Role of FMRP in AKT/mTOR pathway-mediated hippocampal autophagy in fragile X syndrome.

Fragile X syndrome (FXS) is caused by epigenetic silencing of the Fmr1 gene, leading to the deletion of the coding protein FMRP. FXS induces abnormal hippocampal autophagy and mTOR overactivation. However, it remains unclear whether FMRP regulates hippocampal autophagy through the AKT/mTOR pathway, which influences the neural behavior of FXS. Our study revealed that FMRP deficiency increased the protein levels of p-ULK-1 and p62 and decreased LC3II/LC3I level in Fmr1 knockout (KO) mice. The mouse hippocampal neuronal cell line HT22 with knockdown of Fmr1 by lentivirus showed that the protein levels of p-ULK-1 and p62 were increased, whereas LC3II/LC3I was unchanged. Further observations revealed that FMRP deficiency obstructed autophagic flow in HT22 cells. Therefore, FMRP deficiency inhibited autophagy in the mouse hippocampus and HT22 cells. Moreover, FMRP deficiency increased reactive oxygen species (ROS) level, decreased the co-localization between the mitochondrial outer membrane proteins TOM20 and LC3 in HT22 cells, and caused a decrease in the mitochondrial autophagy protein PINK1 in HT22 cells and Fmr1 KO mice, indicating that FMRP deficiency caused mitochondrial autophagy disorder in HT22 cells and Fmr1 KO mice. To explore the mechanism by which FMRP deficiency inhibits autophagy, we examined the AKT/mTOR signaling pathway in the hippocampus of Fmr1 KO mice, found that FMRP deficiency caused overactivation of the AKT/mTOR pathway. Rapamycin-mediated mTOR inhibition activated and enhanced mitochondrial autophagy. Finally, we examined whether rapamycin affected the neurobehavior of Fmr1 KO mice. The Fmr1 KO mice exhibited stereotypical behavior, impaired social ability, and learning and memory impairment, while rapamycin treatment improved behavioral disorders in Fmr1 KO mice. Thus, our study revealed the molecular mechanism by which FMRP regulates autophagy function, clarifying the role of hippocampal neuron mitochondrial autophagy in the pathogenesis of FXS, and providing novel insights into potential therapeutic targets of FXS.

TIM4/NLRP3 axis participates in the effects of M1 macrophages on inflammatory factor release, apoptosis and cell adhesion in thyroid follicular cells.

Hashimoto's thyroiditis (HT) is an autoimmune thyroid disorder that predominantly affects women. The role of the T-cell immunoglobulin and mucin domain-containing 4 (TIM4)/ NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) signaling pathway in macrophages has previously been studied, but its effects on macrophage-mediated HT has not yet been reported. Therefore, the aim of the current study was to explore the regulatory role of TIM4/NLRP3 in the effects of M1 macrophages on the inflammation, apoptosis and cell adhesion of thyroid follicular cells. To induce M1 macrophage, 10 ng/ml of LPS and 20 ng/ml IFN-γ were applied for the administration of THP-1 cells for 24 h. After induction, the mRNA expressions of M1 macrophage markers were assessed utilizing reverse transcription-quantitative (RT-q)PCR. Western blotting and immunofluorescence assay were adopted for the appraisement of inducible nitric oxide synthase. Additionally, the expression levels of TIM4 and NLRP3 before or after transfection were tested using RT-qPCR and western blotting. The release of inflammatory cytokines (TNF-α, IL-1ß and IL-6) were estimated using RT-qPCR and western blotting was adopted for the estimation of phosphorylated (p)-p65, p65, I-κB and p-I-κB. Furthermore, the apoptosis level as well as the accompanied proteins was appraised via TUNEL and western blotting. The mRNA and protein expressions of αvß3 were evaluated employing RT-qPCR and western blotting. The results demonstrated that TIM4 silencing decreased NLRP3 expression level in M1 macrophages. Moreover, TIM4 silencing in M1 macrophages reduced the expression levels of TNF-α, IL-6 and IL-1ß, as well as the phosphorylation levels of p65 and IκB in M1 macrophages co-cultured with Nthy-ori 3-1 cells, whereas NLRP3 overexpression significantly reversed these effects. Furthermore, NLRP3 overexpression reversed the decreased apoptotic rate and cell adhesion of Nthy-ori 3-1 cells induced by TIM4 silencing. In summary, the present study demonstrated that TIM4-silencing alleviated the inflammatory damage, apoptosis and cell adhesion of M1 macrophages co-cultured with Nthy-ori 3-1 cells through downregulation of NLRP3. Therefore, the regulation of M1 macrophages via the TIM4/NLRP3 axis may be a potential therapeutic approach for the treatment of patients with HT.

