Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells

Ramulus Cinnamomi （RC）, a traditional Chinese herb, has been used to attenuate inflammatory responses. The purpose of this study was to investigate the effect of RC extract on lipopolysaccharide （LPS）-induced neuroinflammation in BV2 microglial cells and the underlying mechanisms involved. BV2 cells were incubated with normal medium （control group）, LPS, LPS plus 30 pg/mL RC extract, or LPS plus 100 pg/mL RC extract. The BV2 cell morphology was observed under an optical microscope and cell viability was detected by MTT assay. Nitric oxide level in BV2 cells was detected using Griess regents, and the levels of interleukin-6, interleukin-1 β, and tumor necrosis factor u in BV2 cells were determined by ELISA. The expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 proteins were detected by western blot assay. Compared with the LPS group, both 30 and 100 μg/mL RC extract had no significant effect on the viability of BV2 cells. The levels of nitric oxide, interleukin-6, interleukin-1β and tumor necrosis factor ct in BV2 cells were all significantly increased after LPS induction, and the levels were significantly reversed after treatment with 30 and 100 μg/mL RC extract. Furthermore, RC extract significantly inhibited the protein expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 in LPS-induced BV2 cells. Our findings suggest that RC extract alleviates neuroinflammation by downregulating the TLR4/MyD88 signaling pathway.

Comparative transcriptomic analysis reveals the molecular changes of acute pancreatitis in experimental models

BACKGROUND Acute pancreatitis(AP)encompasses a spectrum of pancreatic inflammatory conditions,ranging from mild inflammation to severe pancreatic necrosis and multisystem organ failure.Given the challenges associated with obtaining human pancreatic samples,research on AP predominantly relies on animal models.In this study,we aimed to elucidate the fundamental molecular mechanisms underlying AP using various AP models.AIM To investigate the shared molecular changes underlying the development of AP across varying severity levels.METHODS AP was induced in animal models through treatment with caerulein alone or in combination with lipopolysaccharide(LPS).Additionally,using Ptf1αto drive the specific expression of the hM3 promoter in pancreatic acinar cells transgenic C57BL/6J-hM3/Ptf1α(cre)mice were administered Clozapine N-oxide to induce AP.Subsequently,we conducted RNA sequencing of pancreatic tissues and validated the expression of significantly different genes using the Gene Expression Omnibus(GEO)database.RESULTS Caerulein-induced AP showed severe inflammation and edema,which were exacerbated when combined with LPS and accompanied by partial pancreatic tissue necrosis.Compared with the control group,RNA sequencing analysis revealed 880 significantly differentially expressed genes in the caerulein model and 885 in the caerulein combined with the LPS model.Kyoto Encyclopedia of Genes and Genomes enrichment analysis and Gene Set Enrichment Analysis indicated substantial enrichment of the TLR and NOD-like receptor signaling pathway,TLR signaling pathway,and NF-κB signaling pathway,alongside elevated levels of apoptosis-related pathways,such as apoptosis,P53 pathway,and phagosome pathway.The significantly elevated genes in the TLR and NOD-like receptor signaling pathways,as well as in the apoptosis pathway,were validated through quantitative real-time PCR experiments in animal models.Validation from the GEO database revealed that only MYD88 concurred in both mouse pancreatic tissue and human AP peripheral blood,while TLR1,TLR7,RIPK3,and OAS2 genes exhibited marked elevation in human AP.The genes TUBA1A and GADD45A played significant roles in apoptosis within human AP.The transgenic mouse model hM3/Ptf1α(cre)successfully validated significant differential genes in the TLR and NOD-like receptor signaling pathways as well as the apoptosis pathway,indicating that these pathways represent shared pathological processes in AP across different models.CONCLUSION The TLR and NOD receptor signaling pathways play crucial roles in the inflammatory progression of AP,notably the MYD88 gene.Apoptosis holds a central position in the necrotic processes of AP,with TUBA1A and GADD45A genes exhibiting prominence in human AP.

