis were examined using hematoxylin and eosin (H&E) staining, western blot, quantitative real-time PCR (qRT-PCR), dichlorofluorescein diacetate (DCF-DA) probe detection, immunofluorescence staining, immunohistochemistry, and Sirius red staining. RESULTS: Exercise for eight weeks improved polycystic ovarian morphology and decreased the levels of inflammation, OS, and fibrosis in PCOS rats. Hyperandrogen increased ROS production in ovarian cells by inducing endoplasmic reticulum stress (ERS) and activating the inositol-requiring enzyme 1α (IRE1α)-thioredoxin-interacting protein (TXNIP)/ROS-NOD-like receptor family pyrin domain containing 3 (NLRP3) signaling pathway, further enhancing the levels of inflammation. Irisin suppressed the expression of IRE1α and its downstream targets, thus improving the ovarian dysfunction of PCOS rats induced by hyperandrogen. CONCLUSION: Exercise can alleviate various phenotypes of PCOS rats induced by DHEA, and its therapeutic effect may be mediated by secreting beneficial myokines. IRE1α may be an important target of irisin for reducing OS and inflammation, thereby improving ovarian fibrosis.

FcγR-dependent apoptosis regulates tissue persistence of mucosal and connective tissue mast cells.

Rodent mast cells can be divided into two major subtypes: the mucosal mast cell (MMC) and the connective tissue mast cell (CTMC). A decade-old observation revealed a longer lifespan for CTMC compared with MMC. The precise mechanisms underlying such differential tissue persistence of mast cell subsets have not been described. In this study, we have discovered that mast cells expressing only one receptor, either FcγRIIB or FcγRIIIA, underwent caspase-independent apoptosis in response to IgG immune complex treatment. Lower frequencies of CTMC in mice that lacked either FcγRIIB or FcγRIIIA compared with WT mice were recorded, especially in aged mice. We proposed that this paradigm of FcγR-mediated mast cell apoptosis could account for the more robust persistence of CTMC, which express both FcγRIIB and FcγRIIIA, than MMC, which express only FcγRIIB. Importantly, we reproduced these results using a mast cell engraftment model, which ruled out possible confounding effects of mast cell recruitment or FcγR expression by other cells on mast cell number regulation. In conclusion, our work has uncovered an FcγR-dependent mast cell number regulation paradigm that might provide a mechanistic explanation for the long-observed differential mast cell subset persistence in tissues.

Pharmacodynamic substances and mechanism of Chelidonii Herba-Corydalis Rhizoma against estrogen receptor-positive breast cancer / 中国药房

OBJECTIVE To analyze the main components of Chelidonii Herba-Corydalis Rhizoma （CHCR）， and to predict pharmacodynamic substances against estrogen receptor （ER） -positive breast cancer and their potential targets and signaling pathways， followed by verifying experiments. METHODS The ethanol extract of CHCR was analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS/MS）. The network pharmacology analysis was performed for the screened components. The network diagram of CHCR “active components-target-pathway” was constructed， and the enrichment pathway in vitro was validated. RESULTS A total of 58 chemical components were identified， including 57 alkaloids and 1 organic acid. A total of 38 active ingredients were screened from the network pharmacology， and 38 core targets were found in the protein-protein interaction network of “component-disease” intersection targets； 258 gene ontology entries and 137 Kyoto encyclopedia of genes and genomics pathways were obtained， mainly including estrogen signal pathway， phosphatidylinositol-3-kinase/protein kinase B （PI3K/Akt） signal pathway， etc. The results of validation test showed that the median inhibitory concentration of CHCR to MCF-7 cells was 693 μg/mL； 150， 300， 600 μg/mL CHCR could significantly reduce the expressions of phosphorylated PI3K， phosphorylated Akt， ERα protein and ESR1 mRNA （P＜0.01）. CONCLUSIONS The anti-ER-positive breast cancer effect of CHCR may be related to the regulation of ER and PI3K/Akt pathways， which has the characteristics of multi-component and multi-target effects.

