Levels Of Pro-inflammatory Factors Research Articles

Median nerve electrical stimulation improves traumatic brain injury by reducing TACR1 to inhibit nuclear factor-κB and CCL7 activation in microglia.

The existing report elucidates that median nerve electrical stimulation (MNS) plays a role in treating traumatic brain injury (TBI). Herein, we explored the mechanism of MNS in TBI. A TBI-induced coma model (skull was hit by a cylindrical impact hammer) was established in adult Sprague-Dawley rats. Microglia were isolated from newborn Sprague-Dawley rats and was injured by lipopolysaccharide (LPS; 10 ng/mL). Consciousness was assessed by sensory and motor functions. Brain tissue morphology was detected using hematoxylin-eosin staining assay. Ionized calcium binding adapter molecule 1, NeuN and tachykinin receptor 1 (TACR1) level were detected by immunohistochemical assay. Levels of pro-inflammatory and anti-inflammatory factors were measured by enzyme linked immune sorbent assay (ELISA). Levels of TACR1, C-C motif chemokine 7 (CCL7), phosphorylation (p)-P65 and P65 were assessed by quantitative real time polymerase chain reaction (qRT-PCR) and western blot. M1 markers (inducible nitric oxide synthase and CD86) and M2 markers (arginase-1 (Arg1) and chitinase 3-like 3 (YM1)) of microglia as well as the transfection efficiency of short hairpin TACR1 (shTACR1) were assessed by qRT-PCR. Immunofluorescence and flow cytometry assay were used to detect microglia morphology and neuron apoptosis. MNS reduced neuron injury and microglia activation in the TBI-induced rat coma model. MNS reversed the effects of TBI on levels of inflammation-related factors, M1/M2 microglia markers, TACR1, p-P65/P65 and CCL7 in rats. shTACR1 reversed the effects of LPS on inflammation-related factors, M1/M2 microglia markers, microglia activation, neuron apoptosis, p-P65/P65 value and CCL7 level. Our results revealed that MNS improved TBI by reducing TACR1 to inhibit nuclear factor-κB (NF-κB) and CCL7 activation in microglia.

Leucine Supplementation Modulates Lipid Metabolism and Inflammation in Early Weaning Piglets.

Early weaning can induce the programmed dysregulation of glycolipid metabolism and inflammation in adult animals. The primary objective of this study was to evaluate the efficacy of leucine supplementation administered promptly after early weaning in mitigating these adverse effects in piglets. At day 21, 24 piglets were randomly selected and divided into 3 groups: EW group where the piglets were weaned at day 21 and fed basal diet, EWL group where the piglets were weaned at day 21 and fed the basal diet with supplementation of 1% leucine, and C group where the piglets were fed basal diet and weaned at 28 days. Each group contained eight replicates, with one piglet per replicate. The results indicated that early weaning had an impact on gut health and could activate the inhibitor of the kappa B kinase gamma/inhibitor kappa B alpha/NF-kappa-B (IKKγ/IκBα/NF-κB) signaling pathway to ameliorate pro-inflammatory factor and apoptosis levels. Furthermore, early weaning reduced the activity of fatty acid β oxidation (FAβO) and affected genes linked with lipid metabolism. Supplementing with leucine can improve the effects of these factors. In summary, leucine may alleviate the influences of early weaning on the lipid metabolism and inflammation in piglets.

Skeletal muscle metabolic dysfunction with circulating carboxymethyl-lysine in dietary food additive-induced leaky gut.

Impaired intestinal permeability induces systemic inflammation and metabolic disturbance. The effect of a leaky gut on metabolism in skeletal muscle, a major nutrient consumer, remains unclear. In this study, we aimed to investigate the glucose metabolic function of the whole body and skeletal muscles in a mouse model of diet-induced intestinal barrier dysfunction. At Week 2, we observed higher intestinal permeability in mice fed a titanium dioxide (TiO2)-containing diet than that of mice fed a normal control diet. Subsequently, systemic glucose and insulin tolerance were found to be impaired. In the skeletal muscle, glucose uptake and phosphorylation levels in insulin signaling were lower in the TiO2 group than those in the control group. Additionally, the levels of pro-inflammatory factors were higher in TiO2-fed mice than those in the control group. We observed higher carboxymethyl-lysin (CML) levels in the plasma and intestines of TiO2-fed mice and lower insulin-dependent glucose uptake in CML-treated cultured myotubes than those in the controls. Finally, soluble dietary fiber supplementation improved glucose and insulin intolerance, suppressed plasma CML, and improved intestinal barrier function. These results suggest that an impaired intestinal barrier leads to systemic glucose intolerance, which is associated with glucose metabolism dysfunction in the skeletal muscles due to circulating CML derived from the intestine. This study highlights that the intestinal condition regulates muscle and systemic metabolic health.