Mechanical Study of Jian-Gan-Xiao-Zhi Decoction on Nonalcoholic Fatty Liver Disease Based on Integrated Network Pharmacology and Untargeted Metabolomics.

Jian-Gan-Xiao-Zhi decoction (JGXZ) has demonstrated beneficial effects on nonalcoholic fatty liver disease (NAFLD). However, the mechanisms by which JGXZ improve NAFLD are still unclear. Methods. In this study, we first used a high-fat diet (HFD) to establish a NAFLD rat model to clarify the therapeutic effect of JGXZ on NAFLD. Secondly, we used network pharmacology to predict the potential targets of JGXZ on NAFLD, and then the key targets obtained from network pharmacology were verified. Finally, we used untargeted metabolomics to study the metabolic regulatory mechanism of JGXZ. Results. JGXZ treatment could decrease body weight and ameliorate dyslipidemia in NAFLD model rats. H&E and oil red O staining indicated that JGXZ reduced steatosis and infiltration of inflammatory cells in the liver. In addition, network pharmacology research found that the potential targets of JGXZ on NAFLD pathway were mainly associated with improving oxidative stress, apoptosis, inflammation, lipid metabolism disorders, and insulin resistance. Further experimental verification confirmed that JGXZ could inhibit inflammation and improve oxidative stress, insulin resistance, and lipid metabolism disorders. Serum untargeted metabolomics analyses indicated that the JGXZ in the treatment of NAFLD may work through the linoleic acid metabolism, alpha-linolenic acid metabolism, tryptophan metabolism, and glycerophospholipid metabolism pathways. Conclusions. In conclusion, this study found that JGXZ has an ameliorative effect on NAFLD, and JGXZ alleviates the inflammatory response and oxidative stress and lipid metabolism disorders in NAFLD rats. The mechanism of action of JGXZ in the treatment of NAFLD may be related to the regulation of linoleic acid metabolism, tryptophan metabolism, alpha-linolenic acid metabolism, and glycerophospholipid metabolism.

Diosgenin Inhibits Excessive Proliferation and Inflammatory Response of Synovial Fibroblasts in Rheumatoid Arthritis by Targeting PDE3B.

Rheumatoid arthritis (RA) is a chronic inflammation that can lead to loss of range of joint abnormalities in severe cases. Diosgenin has anti-inflammatory effects. This paper discussed the effect and mechanism of diosgenin on excessive proliferation and inflammatory response of synovial cells in RA. CCK-8 detected the cell viability, TUNEL assay detected the apoptosis of cells and western blot detected the expression of apoptosis-related proteins. Wound healing was used to detect cell migration and western blot detected the expression of migration-related proteins. ELISA kits were used to detect the levels of inflammatory cytokines in cells. Diosgenin can inhibit the proliferation and migration of RA synovial cells. At the same time, diosgenin could reduce the inflammatory response of RA synovial cells, during which the expression of PDE3B was significantly decreased. By overexpressing PDE3B, we found that diosgenin inhibited the proliferation, migration, and inflammatory response of RA synovial cells by downregulating PDE3B. Diosgenin can inhibit excessive proliferation and inflammatory response of synovial fibroblasts by targeting PDE3B.

Therapeutic effect of mesenchymal stem cell on Hashimoto's thyroiditis in a rat model by modulating Th17/Treg cell balance.

The autoimmune condition Hashimoto's thyroiditis (HT) is a disease wherein lymphocytes mediate the autoimmune damage and destruction of the thyroid gland. There are currently no effective means of treating HT, with the primary strategies of thyroid hormone therapy, surgery, or immunomodulatory therapy being associated with serious risks and side effects. There is thus a clear and urgent need to identify novel treatments for HT. In this study, we utilize female SD rats induced HT to evaluated the ability of transplanted MSCs to regulate Th17/Treg interactions in a rat Hashimoto's thyroiditis (HT) model system. The results showed that Rats in the HT model group exhibited increased thyroid autoantibody levels consistent with successful model development, whereas these levels were lower in rats treated with MSCs. There were also fewer thyroid lesions and less lymphoid infiltration of the thyroid in MSC-treated rats relative to HT model rats, as well as fewer Th17 cells and more Treg cells - an observation consistent with the cytokine analyses. All of these showed that MSCs can regulate Th17/Treg interactions in a rat Hashimoto's thyroiditis (HT) model system. It suggested that transplanted MSCs could be a potential immunotherapy strategy for the treatment of Hashimoto's thyroiditis.