Role of B Lymphocytes in the Pathogenesis of NAFLD: A 2022 Update.

Non-alcoholic fatty liver disease and its related complications are becoming one of the most important health problems globally. The liver functions as both a metabolic and an immune organ. The crosstalk between hepatocytes and intrahepatic immune cells plays a key role in coordinating a dual function of the liver in terms of the protection of the host from antigenic overload as a result of receiving nutrients and gut microbiota antigenic stimulation via facilitating immunologic tolerance. B cells are the most abundant lymphocytes in the liver. The crucial role of intrahepatic B cells in energy metabolism under different immune conditions is now emerging in the literature. The accumulating evidence has demonstrated that the antibodies and cytokines produced by B cells in the microenvironment play key and distinct roles in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Herein, we have aimed to consolidate and update the current knowledge about the pathophysiological roles of B cells as well as the underlying mechanisms in energy metabolism. Understanding how B cells can exacerbate and suppress liver damage by exploiting the antibodies and cytokines they produce will be of great importance for designing B-cell targeting therapies to treat various liver diseases.

S100A4 exerts robust mucosal adjuvant activity for co-administered antigens in mice.

The lack of clinically applicable mucosal adjuvants is a major hurdle in designing effective mucosal vaccines. We hereby report that the calcium-binding protein S100A4, which regulates a wide range of biological functions, is a potent mucosal adjuvant in mice for co-administered antigens, including the SARS-CoV-2 spike protein, with comparable or even superior efficacy as cholera toxin but without causing any adverse reactions. Intranasal immunization with recombinant S100A4 elicited antigen-specific antibody and pulmonary cytotoxic T cell responses, and these responses were remarkably sustained for longer than 6 months. As a self-protein, S100A4 did not stimulate antibody responses against itself, a quality desired of adjuvants. S100A4 prolonged nasal residence of intranasally delivered antigens and promoted migration of antigen-presenting cells. S100A4-pulsed dendritic cells potently activated cognate T cells. Furthermore, S100A4 induced strong germinal center responses revealed by both microscopy and mass spectrometry, a novel label-free technique for measuring germinal center activity. Importantly, S100A4 did not induce olfactory bulb inflammation after nasal delivery, which is often a safety concern for nasal vaccination. In conclusion, S100A4 may be a promising adjuvant in formulating mucosal vaccines, including vaccines against pathogens that infect via the respiratory tract, such as SARS-CoV-2.

Mast Cells Are Identified in the Lung Parenchyma of Wild Mice, Which Can Be Recapitulated in Naturalized Laboratory Mice.

Background: It is well documented that laboratory mice bred and maintained in ultra-hygienic specific pathogen-free (SPF) barriers display reduced richness and complexity of microbiota compared with wild mice. The laboratory mice profoundly lack lung parenchymal mast cells. Hence, we aimed to investigate the lung distribution of mast cells in free-living wild mice. Methods: Wild house mice were trapped in South-Eastern Norway and Hemtabad, West Bengal, India. C57BL/6 laboratory mice were bred in a purposefully built, closed environment with bedding material obtained from the natural environment in order to normalize the gut microbiota of these laboratory mice to that of the wild mice, and the offspring were collected for study at eight weeks of age. Results: Mast cells were easily identified at a substantial density in the lung parenchymal tissues of wild mice from both Norway and India, which stands in clear contrast to the rare distribution of lung parenchymal mast cells in the conventional laboratory SPF mice. Consistently, wild mice also expressed higher pulmonary levels of stem cell factor, a critical growth factor for mast cell survival. Higher levels of histamine were recorded in the lung tissues of the wild mice. Interestingly, "naturalized" C57BL/6 laboratory mice which spent their entire life in a semi-natural environment developed lung parenchymal mast cells at an appreciable density. Conclusion: Our observations support that environmental factors, possibly through modulation of microbiota, may impact the tissue distribution of mast cells in mouse lung parenchyma.

