Chaihu Longgu Muli Decoction relieving temporal lobe epilepsy in rats by inhibiting TLR4 signaling pathway through miR-146a-3p and miR-146a-5p

Objective To explore the effect and mechanism of Chaihu Longgu Muli Decoction(柴胡龙骨牡蛎汤,CHLGMLD)in rats with temporal lobe epilepsy(TLE).Methods A total of 80 Sprague-Dawley(SD)male rats were randomized into control(CON),model(MOD),carbamazepine(CBZ,0.1 g/kg),CHLGMLD low dose(CHLGMLD-L,12.5 g/kg),and high dose(CHLGMLD-H,25 g/kg)groups,with 16 rats in each group.TLE rat models were established in the four groups with the use of lithium-pilocarpine except for the CON group.After the successful establishment of TLE models,all drugs were administered through gavage,and distilled water was given to rats in the CON and MOD groups for four weeks.The frequency and duration of seizures before and after treatment were recorded for the evaluation of the alleviation degree.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-146a-3p and miR-146a-5p.The expression levels of toll-like receptor 4(TLR4),interleukin-1 receptor-associated kinase 1(IRAK1),tumor necrosis factor(TNF)receptor-associated factor 6(TRAF6),TAK1-binding protein(TAB),nuclear factor-kappa B(NF-κB),and interleukin-1 beta(IL-1β)in hippocampus were tested by immunofluorescence assay.Correlation analysis between the above factors and expressions of miR-146a-3p and miR-146a-5p were performed separately.Results CHLGMLD decreased the frequency(P<0.05)and duration(P<0.01)of seizures in rats.CHLGMLD down-regulated the expression levels of miR-146a-5p and miR-146a-3p(P<0.05),and inhibited the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β(P<0.01).The correlation analysis revealed that the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β were positively correlated with the expression levels of miR-146a-3p and miR-146a-5p detected by qRT-PCR,respectively(P<0.01).Conclusion CHLGMLD can inhibite the TLR4 signaling pathway by lowering the expression levels of miR-146a-3p and miR-146a-5p to alleviate hippocampal dentate gyrus inflammation in TLE rats,thus relieving seizures.

MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.

基于HSPA5/GPX4信号通路探讨针刀干预膝关节骨关节炎兔软骨细胞铁死亡的作用机制

目的:观察针刀疗法对膝关节骨关节炎(KOA)兔膝关节软骨细胞热休克蛋白A家族成员5(HSPA5)/谷胱甘肽过氧化物酶4(GPX4)信号通路的影响,探讨针刀干预KOA软骨细胞铁死亡的作用机制。方法:将27只新西兰兔随机分为正常组、模型组和针刀组,每组9只。采用改良Videman法连续固定兔左侧后肢6周建立KOA模型。造模后,针刀组给予针刀干预,每周1次,连续3周。运用奎森功能障碍指数(Lequesne MG评分)评估兔膝关节局部症状、体征及功能;HE染色法与番红-固绿染色法分别观察兔软骨细胞与组织形态;透射电镜观察兔软骨细胞线粒体结构;运用铁离子试剂盒检测兔软骨组织铁含量;流式细胞仪检测兔软骨组织线粒体膜电位(Δψm)和活性氧(ROS)水平,并计算线粒体损伤率;实时荧光定量PCR检测兔软骨组织HSPA5、GPX4、Ⅱ型胶原α1链(COL2A1)、基质金属蛋白酶(MMP)3、MMP13 mRNA表达;Western blot法检测兔软骨组织HSPA5、GPX4、Ⅱ型胶原蛋白(COL-Ⅱ)、MMP3、MMP13蛋白表达;免疫荧光法检测兔软骨组织HSPA5、GPX4平均荧光强度。结果:干预前,与正常组比较,模型组和针刀组兔膝关节Lequesne MG评分升高(P<0.01);干预后,与模型组比较,针刀组兔膝关节Lequesne MG评分降低(P<0.01)。与正常组比较,模型组兔膝关节软骨细胞数量减少且排列紊乱,软骨结构层次不清,潮线紊乱、模糊不清,线粒体皱缩、线粒体嵴减少甚至消失;与模型组比较,针刀组软骨细胞数量较多,软骨结构层次清楚,潮线恢复,线粒体数量较多,结构正常,有较多的嵴。与正常组比较,模型组软骨组织铁含量增加(P<0.01),细胞呈低Δψm,线粒体损伤率增高(P<0.01),ROS平均荧光强度升高(P<0.01),软骨组织HSPA5、GPX4、COL2A1 mRNA及相应蛋白表达降低(P<0.01),软骨组织MMP3、MMP13 mRNA及蛋白表达升高(P<0.01),软骨组织HSPA5、GPX4平均荧光强度降低(P<0.01);与模型组比较,针刀组软骨组织铁含量减少(P<0.01),软骨细胞Δψm升高,线粒体损伤率降低(P<0.01),ROS平均荧光强度降低(P<0.01),软骨组织HSPA5、GPX4、COL2A1 mRNA及相应蛋白表达升高(P<0.01),软骨组织MMP3、MMP13 mRNA和蛋白表达降低(P<0.01),软骨组织HSPA5、GPX4平均荧光强度升高(P<0.01)。结论:针刀干预能够缓解KOA模型兔膝关节软骨损伤,其机制可能与调节HSPA5/GPX4信号通路维持关节软骨铁稳态,从而抑制软骨细胞铁死亡,缓解细胞外基质(ECM)降解有关。