Self-Assembled Multilayer-Modified Needles Simulate Acupuncture and Diclofenac Sodium Delivery for Rheumatoid Arthritis.

A novel therapeutic approach combining acupuncture and diclofenac sodium (DS) administration was established for the potential treatment for rheumatoid arthritis (RA). DS is a commonly used anti-inflammatory and analgesic drug but has short duration and adverse effects. Acupoints are critical linkages in the meridian system and are potential candidates for drug delivery. Herein, we fabricated a DS-loaded multilayer-modified acupuncture needle (DS-MMAN) and investigated its capacity for inhibiting RA. This DS-MMAN possesses sustained release properties and in vitro anti-inflammatory effects. Experimental results showed that the DS-MMAN with microdoses can enhance analgesia and efficiently relieve joint swelling compared to the oral or intra-articular administration of DS with gram-level doses. Moreover, the combination of acupoint and DS exerts a synergistic improvement in inflammation and joint damage. Cytokine and T cell analyses in the serum indicated that the application of DS-MMAN suppressed the levels of pro-inflammatory factors and increased the levels of anti-inflammatory factors. Furthermore, the acupoint administration via DS-MMAN could decrease the accumulation of DS in the liver and kidneys, which may express better therapeutic efficiency and low toxicity. The present study demonstrated that the acupuncture needle has the potential to build a bridge between acupuncture and medication, which would be a promising alternative to the combination of traditional and modern medicine.

Serum long non-coding Ribonucleic Acid H19 serves as a biomarker for systemic lupus erythematosus and participates in the disease progression.

This study aimed to investigate the expression of H19 and its possible molecular mechanism in systemic lupus erythematosus (SLE). The expression of H19 and miR-19b in serum and peripheral blood mononuclear cells (PBMCs) were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Receiver operator characteristic (ROC) curve was constructed to evaluate the diagnostic value of serum H19 in SLE. Pearson correlation coefficient was used to analyze the correlation between serum levels of H19 and miR-19b. Flow cytometry and Cell counting kit-8 (CCK-8) assay were performed to detect cell apoptosis and viability. The levels of pro-inflammatory and anti-inflammatory factors were measured by enzyme-linked immunosorbent assay (ELISA). Luciferase reporter gene assay was conducted to verify the interaction between H19 and miR-19b. The expression of H19 and miR-19b in SLE group were up-regulated and down-regulated, respectively. Serum H19 has certain clinical diagnostic value in SLE. In in vitro studies, overexpression of H19 can significantly inhibit the viability of PBMCs and promote apoptosis and inflammatory response of PBMCs by interacting with miR-19b. The expression of H19 is upregulated in patients with SLE and plays a role in cell function and inflammation by targeting miR-19b in PBMCs, which may be one of the pathological mechanisms of SLE.

Erjingpill bionic cerebrospinal fluid alleviates LPS-induced inflammatory response in BV2 cells by inhibiting glycolysis via mTOR