Silencing of METTL3 effectively hinders invasion and metastasis of prostate cancer cells.

Background: Since primary prostate cancer (PCa) can advance to the life-threatening metastatic PCa, exploring the molecular mechanisms underlying PCa metastasis is crucial for developing the novel targeted preventive strategies for decreasing the mortality of PCa. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and its specific roles in PCa progression remains elusive. Methods: Western blotting, quantitative real-time PCR and immunohistochemical analyses were used to detect target gene expression in PCa cells in vitro and prostate tissues from patients. RNA immunoprecipitation was conducted to analyze the specific binding of mRNA to the target protein. Migration and invasion assays were used to assess the migratory capacities of cancer cells. The correlation between target gene expression and survival rate of PCa patients was analyzed based the TCGA database. Results: We found that total RNA N6-methyladenosine (m6A) modification levels were markedly upregulated in human PCa tissues due to increased expression of methyltransferase like 3 (METTL3). Further studies revealed that the migratory and invasive capacities of PCa cells were markedly suppressed upon METTL3 knockdown. Mechanistically, METTL3 mediates m6A modification of USP4 mRNA at A2696, and m6A reader protein YTHDF2 binds to and induces degradation of USP4 mRNA by recruiting RNA-binding protein HNRNPD to the mRNA. Decrease of USP4 fails to remove the ubiquitin group from ELAVL1 protein, resulting in a reduction of ELAVL1 protein. Lastly, downregulation of ELAVL1 in turn increases ARHGDIA expression, promoting migration and invasion of PCa cells. Conclusions: Our findings highlight the role of METTL3 in modulating invasion and metastasis of PCa cells, providing insight into promising therapeutic strategies for hindering PCa progressing to deadly metastases.

S100A4 Is Critical for a Mouse Model of Allergic Asthma by Impacting Mast Cell Activation.

Background: The calcium-binding protein S100A4 demonstrates important regulatory roles in many biological processes including tumorigenesis and inflammatory disorders such as allergy. However, the specific mechanism of the contribution of S100A4 to allergic diseases awaits further clarification. Objective: To address the effect of S100A4 on the regulation of mast cell activation and its impact on allergy. Methods: Bone marrow-derived cultured mast cells (BMMCs) were derived from wild-type (WT) or S100A4-/- mice for in vitro investigation. WT and S100A4-/- mice were induced to develop a passive cutaneous anaphylaxis (PCA) model, a passive systemic anaphylaxis (PSA) model, and an ovalbumin (OVA)-mediated mouse asthma model. Results: Following OVA/alum-based sensitization and provocation, S100A4-/- mice demonstrated overall suppressed levels of serum anti-OVA IgE and IgG antibodies and proinflammatory cytokines in serum, bronchoalveolar lavage fluid (BALF), and lung exudates. S100A4-/- mice exhibited less severe asthma signs which included inflammatory cell infiltration in the lung tissue and BALF, and suppressed mast cell recruitment in the lungs. Reduced levels of antigen reencounter-induced splenocyte proliferation in vitro were recorded in splenocytes from OVA-sensitized and challenged mice that lacked S100A4-/-. Furthermore, deficiency in the S100A4 gene could dampen mast cell activation both in vitro and in vivo, evidenced by reduced ß-hexosaminidase release and compromised PCA and PSA reaction. We also provided evidence supporting the expression of S100A4 by mast cells. Conclusion: S100A4 is required for mast cell functional activation, and S100A4 may participate in the regulation of allergic responses at least partly through regulating the activation of mast cells.

HIPK2 phosphorylates HDAC3 for NF-κB acetylation to ameliorate colitis-associated colorectal carcinoma and sepsis.