IRF family proteins and type I interferon induction in dendritic cells

Dendritic cells （DC）, although a minor population in hematopoietic cells, produce type I interferons （IFN） and other cytokines and are essential for innate immunity. They are also potent antigen presenters and regulate adaptive immunity. Among DC subtypes plasmacytoid DC （pDC） produce the highest amounts of type I IFN. In addition, pro- and anti-inflammatory cytokines such as IL-12 and IL-10 are induced in DC in response to Toll like receptor （TLR） signaling and upon viral infection. Proteins in the IRF family control many aspects of DC activity. IRF-8 and IRF-4 are essential for DC development. They differentially control the development of four DC subsets. IRF-8^-/- mice are largely devoid of pDC and CD8α^＋ DC, while IRF-4^-/- mice lack CD4^＋ DC. IRF-8^-/-, IRF4^-/-, double knock-out mice have only few CD8α CD4^-DC that lack MHC Ⅱ. IRF proteins also control type Ⅰ IFN induction in DC. IRF-7, activated upon TLR signaling is required for IFN induction not only in pDC, but also in conventional DC （cDC） and non-DC cell types. IRF-3, although contributes to IFN induction in fibroblasts, is dispensable in IFN induction in DC. Our recent evidence reveals that type Ⅰ IFN induction in DC is critically dependent on IRF-8, which acts in the feedback phase of IFN gene induction in DC. Type Ⅰ IFN induction in pDC is mediated by MyD88 dependent signaling pathway, and differs from pathways employed in other cells, which mostly rely on TLR3 and RIG-Ⅰ family proteins. Other pro-inflammatory cytokines are produced in an IRF-5 dependent manner. However, IRF-5 is not required for IFN induction, suggesting the presence of separate mechanisms for induction of type Ⅰ IFN and other pro-inflammatory cytokines. IFN and other cytokines produced by activated DC in turn advance DC maturation and change the phenotype and function of DC. These processes are also likely to be governed by IRF family proteins.

Protective effects of VMY-2-95 on corticosterone-induced injuries in mice and cellular models

Nicotinicα4β2 receptor antagonists have drawn increasing attention in the development of new antidepressants.In this study,we aimed to investigate the protective effect of VMY-2-95,the new selective antagonist ofα4β2 nicotinic acetylcholine receptor(nAChR)on corticosterone(CORT)injured mice and cellular models.Fluoxetine was applied as a positive control,and the effects of VMY-2-95 were investigated with three different doses or concentrations(1,3,10 mg/kg in mice,and 0.003,0.03,0.1μmol/L in cells).As a result,VMY-2-95 showed significant antidepressant-like effects in the CORT injured mice by improving neuromorphic function,promoting hippocampal nerve proliferation,and regulating the contents of monoamine transmitters.Meanwhile,VMY-2-95 exhibited protective effects on cell viability,cell oxidant,cell apoptosis,and mitochondrial energy metabolism on corticosterone-impaired SH-SY5 Y cells.Also,the PKA-CREB-BDNF signaling pathway was up-regulated by VMY-2-95 both in vitro and in vivo,and pathway blockers were also combined with VMY-2-95 to verify the effects furtherly.Therefore,we preliminarily proved that VMY-2-95 had protective effects in depressed mice and SH-SY5 Y cells against injuries induced by corticosterone.This work indicated that the application of VMY-2-95 is a potential pharmacological solution for depression.This study also supported the development ofα4β2 nAChR antagonists towards neuropsychiatric dysfunctions.