Ethnopharmacological relevanceErjingpill, a well-known prescription documented in the classic Chinese medical text “Shengji Zonglu,” has been proven to have effective alleviating effects on neuroinflammation in Alzheimer's disease (AD). Although the alterations in microglial cell glycolysis are known to play a crucial role in the development of neuroinflammation, it remains unclear whether the anti-neuroinflammatory effects of Erjingpill are associated with its impact on microglial cell glycolysis. Aim of the studyThis study aims to determine whether Erjingpill exerts anti-neuroinflammatory effects by influencing microglial cell glycolysis. Materials and methodsFirstly, Erjingpill decoction was prepared into an Erjingpill bionic cerebrospinal fluid (EBCF) through a process of in vitro intestinal absorption, hepatocyte incubation, and blood-brain barrier (BBB) transcytosis. Subsequently, UPLC/Q-TOF-MS/MS technology was used to analyze the compounds in Erjingpill and EBCF. Next, an in vitro neuroinflammation model was established by LPS-induced BV2 cells. The impact of EBCF on BV2 cell proliferation activity was evaluated using the CCK-8 assay, while the NO release was assessed using the Griess assay. Additionally, mRNA levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α, and COX-2), anti-inflammatory factors (IL-10, IL-4, Arg-1, and TGF-β), M1 microglial markers (iNOS, CD86), M2 microglial markers (CD36, CD206), and glycolytic enzymes (HK2, GLUT1, PKM, and LDHA) were measured using qPCR. Furthermore, protein expression of microglial activation marker Iba-1, M1 marker iNOS, and M2 marker CD206 were identified through immunofluorescence, while concentrations of pro-inflammatory cytokines IL-1β and TNF-α were measured using ELISA. Enzymatic activity of glycolytic enzymes (HK, PK, and LDH) was assessed using assay kits, and the protein levels of pro-inflammatory factors (IL-1β, iNOS, and COX-2), anti-inflammatory factors (IL-10 and Arg-1), and key glycolytic proteins GLUT1 and PI3K/AKT/mTOR were detected by Western blot. ResultsThrough the analysis of Erjingpill and EBCF, 144 compounds were identified in Erjingpill and 40 compounds were identified in EBCF. The results demonstrated that EBCF effectively inhibited the elevation of inflammatory factors and glycolysis levels in LPS-induced BV2 cells, promoted polarization of M1 microglial cells towards the M2 phenotype, and suppressed the PI3K/AKT/mTOR inflammatory pathway. Moreover, EBCF alleviated LPS-induced BV2 cell inflammatory response by modulating mTOR to inhibit glycolysis. ConclusionsEBCF exhibits significant anti-neuroinflammatory effects, likely attributed to its modulation of mTOR to inhibit microglial cell glycolysis. This study furnishes experimental evidence supporting the clinical utilization of Erjingpill for preventing and treating AD.

Multi-omics and multi-stages integration identified a novel variant associated with silicosis risk.

Assessing the association between candidate single-nucleotide polymorphisms (SNPs) identified by multi-omics approaches and susceptibility to silicosis. RNA-seq analysis was performed to screen the differentially expressed mRNAs in the fibrotic lung tissues of mice exposed to silica particles. Following this, we integrated the SNPs located in the above human homologenes with the silicosis-related genome-wide association study (GWAS) data to select the candidate SNPs. Then, expression quantitative trait locus (eQTL)-SNPs were identified by the GTEx database. Next, we validated the associations between the functional eQTL-SNPs and silicosis susceptibility by additional case-control study. And the contribution of the identified SNP and its host gene in the fibrosis process was further validated by functional experiments. A total of 12 eQTL-SNPs were identified in the screening stage. The results of the validation stage suggested that the variant T allele of rs419540 located in IL12RB1 significantly increased the risk of developing silicosis [additive model: odds ratio (OR) = 1.78, 95% confidence interval (CI) 1.11-2.85, P = 0.017]. Furthermore, the combination of GWAS and the results of validation stage also indicated that the variant T allele of rs419540 in IL12RB1 was associated with increased silicosis risk (additive model: OR = 2.07, 95% CI 1.38-3.12, P < 0.001). Additionally, after knockdown or overexpression of IL12RB1, the levels of pro-inflammatory factors, such as IL-12, IFN-γ, and other pro-inflammatory factors, were correspondingly decreased or increased. The novel eQTL-SNP, rs419540, might increase the risk of silicosis by modulating the expression levels of IL12RB1.

Therapeutic effects of orexin-A in sepsis-associated encephalopathy in mice

BackgroundSepsis-associated encephalopathy (SAE) causes acute and long-term cognitive deficits. However, information on the prevention and treatment of cognitive dysfunction after sepsis is limited. The neuropeptide orexin-A (OXA) has been shown to play a protective role against neurological diseases by modulating the inflammatory response through the activation of OXR1 and OXR2 receptors. However, the role of OXA in mediating the neuroprotective effects of SAE has not yet been reported.MethodsA mouse model of SAE was induced using cecal ligation perforation (CLP) and treated via intranasal administration of exogenous OXA after surgery. Mouse survival, in addition to cognitive and anxiety behaviors, were assessed. Changes in neurons, cerebral edema, blood-brain barrier (BBB) permeability, and brain ultrastructure were monitored. Levels of pro-inflammatory factors (IL-1β, TNF-α) and microglial activation were also measured. The underlying molecular mechanisms were investigated by proteomics analysis and western blotting.ResultsIntranasal OXA treatment reduced mortality, ameliorated cognitive and emotional deficits, and attenuated cerebral edema, BBB disruption, and ultrastructural brain damage in mice. In addition, OXA significantly reduced the expression of the pro-inflammatory factors IL-1β and TNF-α, and inhibited microglial activation. In addition, OXA downregulated the expression of the Rras and RAS proteins, and reduced the phosphorylation of P-38 and JNK, thus inhibiting activation of the MAPK pathway. JNJ-10,397,049 (an OXR2 blocker) reversed the effect of OXA, whereas SB-334,867 (an OXR1 blocker) did not.ConclusionThis study demonstrated that the intranasal administration of moderate amounts of OXA protects the BBB and inhibits the activation of the OXR2/RAS/MAPK pathway to attenuate the outcome of SAE, suggesting that OXA may be a promising therapeutic approach for the management of SAE.

