Pituitary P62 deficiency leads to female infertility by impairing luteinizing hormone production

P62 is a protein adaptor for various metabolic processes. Mice that lack p62 develop adult-onset obesity. However, investigations on p62 in reproductive dysfunction are rare. In the present study, we explored the effect of p62 on the reproductive system. P62 deficiency-induced reproductive dysfunction occurred at a young age (8 week old). Young systemic p62 knockout (p62-/-) and pituitary-specific p62 knockout (p62flox/flox αGSUcre) mice both presented a normal metabolic state, whereas they displayed infertility phenotypes (attenuated breeding success rates, impaired folliculogenesis and ovulation, etc.) with decreased luteinizing hormone (LH) expression and production. Consistently, in an infertility model of polycystic ovary syndrome (PCOS), pituitary p62 mRNA was positively correlated with LH levels. Mechanistically, p62-/- pituitary RNA sequencing showed a significant downregulation of the mitochondrial oxidative phosphorylation (OXPHOS) pathway. In vitro experiments using the pituitary gonadotroph cell line LβT2 and siRNA/shRNA/plasmid confirmed that p62 modulated LH synthesis and secretion via mitochondrial OXPHOS function, especially Ndufa2, a component molecule of mitochondrial complex I, as verified by Seahorse and rescue tests. After screening OXPHOS markers, Ndufa2 was found to positively regulate LH production in LβT2 cells. Furthermore, the gonadotropin-releasing hormone (GnRH)-stimulating test in p62flox/flox αGSUcre mice and LβT2 cells illustrated that p62 is a modulator of the GnRH-LH axis, which is dependent on intracellular calcium and ATP. These findings demonstrated that p62 deficiency in the pituitary impaired LH production via mitochondrial OXPHOS signaling and led to female infertility, thus providing the GnRH-p62-OXPHOS(Ndufa2)-Ca2+/ATP-LH pathway in gonadotropic cells as a new theoretical basis for investigating female reproductive dysfunction.

shRNA targeting nonstructural protein inhibits the replication of severe fever with thrombocytopenia syndrome virus

Aim: To explore whether shRNA targeting nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) could inhibit SFTSV replication in Vero cells. Materials & methods: SFTSV used in this experiment was propagated in Vero cells and stored at -20°C. shRNA plasmid against NSs of SFTSV was transfected to Vero cells and infected with SFTSV, after which western blotting and tissue culture infective dose (TCID50) were used to measure the virus titers. Results: shRNA against NSs protein decreased the expression of NSs and inhibited the replication of SFTSV. Conclusion: The constructed SFTSV NSs-shRNA plasmid could inhibit the replication of SFTSV. It was concluded that SFTSV NSs-shRNA could inhibit virus replication for at least 72 h. shRNA-mediated antiviral effects were dose-dependent.

Study on the Mechanism of Olfactomedin 4-shRNA Plasmid Chitosan Magnetic Nanoparticles in Intestinal Metaplasia of Chronic Atrophic Gastritis

Intestinal metaplasia (IM) refers to the replacement of gastric epithelial cells by intestinal epithelial cells. This is often accompanied by chronic atrophic gastritis (CAG), which is a precancerous lesion of gastric cancer. The incidence rates of CAG and IM are increasing gradually, which generates an enormous economic burden to individuals and society. To explore the pathogenesis of CAG with IM, we screened out the differentially expressed gene olfactomedin 4 (OLFM4) by bioinformatics and constructed OLFM4-shRNA plasmid chitosan magnetic nanoparticles to knockdown this gene. This caused a downregulation of caudal type homeobox 2 (CDX2), a marker of IM, suggesting that knocking down OLFM4 may slow the pathological process of IM, thus providing putative relevant targets for the treatment of CAG with IM.