Although inflammation is critical for the clearance of pathogens, uncontrolled inflammation also contributes to the development of multiple diseases such as cancer and sepsis. Since NF-κB-mediated transactivation in the nucleus is pivotal downstream of various stimuli to induce inflammation, searching the nuclear-localized targets specifically regulating NF-κB activation will provide important therapeutic application. Here, we have identified that homeodomain-interacting protein kinase 2 (HIPK2), a nuclear serine/threonine kinase, increases its expression in inflammatory macrophages. Importantly, HIPK2 deficiency or overexpression could enhance or inhibit inflammatory responses in LPS-stimulated macrophages, respectively. HIPK2-deficient mice were more susceptible to LPS-induced endotoxemia and CLP-induced sepsis. Adoptive transfer of Hipk2+/- bone marrow cells (BMs) also aggravated AOM/DSS-induced colorectal cancer. Mechanistically, HIPK2 bound and phosphorylated histone deacetylase 3 (HDAC3) at serine 374 to inhibit its enzymatic activity, thus reducing the deacetylation of p65 at lysine 218 to suppress NF-κB activation. Notably, the HDAC3 inhibitors protected wild-type or Hipk2-/- BMs-reconstituted mice from LPS-induced endotoxemia. Our findings suggest that the HIPK2-HDAC3-p65 module in macrophages restrains excessive inflammation, which may represent a new layer of therapeutic mechanism for colitis-associated colorectal cancer and sepsis.

Induction of the apoptosis, degranulation and IL-13 production of human basophils by butyrate and propionate via suppression of histone deacetylation.

Allergic diseases are caused by dysregulated Th2 immune responses involving multiple effector cells including basophils. Short chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, exert immunomodulatory functions via activation of its receptors GPR41 and GPR43, and inhibition of the histone deacetylases (HDACs) activity. In allergic diseases, SCFAs suppress the activity of mast cells, eosinophils and type 2 innate lymphoid cells (ILC2) but enhance the function of Th2 cells. Here, we aimed to elucidate the function of SCFAs on human basophils. Human basophils were purified from healthy donors by flow cytometric sorting. The surface proteins, apoptosis and degranulation of basophils were analyzed by flow cytometric analysis. The mRNA expression was assayed using real-time PCR. Interleukin 4 (IL-4) and IL-13 were measured by ELISA. Histone acetylation was examined by western blot. GPR41 was expressed by basophils and was enhanced by IL-3. Acetate induced intracellular calcium influx in basophils which was suppressed by blocking GPR41. Propionate and butyrate, but not acetate, induced the expression of CD69 and IL-13. In addition, propionate and butyrate enhanced IgE-mediated basophil degranulation but inhibited basophil survival and IL-4 secretion. Propionate and butyrate induced histone acetylation of basophils and suppression of HDACs activity mimicked the effects of propionate and butyrate on human basophils. Our findings demonstrate that propionate and butyrate may play a complex role in regulating basophil apoptosis, activation and degranulation via inhibiting HDACs activity. The in vivo effects of SCFAs on the regulation of basophil-associated allergic diseases need to be further explored.

Correction: Insulin resistance enhances the mitogen-activated protein kinase signaling pathway in ovarian granulosa cells.

[This corrects the article DOI: 10.1371/journal.pone.0188029.].

Effects of different vibration frequencies on muscle strength, bone turnover and walking endurance in chronic stroke.

This randomized controlled trial aimed to evaluate the effects of different whole body vibration (WBV) frequencies on concentric and eccentric leg muscle strength, bone turnover and walking endurance after stroke. The study involved eighty-four individuals with chronic stroke (mean age = 59.7 years, SD = 6.5) with mild to moderate motor impairment (Fugl-Meyer Assessment lower limb motor score: mean = 24.0, SD = 3.5) randomly assigned to either a 20 Hz or 30 Hz WBV intervention program. Both programs involved 3 training sessions per week for 8 weeks. Isokinetic knee concentric and eccentric extension strength, serum level of cross-linked N-telopeptides of type I collagen (NTx), and walking endurance (6-min walk test; 6MWT) were assessed at baseline and post-intervention. An intention-to-treat analysis revealed a significant time effect for all muscle strength outcomes and NTx, but not for 6MWT. The time-by-group interaction was only significant for the paretic eccentric knee extensor work, with a medium effect size (0.44; 95% CI: 0.01, 0.87). Both WBV protocols were effective in improving leg muscle strength and reducing bone resorption. Comparatively greater improvement in paretic eccentric leg strength was observed for the 30 Hz protocol.