Complanatuside A ameliorates 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice by regulating the Th17/Treg balance via the JAK2/STAT3 signaling pathway.

Immunity imbalance of T helper 17 (Th17)/regulatory T (Treg) cells is involved in the pathogenesis of Crohn's disease (CD). Complanatuside A (CA), a flavonol glycoside, exerts anti-inflammatory activities and our study aimed to identify its effect on TNBS-induced colitis and the possible mechanisms. We found that CA alleviated the symptoms of colitis in TNBS mice, as demonstrated by prevented weight loss and colon length shortening, as well as decreased disease activity index scores, inflammatory scores, and levels of proinflammatory factors. Flow cytometry analysis showed that CA markedly reduced the percentage of Th17 cells while increasing the percentage of Treg cells in TNBS mice. Under Th17 cell polarizing conditions, CA inhibited the differentiation of Th17 cells while the Treg cell differentiation was elevated under Treg cell polarizing conditions. Furthermore, it was observed that JAK2 interacted with CA through six hydrogen bonds via molecular docking. The phosphorylation of JAK2/STAT3 was reduced by CA, which might be correlated with the protective effect of CA on colitis. In conclusion, CA reduced the imbalance of Th17/Treg cells by inhibiting the JAK2/STAT3 signaling pathway in TNBS-induced colitis, which may provide novel strategies for CD treatment.

Decoction derived from Allium ascalonicum L. bulbs and Sojae Semen Praeparatum alleviates wind-cold-type common cold via Nrf2/HO-1 pathway and modulation of Lactobacillus murinus level.

Cong-Chi decoction (CCD) is made using Allium ascalonicum L. (shallot) bulbs and Sojae Semen Praeparatum (SSP). Shallot bulbs and SSP are both used regularly in traditional Chinese medicine; however, there are no recent pharmacological studies on their synergistic effects. Despite their roles in the treatment of the common cold for thousands of years, their pharmacological mechanisms of action against wind-cold-type common cold are yet to be explored comprehensively. A mouse model was standardized using wind-cold modeling equipment to study the anti-inflammatory, antioxidant, and antiapoptotic effects of CCD. Then, 16S rRNA sequencing was employed to analyze the association between Lactobacillus murinus and changes in body temperature. Additionally, the antipyretic effects of L. murinus were validated via animal experiments. The results indicate that CCD improves the symptoms of wind-cold by reducing fever, levels of pro-inflammatory factors, and cellular apoptosis, as well as increasing the blood leukocyte and lymphocyte counts, thereby alleviating lung tissue damage. The effects of CCD are mediated by upregulation of pulmonary Nrf2 and HO-1 expressions, thereby reducing oxidative damage in the lungs, in addition to other anti-inflammatory mechanisms. Furthermore, CCD increases the abundance of L. murinus in the intestinal tract. The animal experiments confirm that L. murinus ameliorates fever in mice. CCD exhibits remarkable antioxidant and anti-inflammatory properties for effectively treating wind-cold-type common cold. Furthermore, its regulatory effects on L. murinus represent a novel mechanism for product development.