CXCL1 contributes to IL-6 expression in osteoarthritis and rheumatoid arthritis synovial fibroblasts by CXCR2, c-Raf, MAPK, and AP-1 pathway

Background Osteoarthritis (OA) and rheumatoid arthritis (RA) are common joint disorders that are considered to be different diseases due to their unique molecular mechanisms and pathogenesis. Chemokines and their corresponding receptors have been well characterized in RA progression, but less so in OA pathogenesis. Methods The human primary synovial fibroblasts (SFs) were obtained from human OA and RA tissue samples. The Western blot and qPCR were performed to analyze the expression levels of CXCL1, as well as CXCL-promoted IL-6 expression in both OASFs and RASFs. The signal cascades that mediate the CXCL1-promoted IL-6 expression were identified by using chemical inhibitors, siRNAs, and shRNAs. Results Here, we found that both diseases feature elevated levels of CXCL1 and interleukin (IL)-6, an important proinflammatory cytokine that participates in OA and RA pathogenesis. In OASFs and RASFs, CXCL1 promoted IL-6 expression in a dose- and time-dependent manner. In OASFs and RASFs overexpressing CXCL1 or transduced with shRNA plasmid, IL-6 expression was markedly upregulated. CXCR2, c-Raf, and MAPKs were found to regulate CXCL1-induced IL-6 expression in OASFs and RASFs. Finally, CXCL1 triggered the transcriptional activities of c-Jun (which regulates the expression of proinflammatory proteins) in OASFs and RASFs. Conclusions Our present work suggests that the CXCL1/CXCR2 axis helps to orchestrate inflammatory responses in OA and RA SFs.

GPAT Gene Silencing in Muscle Reduces Diacylglycerols Content and Improves Insulin Action in Diet-Induced Insulin Resistance

Skeletal muscle is an important tissue responsible for glucose and lipid metabolism. High-fat diet (HFD) consumption is associated with the accumulation of bioactive lipids: long chain acyl-CoA, diacylglycerols (DAG) and ceramides. This leads to impaired insulin signaling in skeletal muscle. There is little data on the involvement of DAG in the development of these disorders. Therefore, to clarify this enigma, the gene encoding glycerol-3-phosphate acyltransferase enzyme (GPAT, responsible for DAG synthesis) was silenced through shRNA interference in the gastrocnemius muscle of animals with diet-induced insulin resistance. This work shows that HFD induces insulin resistance, which is accompanied by an increase in the concentration of plasma fatty acids and the level of bioactive lipids in muscle. The increase in these lipids inhibits the insulin pathway and reduces muscle glucose uptake. GPAT silencing through electroporation with shRNA plasmid leads to a reduction in DAG and triacylglycerol (TAG) content, an increase in the activity of the insulin pathway and glucose uptake without a significant effect on ceramide content. This work clearly shows that DAG accumulation has a significant effect on the induction of muscle insulin resistance and that inhibition of DAG synthesis through GPAT modulation may be a potential target in the treatment of insulin resistance.

CXCL1 contributes to IL-6 expression in osteoarthritis and rheumatoid arthritis synovial fibroblasts by CXCR2, c-Raf, MAPK and AP-1 pathway

Background: Osteoarthritis (OA) and rheumatoid arthritis (RA) are common joint disorders that are considered to be different diseases due to their unique molecular mechanisms and pathogenesis. Chemokines and their corresponding receptors have been well-characterized in RA progression, but less so in OA pathogenesis.Methods: The human primary synovial fibroblasts (SFs) were obtained from human OA and RA tissue samples. The Western blot and qPCR were performed to analyze expression levels of CXCL1, as well as CXCL-promoted IL-6 expression in both OASFs and RASFs. The signal cascades that mediate the CXCL1-promoted IL-6 expression were identified by using chemical inhibitors, siRNAs and shRNAs.Results: Here, we found that both diseases feature elevated levels of CXCL1 and interleukin (IL)-6, an important proinflammatory cytokine that participates in OA and RA pathogenesis. In OASFs and RASFs, CXCL1 promoted IL-6 expression in a dose- and time-dependent manner. In OASFs and RASFs overexpressing CXCL1 or transduced with shRNA plasmid, IL-6 expression was markedly upregulated. CXCR2, c-Raf and MAPKs was found to regulate CXCL1-induced IL-6 expression in OASFs and RASFs. Finally, CXCL1 triggered the transcriptional activities of c-Jun (which regulates the expression of proinflammatory proteins) in OASFs and RASFs.Conclusions: Our present work suggests that the CXCL1/CXCR2 axis helps to orchestrate inflammatory responses in OA and RA SFs.