NLRP3 inflammasome activation in alveolar epithelial cells promotes myofibroblast differentiation of lung-resident mesenchymal stem cells during pulmonary fibrogenesis.

Idiopathic pulmonary fibrosis (IPF) is a lethal and agnogenic interstitial lung disease, which has limited therapeutic options. Recently, the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome has been demonstrated as an important contributor to various fibrotic diseases following its persistent activation. However, the role of NLRP3 inflammasome in pulmonary fibrogenesis still needs to be further clarified. Here, we found that the activation of the NLRP3 inflammasome was raised in fibrotic lungs. In addition, the NLRP3 inflammasome was found to be activated in alveolar epithelial cells (AECs) in the lung tissue of both IPF patients and pulmonary fibrosis mouse models. Further research revealed that epithelial cells, following activation of the NLRP3 inflammasome, could induce the myofibroblast differentiation of lung-resident mesenchymal stem cells (LR-MSCs). In addition, inhibiting the activation of the NLRP3 inflammasome in epithelial cells promoted the expression of dickkopf-1 (DKK1), a secreted Wnt antagonist. DKK1 was capable of suppressing the profibrogenic differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis. In conclusion, this study not only provides a further in-depth understanding of the pathogenesis of pulmonary fibrosis, but also reveals a potential therapeutic strategy for disorders associated with pulmonary fibrosis.

Alveolar epithelial cell-derived Sonic hedgehog promotes pulmonary fibrosis through OPN-dependent alternative macrophage activation.

The alternative activation of macrophages in the lungs has been considered as a major factor promoting pulmonary fibrogenesis; however, the mechanisms underlying this phenomenon are still elusive. In this study, we investigated the interaction between macrophages and fibrosis-associated alveolar epithelial cells using a bleomycin-induced mouse pulmonary fibrosis model and a coculture system. We demonstrated that fibrosis-promoting macrophages are spatially proximate to alveolar type II (ATII) cells, permissive for paracrine-induced macrophage polarization. Importantly, we revealed that fibrosis-associated ATII cells secrete Sonic hedgehog (Shh), a hedgehog pathway ligand, and that ATII cell-derived Shh promotes the development of pulmonary fibrosis by osteopontin (OPN)-mediated macrophage alternative activation. Mechanistically, Shh promotes the secretion of OPN in macrophages via Shh/Gli signaling cascade. The secreted OPN acts on the surrounding macrophages in an autocrine or paracrine manner and induces macrophage alternative activation through activating the JAK2/STAT3 signaling pathway. Tissue samples from idiopathic pulmonary fibrosis patients confirmed the increased expression of Shh and OPN in ATII cells and macrophages, respectively. Together, our study illustrated an alveolar epithelium-dependent mechanism for macrophage M2 polarization and pulmonary fibrogenesis and suggested that targeting Shh may offer a selective and efficient therapeutic strategy for the development and progression of pulmonary fibrosis.

PRCC-TFE3 fusion-mediated PRKN/parkin-dependent mitophagy promotes cell survival and proliferation in PRCC-TFE3 translocation renal cell carcinoma.