MTOR promotes an inflammatory response through the HIF1 signaling pathway in ulcerative colitis

The imbalance between T helper cell 17 （Th17）and regulatory T cells (Treg) cells leading to inflammation has an important role in the pathogenesis of ulcerative colitis (UC). Mammalian target of rapamycin (mTOR) can regulate the differentiation of T cells, but the specific pathway leading mTOR to regulate Th17/Treg cells in UC remains unclear. Our aim with this study was to investigate the effects of mTOR overexpression and silencing on the hypoxia inducible factor-1α (HIF-1α) − Th17/Treg signaling pathway. To mimic a human study, we established a colon cancer epithelial cell line (HT-29) co-culture system with human CD4+ T cells, and we treated the cells with TNF-α. We observed the effects of mTOR on the HIF-Th17/Treg signaling pathway to determine whether mTOR is involved in the regulatory mechanism.Under the stimulation of TNF-α, the levels of HIF-1α in CD4+T cells were increased in the HT-29 co-culture with CD4+ T cells, promoting glycolysis, increasing the Th17 proportion, decreasing the Treg proportion, increasing the pro-inflammatory factors levels, and decreasing the anti-inflammatory factors levels. Moreover, after mTOR silencing, the HIF-1α level and cell glycolysis levels decreased, Th17 cell differentiation decreased, the pro-inflammatory factor levels decreased, and the anti-inflammatory factor levels increased. In contrast, mTOR overexpression lead to the opposite results.mTOR promotes inflammation by regulating the HIF signaling pathway during UC, and silencing mTOR may alleviate inflammation. An mTOR inhibitor is a potential therapeutic target for UC treatment.

Dual targeting total saponins of Pulsatilla of natural polymer crosslinked gel beads with multiple therapeutic effects for ulcerative colitis

Oral colon targeted drug delivery system (OCTDDS) is desirable for the treatment of ulcerative colitis (UC). In this study, we designed a partially oxidized sodium alginate-chitosan crosslinked microsphere for UC treatment. Dissipative particle dynamics (DPD) was used to study the formation and enzyme response of gel beads from a molecular perspective. The formed gel beads have a narrow particle size distribution, a compact structure, low cytotoxicity and great colon targeting in vitro and in vivo. Animal experiments demonstrated that gel beads promoted colonic epithelial barrier integrity, decreased the level of pro-inflammatory factors, accelerated the recovery of intestinal microbial homeostasis in UC rats and restored the intestinal metabolic disorders. In conclusion, our gel bead is a promising approach for the treatment of UC and significant for the researches on the pathogenesis and treatment mechanism of UC.

Scutellarein alleviates osteoarthritis progression through the PI3K/Akt/NF-kappaB signaling pathway: In vitro and in vivo studies.

Osteoarthritis (OA), a joint disease that is associated with inflammatory processes is involved in joint destruction. Scutellarein (Scu), a component of the medicinal herbs Scutellaria barbata D. Don and Erigeron breviscapus (vant) Hand Mass, has anti-inflammatory effects. We explored the role of Scu in the development of OA and the underlying mechanisms. CCK-8 assays, Calcein-AM/PI and EdU staining were used to determine chondrocyte viability after Scu exposure. Western blot, qPCR, as well as ELISA were utilized to measure extracellular matrix (ECM) degradation and inflammation. Immunofluorescence (IF), western blot and luciferase assays were used to examine the NF-kappaB (NF-κB) pathway. Scu interacting proteins were predicted using network pharmacology analysis and molecular docking. X-ray, H&E, Safranin O-Fast Green(S-O), toluidine blue, and immunohistochemistry analysis were used to examine the therapeutic effects of Scu in OA using destabilization of medial meniscus (DMM) models. Scu demonstrated inhibitory effects on ECM degradation and pro-inflammatory factor levels in chondrocytes treated with IL-1β. Mechanistically, Scu inhibited the IL-1β-induced activation of the PI3K/Akt/ NF-κB signaling pathway cascades. Furthermore, Scu has been shown to have significant binding capacities to PI3K. Additionally, Scu ameliorated the OA progression in DMM models. Our findings suggest that Scu may contribute to the amelioration of OA progression by targeting the PI3K/Akt/NF-κB signaling pathway, implying Scu possesses promising therapeutic potential for the treatment of OA.

Mechanism of Si Ni San Combined with Astragalus in Treating Hepatic Fibrosis: A Network Pharmacology and Molecular Docking Study.