CXCL1 contributes to IL-6 expression in osteoarthritis and rheumatoid arthritis synovial fibroblasts by CXCR2, c-Raf, MAPK and AP-1 pathway

Background: Osteoarthritis (OA) and rheumatoid arthritis (RA) are common joint disorders that are considered to be different diseases due to their unique molecular mechanisms and pathogenesis. Chemokines and their corresponding receptors have been well-characterized in RA progression, but less so in OA pathogenesis. Methods: The human primary synovial fibroblasts (SFs) were obtained from human OA and RA tissue samples. The Western blot and qPCR were performed to analyze expression levels of CXCL1, as well as CXCL-promoted IL-6 expression in both OASFs and RASFs. The signal cascades that mediate the CXCL1-promoted IL-6 expression were identified by using chemical inhibitors, siRNAs and shRNAs. Results: Here, we found that both diseases feature elevated levels of CXCL1 and interleukin (IL)-6, an important proinflammatory cytokine that participates in OA and RA pathogenesis. In OASFs and RASFs, CXCL1 promoted IL-6 expression in a dose- and time-dependent manner. In OASFs and RASFs overexpressing CXCL1 or transduced with shRNA plasmid, IL-6 expression was markedly upregulated. CXCR2, c-Raf and MAPKs was found to regulate CXCL1-induced IL-6 expression in OASFs and RASFs. Finally, CXCL1 triggered the transcriptional activities of c-Jun (which regulates the expression of proinflammatory proteins) in OASFs and RASFs. Conclusions: Our present work suggests that the CXCL1/CXCR2 axis helps to orchestrate inflammatory responses in OA and RA SFs.

CXCL1 contributes to IL-6 expression in osteoarthritis and rheumatoid arthritis synovial fibroblasts by CXCR2, c-Raf, MAPK and AP-1 pathway

Background Osteoarthritis (OA) and rheumatoid arthritis (RA) are common joint disorders that are considered to be different diseases due to their unique molecular mechanisms and pathogenesis. Chemokines and their corresponding receptors have been well-characterized in RA progression, but less so in OA pathogenesis. Methods The human primary synovial fibroblasts (SFs) were obtained from human OA and RA tissue samples. The Western blot and qPCR were performed to analyze expression levels of CXCL1, as well as CXCL-promoted IL-6 expression in both OASFs and RASFs. The signal cascades that mediate the CXCL1-promoted IL-6 expression were identified by using chemical inhibitors, siRNAs and shRNAs. Results Here, we found that both diseases feature elevated levels of CXCL1 and interleukin (IL)-6, an important proinflammatory cytokine that participates in OA and RA pathogenesis. In OASFs and RASFs, CXCL1 promoted IL-6 expression in a dose- and time-dependent manner. In OASFs and RASFs overexpressing CXCL1 or transduced with shRNA plasmid, IL-6 expression was markedly upregulated. CXCR2, c-Raf and MAPKs was found to regulate CXCL1-induced IL-6 expression in OASFs and RASFs. Finally, CXCL1 triggered the transcriptional activities of c-Jun (which regulates the expression of proinflammatory proteins) in OASFs and RASFs. Conclusions Our present work suggests that the CXCL1/CXCR2 axis helps to orchestrate inflammatory responses in OA and RA SFs.