TFE3 (transcription factor binding to IGHM enhancer 3) nuclear translocation and transcriptional activity has been implicated in PINK1-PRKN/parkin-dependent mitophagy. However, the transcriptional control governing the mitophagy in TFE3/Xp11.2 translocation renal cell carcinoma (TFE3 tRCC) is largely unknown. Here, we investigated the role and mechanisms of PRCC-TFE3 fusion protein, one of TFE3 fusion types in TFE3 tRCC, in governing mitophagy to promote development of PRCC-TFE3 tRCC. We observed and analyzed mitophagy, transcriptional control of PRCC-TFE3 on PINK1-PRKN-dependent mitophagy, PRCC-TFE3 fusions nuclear translocation, cancer cell survival and proliferation under mitochondrial oxidative damage in PRCC-TFE3 tRCC cell line. We found that nuclear-aggregated PRCC-TFE3 fusions constitutively activated expression of the target gene E3 ubiquitin ligase PRKN, leading to rapid PINK1-PRKN-dependent mitophagy that promoted cell survival under mitochondrial oxidative damage as well as cell proliferation through decreasing mitochondrial ROS formation. However, nuclear translocation of TFE3 fusions escaped from PINK1-PRKN-dependent mitophagy. Furthermore, we confirmed that PRCC-TFE3 fusion accelerated mitochondrial turnover by activating PPARGC1A/PGC1α-NRF1. In conclusion, our findings indicated a major role of PRCC-TFE3 fusion-mediated mitophagy and mitochondrial biogenesis in promoting proliferation of PRCC-TFE3 tRCC.

m6A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells.

Macroautophagy/autophagy is indispensable for testosterone synthesis in Leydig cells (LCs), and here we report a negative association between m6A modification and autophagy in LCs during testosterone synthesis. A gradual decrease of METTL14 (methyltransferase like 14) and an increase of ALKBH5 (alkB homolog 5, RNA demethylase) were observed in LCs during their differentiation from stem LCs to adult LCs. These events led to reduced mRNA methylation levels of N6-methyladenosine (m6A) and enhanced autophagy in LCs. Similar regulation of METTL14, ALKBH5, and m6A was also observed in LCs upon treatment with human chorionic gonadotropin (HsCG). Mechanistically, m6A modification promoted translation of PPM1A (protein phosphatase 1A, magnesium dependent, alpha isoform), a negative AMP-activated protein kinase (AMPK) regulator, but decreased expression of CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, beta), a positive AMPK regulator, by reducing its RNA stability. Thus, m6A modification resulted in reduced AMPK activity and subsequent autophagy inhibition. We further demonstrated that ALKBH5 upregulation by HsCG was dependent on enhanced binding of the transcriptional factor CEBPB (CCAAT/enhancer binding protein [C/EBP], beta) and the TFEB (transcription factor EB) to its gene promoter. Moreover, HsCG treatment decreased METTL14 by reducing its stability. Collectively, this study highlights a vital role of m6A RNA methylation in the modulation of testosterone synthesis in LCs, providing insight into novel therapeutic strategies by exploiting m6A RNA methylation as targets for treating azoospermatism and oligospermatism patients with reduction in serum testosterone.Abbreviations: 3-MA: 3-methyladenine; ACTB: Actin, beta; ALKBH5: alkB homolog 5, RNA demethylase; AMPK: AMP-activated protein kinase; BafA1: bafilomycin A1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CEBPB: CCAAT/enhancer-binding protein (C/EBP), beta; ChIP: chromatin immunoprecipitation; FTO: fat mass and obesity associated; HsCG: human chorionic gonadotropin; HSD3B: 3ß-hydroxysteroid dehydrogenase; LCs: Leydig cells; m6A: N6-methyladenosine; METTL14: methyltransferase like 14; METTL3: methyltransferase like 3; MTOR: mechanistic target of rapamycin kinase; PPM1A: protein phosphatase 1A, magnesium dependent, alpha isoform; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; ULK1: unc-51-like kinase 1; WTAP: Wilms tumor 1-associating protein; YTHDF: YTH N6-methyladenosine RNA binding protein.