Si Ni San combined with Astragalus (SNSQ) has demonstrated significant efficacy in the treatment of hepatic fibrosis (HF), as confirmed by clinical practice. However, its pharmacological mechanism remains unclear. This study employs network pharmacology to identify key targets and proteins for molecular docking. Additionally, animal experiments were conducted to validate the network pharmacology results, providing further insights into the mechanism of SNSQ in treating HF. Effective compounds of SNSQ were screened from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Encyclopedia of Traditional Chinese Medicine (ETCM) databases. Molecular formula structures of these effective compounds were obtained from the PubChem database. Partial target proteins with a probability greater than 0.6 were sourced from the SWISS database. Uniprot IDs corresponding to these target proteins were retrieved from the SUPERPRED database. The remaining target proteins of the compounds were obtained from the Uniprot database based on the Uniprot IDs. The drug target proteins were then summarized. Target points related to HF were selected from the GeneCards and OMIM databases. Common target points were identified in the Venn diagram and imported into Cytoscape 3.9.1 software to construct the "SNSQ-effective compound-target pathway-HF" network. AutoDock software was used for molecular docking of compounds and target proteins with high-degree values. The common target points underwent GO function enrichment and KEGG pathway enrichment analysis using the DAVID database. An HF rat model was established, and serum AST and ALT activities were measured. The Hyp assay kit was utilized to detect the Hyp content in liver tissue. To the transcription levels of pro-inflammatory factors (IL-1β, TNF-α, IL-6) and anti-inflammatory factors (IL-10, TGF-β1, IL-4) in rat serum and liver.IL-1β, TNF-α, IL-10, and TGF-β1 were chosen for validation through ELISA. Western blotting and qRT-PCR were used to assess the expression of related proteins, namely NFKB1, NF-κBp65, NF-κBp50, α-SMA, and Col-1 in liver tissue. qRT-PCR was also employed to study the expression of ECM synthesis and proliferation-related genes, including Cyclin D1, TIMP1, COL1A1 in HSC-T6 cells and rat liver tissue, as well as the inhibition of the ECM-related gene MMP13 in HSC-T6 cells and rat liver tissue. A total of 16 valid compounds were predicted, with kaempferol, sitosterol, and isorhamnetin exhibiting high-degree values. KEGG enrichment analysis revealed that the target genes of SNSQ were enriched in multiple pathological pathways, with the NF-Kappa B signaling pathway being predominant. Molecular docking simulations indicated strong affinities between SNSQ's primary components-kaempferol, sitosterol, isorhamnetin-and NFKB1. Experimental results demonstrated significant reductions in AST, ALT, and Hyp levels in the SNSQ group. Pro-inflammatory factors (IL-1β, TNF-ɑ) were markedly reduced, while anti-inflammatory factors (IL-10, TGF-β1) were substantially increased. The protein expression and transcription levels of α-SMA and Col-1 were significantly decreased, whereas those of NFKB1, NF-κBp65, and NF-κBp50 were notably elevated. mRNA expression levels of Cyclin D1, TIMP1, COL1A1 in HSC-T6 cells and rat liver tissue were significantly decreased, whereas MMP13 mRNA expression level was significantly increased. Treatment of HF with SNSQ involves multiple targets and pathways, with a close association with the overexpression of NFKB1 and activation of the NF-Kappa B signaling pathway. Its mechanism is closely linked to the activation of inflammatory responses, HSC activation, and proliferation.

FBXO28 reduces high-fat diet-induced hyperlipidemia in mice by alleviating abnormal lipid metabolism and inflammatory responses.

Hyperlipidemia is a lipid metabolism disorder with increasing incidence and prevalence worldwide. Abnormal lipid metabolism and inflammation are two significant characteristics of hyperlipidemia. The purpose of this study was to explore the role and mechanism of F-box only protein 28 (FBXO28) in hyperlipidemia. Mice were fed with high-fat diet (HFD) to elicit obesity, and 3T3-L1 preadipocytes were stimulated with MDI cocktail (IBMX, DEX and insulin) to evoke differentiation. In vivo and in vitro role of FBXO28 in hyperlipidemia was investigated by hematoxylin-eosin and oil Red O staining, the lipid biochemistry measurement, enzyme-linked immunosorbent assay, reverse transcription quantitative polymerase chain reaction and western blotting assays. The mechanism of FBXO28 explored by co-immunoprecipitation, immunofluorescence, ubiquitination and cycloheximide assays. Low expression of FBXO28 was found in hyperlipidemia in silico, in vivo and in vitro. Upregulation of FBXO28 declined the body weight, fat accumulation, and serum lipid content in HFD-fed mice. Abnormal lipid accumulation, and the level of liposynthetic genes and beta-oxidation related genes were improved by overexpression of FBXO28 both in HFD-elicited mice and MDI-treated 3T3-L1 preadipocytes. Besides, overexpression of FBXO28 declined HFD-induced the level of proinflammatory factors and F4/80. Mechanically, FBXO28 directly bound RAB27A and promoted its ubiquitinated degradation. Thus, upregulation of RAB27A inverted the improved role of FBXO28 in abnormal lipid metabolism and inflammation in vivo and in vitro. FBXO28 ameliorated abnormal lipid metabolism and inflammation through the ubiquitinated degradation of RAB27A, thereby attenuating HFD-induced hyperlipidemia. The results could promote the treatment of hyperlipidemia, and the relevant diseases.