Research Progress and Research Ideas on Anti-hepatic Fibrosis and Promoting Blood Circulation and Removing Blood Stasis of the National Drug Plumbapin Based on TLR4 Signal Pathway

Hepatic fibrosis is a reversible pathological phenomenon in the early and middle stages,but but no satisfactory intervention drugs have been available so far.Recent studies have suggested that microcirculation disturbance of liver is one of the important pathogenesis of chronic liver disease,the improvement of microcirculation is beneficial to the recovery of liver function and the delay of liver fibrosis.Hepatic stellate cells are the core cells of hepatic fibrosis,and also the most critical cells that affect the microcirculation of the liver.While TLR4/MyD88/NF-κB and TLR4/MyD88/MAPKs which are based on the action of hepatic stellate cells are two pathways that have very important influence on the inflammatory response of liver,the proliferation and apoptosis of hepatic stellate cells,and the secretion of fibrogenic cytokines.It was found that Plumbapin,the active ingredient of Guangxi specialty ethnic medicine,has the definite effect of promoting blood circulation and removing blood stasis and anti-hepatic fibrosis,but its mechanism is not clear.In this study,the research progress of the above problems was reviewed,and further research ideas were derived as follows:the pharmacological effect of Plumbapin on anti-hepatic fibrosis,promoting blood circulation and removing stasis was based on the influence of TLR4/MyD88/NF-κB and MAPKs signal pathway.

Huoxin Pill Reduces Myocardial Ischemia Reperfusion Injury in Rats via TLR4/NFκB/NLRP3 Signaling Pathway

Objective:To explore the protective effect of Huoxin Pill(HXP)on acute myocardial ischemia-reperfusion(MIRI)injury in rats.Methods:Seventy-five adult SD rats were divided into the sham-operated group,model group,positive drug group(diltiazem hydrochloride,DH),high dose group(24 mg/kg,HXP-H)and low dose group(12 mg/kg,HXP-L)of Huoxin Pill(n=15 for every group)according to the complete randomization method.After 1 week of intragastric administration,the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h.Serum was separated and the levels of creatine kinase(CK),creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH),superoxide dismutase(SOD),and malondialdehyde(MDA),hypersensitive C-reactive protein(hs-CRP)and interleukin-1β(IL-1β)were measured.Myocardial ischemia rate,myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride(TTC).Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine(BATMAN)databases were used to screen for possible active compounds of HXP and their potential therapeutic targets;the results of anti-inflammatory genes associated with MIRI were obtained from GeneC ards,Drugbank,Online Mendelian Inheritance in Man(OMIM),and Therapeutic Target Datebase(TTD)databases was performed;Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment were used to analyze the intersected targets;molecular docking was performed using AutoD ock Tools.Western blot was used to detect the protein expression of Toll-like receptor 4(TLR4)/nuclear factor kappa-B(NFκB)/NOD-like receptor protein 3(NLRP3).Results:Compared with the model group,all doses of HXP significantly reduced the levels of LDH,CK and CK-MB(P<0.05,P<0.01);HXP significantly increased serum activity of SOD(P<0.05,P<0.01);all doses of HXP significantly reduced the levels of hs-CRP and IL-1β(P<0.05,P<0.01)and the myocardial infarction rate and myocardial no-reflow rate(P<0.01).GO enrichment analysis mainly involved positive regulation of gene expression,extracellular space and identical protein binding,KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis.Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4,NFκB and NLRP3 molecules.The protein expressions of TLR4,NFκB and NLRP3 were reduced in the HXP group(P<0.01).Conclusions:HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats,and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4/NFκB/NLRP3 signaling pathway.