Homeobox B4 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on endotoxin-associated acute lung injury in rats

BackgroundAlveolar capillary endothelial cell (EC) injury has a pivotal role in driving acute respiratory distress syndrome (ARDS) progression and maintaining endothelial homeostasis. A previous ex vivo study revealed that overexpression of homeobox B4 (HOXB4) in bone marrow mesenchymal stem cells (BMSCs) enhanced protection against lipopolysaccharide (LPS)-induced EC injury by activating the Wnt/β-catenin pathway. This in vivo study was performed to verify whether BMSCs overexpressing HOXB4 exert similar protective effects on LPS-induced acute lung injury (ALI) in an animal model. MethodsThe ALI rat model was established by intraperitoneal injection of LPS. Wildtype BMSCs or BMSCs overexpressing HOXB4 were then injected via the tail vein. The lung characteristics of rats were visualized by computed tomography. Lung histopathological characteristics and collagen deposition were assessed by hematoxylin-eosin and Masson's staining, respectively, which were combined with the lung wet/dry ratio and proinflammatory factor levels in bronchoalveolar lavage fluid to further evaluate therapeutic effects. Expression of β-catenin and VE-cadherin was assessed by western blotting and immunofluorescence. ResultsCompared with wildtype BMSCs, overexpression of HOXB4 optimized the therapeutic effects of BMSCs, which manifested as improvements in lung exudation and histopathological features, reduced lung collagen deposition, amelioration of lung permeability, attenuation of lung inflammation, and enhanced expression of β-catenin and VE-cadherin proteins. ConclusionsHOXB4-overexpressing BMSCs optimized the protective effect against LPS-induced ALI by partially activating Wnt/β-catenin signaling.

Oxypeucedanin hydrate alleviates rheumatoid arthritis by inhibiting the TLR4-MD2/NF-κB/MAPK signaling axis.

Rheumatoid arthritis (RA) is an idiopathic and chronic autoimmune disease for which there are currently no effective treatments. Oxypeucedanin hydrate (OXH) is a natural coumarin known for its potent anti-inflammatory properties. However, further investigations are needed to determine its therapeutic efficacy in treating RA. In this study, we evaluate the anti-inflammatory activity of OXH by treating LPS-induced RAW264.7 macrophages. Our results show that OXH treatment reverses the changes in iNOS, COX-2, IL-1β, IL-6, and TNF-α levels. Additionally, OXH reduces ROS production. Further analysis reveals that OXH suppresses the activation of the NF-κB/MAPK pathway. CETSA results show that OXH competes with LPS for binding to the TLR4/MD2 complex. MST experiments demonstrate the specific affinity of OXH for the TLR4/MD2 complex, with a Kd value of 33.7 μM. Molecular docking analysis suggests that OXH binds to the pocket of the TLR4/MD2 complex and interacts with specific amino acids, such as GLY-343, LYS-388, and PHE-345. Molecular dynamics simulations further confirm this conclusion. Finally, we investigate the potential of OXH in treating RA using a collagen-induced arthritis (CIA) model in rats. OXH effectively ameliorates the symptoms of CIA, including improving body weight, reducing swelling and redness, increasing talus volume, and decreasing bone erosion. OXH also decreases the mRNA levels of pro-inflammatory factors in synovial tissue. Transcriptome enrichment analysis and western blot analysis confirm that OXH suppresses the NF-κB/MAPK pathway, which is consistent with our in vitro findings.