Albiflorin attenuates inflammatory injury by regulating the TLR4 signaling pathway and its negative regulating factor Tollip in experimental models of ulcerative colitis

Albiflorin （AF） is the main active component extracted from Paeoniae Radix Alba. This study investigated the efficacy of AF in attenuating inflammatory injury by regulating the TLR4 signaling pathway and its negative regulating factor Tollip in an experimental ulcerative colitis （UC） model. We administrated trinitrobenzene sulfonic acid for 21 d to induce UC in rats. The efficacy of AF in attenuating UC was assessed using various biochemical markers, such as tumor necrosis factor-α （TNF-α）, interleukin- 1 （IL- 1）, interleukin- 10 （IL- 10）, 5-hydroxytryptamine （5-HT）, and tissue myeloperoxidase （MPO）, along with histopathological studies on toll-like receptor-4 （TLR-4） signaling pathway and its negative regulating factor Tollip. The results showed that AF can significantly downregulate the levels of TNF-α, IL-1, IL-10, and 5-HT. AF decreased the activation of TLR4, MyD88, and NF-κB p65 protein expression by increasing Tollip expression. AF can relieve symptoms of UC by suppressing the activation of the TLR4 signaling pathway and upregulating its negative regulating factor Tollip. Therefore, AF may be a potential natural product for treating UC.

The IL-1R/TLR signaling pathway is essential for efficient CD8+ T-cell responses against hepatitis B virus in the hydrodynamic injection mouse model

The outcome of hepatitis B viral(HBV)infection is determined by the complex interactions between replicating HBV and the immune system.While the role of the adaptive immune system in the resolution of HBV infection has been studied extensively,the contribution of innate immune mechanisms remains to be defined.Here we examined the role of the interleukin-1 receptor/Toll-like receptor(IL-1R/TLR)signaling pathway in adaptive immune responses and viral clearance by exploring the HBV mouse model.Hydrodynamic injection with a replication-competent HBV genome was performed in wild-type mice(WT)and a panel of mouse strains lacking specific innate immunity component expression.We found higher levels of HBV protein production and replication in Tlr2^(−/−),Tlr23479^(−/−),3d/Tlr24^(−/−),Myd88/Trif^(−/−)and Irak4^(−/−)mice,which was associated with reduced HBV-specific CD8+T-cell responses in these mice.Importantly,HBV clearance was delayed for more than 2 weeks in 3d/Tlr24^(−/−),Myd88/Trif^(−/−)and Irak4^(−/−)mice compared to WT mice.HBV-specific CD8+T-cell responses were functionally impaired for producing the cytokines IFN-γ,TNF-αand IL-2 in TLR signaling-deficient mice compared to WT mice.In conclusion,the IL-1R/TLR signaling pathway might contribute to controlling HBV infection by augmenting HBV-specific CD8+T-cell responses.

Inhibition of miR-873 provides therapeutic benefit in lipopolysaccharide-induced Parkinson disease animal model

OBJECTIVE Neuroinflammation plays a critical role in neurodegenerative disorders,although the inflammation may not the initiating factor.Parkinson disease(PD)is characterized pathologically by the accumulation of alpha synuclein(α-syn)and the loss of the dopamine(DA)neurons in the substantia nigra(SN),which has been reported to be induced by the stereotaxic injection of lipopolysaccharide(LPS)to the SN region in rodents.This study is to investigate the therapeutic benefit of the inhibition of miR-873 in PD.METHODS Rats received the right-unilaterally injection with concentrated LV-sponge or LV-EGFP 3 d before LPS treatment,7 or 14 d after LPS treatment.The animals were tested for rotational behavior with the dopaminergic agonist apomorphine dissolved in sterile saline at 21 d after LPS injection.The regulation of miR-873 on the genes related with cholesterol transport and inflammation was assayed in SH-SY5Y cells and U251 cells.RESULTS TLR4-My D88 signaling pathway was involved the regulation of miR-873 by LPS.The luciferase assay showed that HMGCR,ABCA1 and A20 were down-stream genes of miR-873.The transfection of miR-873 decreased the cholesterol levels in cell membrane,but increased in lysosome in SH-SY5Y cells.Compared with the control SH-SY5Y cells,cholesterol levels were higher in lysosome withα-synuclein overexpression or LPS treatment.The transfection of miR-873 increased theα-syn levels in lysosome in cel s withα-synuclein overexpression.The loss of dopaminergic neuorns induced by LPS was significantly respectively decreased by 22.8%,35.6%and 57% after the inhibition of miR-873 at 3 d before LPS treatment,7 or14 d after LPS treatment.Compared with LPS-treated group,the number of the rotation of rats was decreased by 60.4%,33.5%and 13.2%after the inhibition of miR-873 at 3 d before LPS treatment,7or 14 d after LPS treatment.The inhibition of miR-873 significantly decreased accumulation ofα-syn.The m RNA levels of HMGCR,ABCA1 and A20 in SN were decreased by LPS treatment,which was attenuated by the injection of LV-sponge.CONCLUSION The selective regulation of miR-873 can protect the dopaminergic neurons from the LPS-induced damage.The inhibition of miR-873 can attenuate the relocation of cholesterol in lysosome and the accumulation ofα-syn in neurons induced by LPS via the regulation of HMGCR,ABCA1 and A20.