Ropinirole suppresses LPS-induced periodontal inflammation by inhibiting the NAT10 in an ac4C-dependent manner

BackgroundPeriodontitis is a chronic osteolytic inflammatory disease, where anti-inflammatory intervention is critical for restricting periodontal damage and regenerating alveolar bone. Ropinirole, a dopamine D2 receptor agonist, has previously shown therapeutic potential for periodontitis but the underlying mechanism is still unclear.MethodsHuman gingival fibroblasts (HGFs) treated with LPS were considered to mimic periodontitis in vitro. The dosage of Ropinirole was selected through the cell viability of HGFs evaluation. The protective effects of Ropinirole on HGFs were evaluated by detecting cell viability, cell apoptosis, and pro-inflammatory factor levels. The molecular docking between NAT10 and Ropinirole was performed. The interaction relationship between NAT10 and KLF6 was verified by ac4C Acetylated RNA Immunoprecipitation followed by qPCR (acRIP-qPCR) and dual-luciferase reporter assay.ResultsRopinirole alleviates LPS-induced damage of HGFs by promoting cell viability, inhibiting cell apoptosis and the levels of IL-1β, IL-18, and TNF-α. Overexpression of NAT10 weakens the effects of Ropinirole on protecting HGFs. Meanwhile, NAT10-mediated ac4C RNA acetylation promotes KLF6 mRNA stability. Upregulation of KLF6 reversed the effects of NAT10 inhibition on HGFs.ConclusionsTaken together, Ropinirole protected HGFs through inhibiting the NAT10 ac4C RNA acetylation to decrease the KLF6 mRNA stability from LPS injury. The discovery of this pharmacological and molecular mechanism of Ropinirole further strengthens its therapeutic potential for periodontitis.

Effects of silencing NLRP3 gene on proliferation of psoriasis-like HaCaT cells and expressions of cytokines.

We aimed to explore the effects of silencing NOD-like receptor protein 3 (NLRP3) on proliferation of psoriasis-like HaCaT cells and expressions of cytokines. HaCaT cells were treated with human keratinocyte growth factor (KGF) and were divided into KGF group, negative control group, NLRP3-RNAi group and control group. Cells proliferation was detected by CCK8, cell clone formation rate was detected by clone formation assay, distribution of cells cycle was detected by flow cytometry, expressions of cyclin B1 (Cyclin B1), cyclin-dependent kinase 2 (CDK2), Ki67 and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot, and levels of interleukin (IL)-17, IL-23, IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were increased in KGF group, percentage of cells in G0/G1 phase was decreased, percentage of cells in S phase was increased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were increased, and levels of IL-17, IL-23, IL-6 and TNF-α were increased. Compared with negative control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were decreased in NLRP3-RNAi group, percentage of cells in G0/G1 phase was increased, percentage of cells in S phase was decreased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were decreased, and levels of IL-17, IL-23, IL-6 and TNF-α were decreased. Silencing NLRP3 gene can inhibit the proliferation of psoriasis-like HaCaT cells, arrest cell cycle, inhibit the expressions of cell proliferation-related proteins and reduce levels of pro-inflammatory factors.

Effects of sn-2 Palmitic Triacylglycerols and the Ratio of OPL to OPO in Human Milk Fat Substitute on Metabolic Regulation in Sprague-Dawley Rats.

In this study, the influence of total sn-2 palmitic triacylglycerols (TAGs) and ratio of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) to 1,3-dioleoyl-2-palmitoylglycerol (OPO) in human milk fat substitute (HMFS) on the metabolic changes were investigated in Sprague-Dawley rats. Metabolomics and lipidomics profiling analysis indicated that increasing the total sn-2 palmitic TAGs and OPL to OPO ratio in HMFS could significantly influence glycine, serine and threonine metabolism, glycerophospholipid metabolism, glycerolipid metabolism, sphingolipid metabolism, bile acid biosynthesis, and taurine and hypotaurine metabolism pathways in rats after 4 weeks of feeding, which were mainly related to lipid, bile acid and energy metabolism. Meanwhile, the up-regulation of taurine, L-tryptophan, and L-cysteine, and down-regulations of lysoPC (18:0) and hypoxanthine would contribute to the reduction in inflammatory response and oxidative stress, and improvement of immunity function in rats. In addition, analysis of targeted biochemical factors also revealed that HMFS-fed rats had significantly increased levels of anti-inflammatory factor (IL-4), immunoglobulin A (IgA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px), and decreased levels of pro-inflammatory factors (IL-6 and TNF-α) and malondialdehyde (MDA), compared with those of the control fat-fed rats. Collectively, these observations present new in vivo nutritional evidence for the metabolic regulatory effects of the TAG structure and composition of human milk fat substitutes on the host.