ATF4 is directly recruited by TLR4 signaling and positively regulates TLR4-trigged cytokine production in human monocytes

Toll-like receptors （TLRs） are sentinels of the host defense system, which recognize a large number of microbial pathogens. The host defense system may be inefficient or inflammatory diseases may develop if microbial recognition by TLRs and subsequent TLR-triggered cytokine production are deregulated. Activating transcription factor 4 （ATF4）, a member of the ATF/CREB transcription factor family, is an important factor that participates in several pathophysiological processes. In this report, we found that ATF4 is also involved in the TLR-mediated innate immune response, which participates in TLR4 signal transduction and mediates the secretion of a variety of cytokines. We observed that ATF4 is activated and translocates to the nucleus following l ipopolysaccharide （LPS） stimulation via the TLR4-MyD88-dependent pathway. Additionally, a cytokine array assay showed that some key inflammatory cytokines, such as I L-6, I L-8 and RANTES, are positively regulated by ATF4. We also demonstrate that c-Jun directly binds to ATF4, thereby promoting the secretion of inflammatory cytokines. Taken together, these results indicate that ATF4 acts as a positive regulator in TLR4-triggered cytokine production.

Hepatic protective effects of Shenling Baizhu powder, a herbal compound, against inflammatory damage via TLR4/NLRP3 signalling pathway in rats with nonalcoholic fatty liver disease

Objective: High-fat diet(HFD) and inflammation are two key contributors to nonalcoholic fatty liver disease(NAFLD). Shenling Baizhu powder(SLBZP), a classical herbal compound, has been successfully used to alleviate NAFLD. However, its specific mechanisms are not fully understood. In this study, we assessed the anti-NAFLD effect of SLBZP in vivo.Methods: Rats were fed an HFD with or without SLBZP or with probiotics. At the end of week 16, an echo magnetic resonance imaging(EchoMRI) body composition analyser was used to quantitatively analyse body composition;a micro-computed tomography(micro-CT) imaging system was used to evaluate whole body and liver fat;and the Moor full-field laser perfusion imager 2 was used to assess liver microcirculation, after which, all rats were sacrificed. Then, biochemical indicators in the blood and the ultrastructure of rat livers were evaluated. Protein expression related to the liver Toll-like receptor 4(TLR4)/Nod-like receptor family pyrin domain-containing 3(NLRP3) signalling pathway was assessed using Western blot analysis. Further, high-throughput screening of 29 related inflammatory factors in liver tissue was performed using a cytokine array.Results: SLBZP supplementation reduced body weight, serum free fatty acid, and insulin resistance index(P<0.05). It also ameliorated liver microcirculation and ultrastructural abnormalities. EchoMRI and micro-CT quantitative analyses showed that treatment with SLBZP reduced fat mass and visceral fat(P<0.05 and P<0.01, respectively). In addition, SLBZP decreased the expression of lipopolysaccharide(LPS)-activated TLR4/NLRP3 signalling pathway-related proteins and altered the expression levels of some inflammatory cytokines in liver tissues.Conclusion: SLBZP can inhibit NLRP3 inflammasome activation and interleukin-1 b release by suppressing LPS-induced TLR4 expression in rats with HFD-induced NAFLD. Thus, SLBZP may be beneficial for the prevention and treatment of inflammatory damage and associated diseases.