TLR4 Expression Research Articles

Glutathione regulates CIA-activated splenic-lymphocytes via NF-κB/MMP-9 and MAPK/PCNA pathways manipulating immune response

Reduced glutathione (GSH) is an antioxidant involved in redox homeostasis, and recently regarded as an inducer of Reductive stress. Its immune-regulatory effects on lymphocytes have not been extensively studied. This study is based on the finding that much increased GSH level in collagen-induced arthritis (CIA) rat spleen, and aimed to investigate the effects of GSH (0, 1, 10, 100 mM) on normal and immune-stimulated spleen lymphocytes respectively. The elevated GSH level is associated with the increased levels of inflammatory factors; especially the increased DPP1 activity indicated immune-granulocytes activation in CIA rat spleen. Exogenous GSH had different influences on normal and CIA lymphocytes, affecting intracellular levels of GSH, Glutathione-S-transferases (GSTs) and Reactive oxygen species (ROS); as well as the expressions of NF-κB, MMP-9, Bcl-2, GST, P38, PCNA and TLR4. The increased extracellular GSH level disturbed redox homeostasis and induces reductive stress to spleen lymphocytes, which decreased intracellular GSH concentration and influenced the MAPK/PCNA and NF-κB/MMP-9 signaling pathways, as well as cell cycles respectively, leading to cell senescence/ferroptosis /apoptosis. This study also revealed the multiple faces of GSH in regulating spleen lymphocytes, which depends on its levels in tissue or in cells, and the activation status of lymphocytes. These findings indicate the immune-regulatory role of GSH on spleen-lymphocytes, and the high level GSH in CIA rat spleens may contribute to CIA development.

Proanthocyanidins alleviate Henoch-Schönlein purpura by mitigating inflammation and oxidative stress through regulation of the TLR4/MyD88/NF-κB pathway.

Investigate Proanthocyanidins (PCs) efficacy and mechanisms in treating Henoch-Schönlein purpura (HSP)-like rat models, focusing on inflammatory and oxidative stress (OS) responses. An HSP-like rat model was established using ovalbumin (OVA) injection, leading to symptoms mimicking HSP. The study measured inflammatory markers (IL-4, IL-17, TNF-α), OS markers (MDA, SOD, CAT), and assessed the TLR4/MyD88/NF-κB signaling pathway's involvement via histopathological and immunofluorescence analyses. PCs treatment significantly improved HSP-like symptoms, reduced inflammatory cell infiltration, and decreased IgA deposition in renal mesangial areas. Serum analyses revealed that PCs effectively lowered IL-4, IL-17, TNF-α, and MDA levels while increasing SOD and CAT levels (p<0.05). Crucially, PCs also downregulated TLR4, MyD88, and NF-κB expressions, highlighting the blockage of the TLR4-mediated signaling pathway as a key mechanism. PCs show promising therapeutic effects in HSP-like rats by mitigating inflammatory responses and oxidative damage, primarily through inhibiting the TLR4/MyD88/NF-κB pathway. These findings suggest PCs as a potential treatment avenue for HSP, warranting further investigation.

Anti-inflammatory Effect of Novel 2-Phenylphthalazin-2-ium Bromides on LPS-induced RAW264.7 Cells and their Mechanism

Background: Inspired by natural anti-inflammatory quaternary benzo[C]phenanthridine alkaloids, novel 2-phenylphthalazin-2-ium bromides were previously designed and synthesized. Objective: The anti-inflammatory effect of 2-phenylphthalazin-2-ium bromides was evaluated based on inflammatory cytokines, and their possible mechanism was explored through the NF-κB, TLR4 and MAPK signaling pathways. Methods: The tested concentrations of two compounds were assessed using MTT assay in vitro. Griess assay was used to determine the changes in nitric oxide (NO) in the cell culture supernatant. qRT‒PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS. The secretion levels of TNF-α and IL-1β were detected by ELISA. Western blot test was used to detect the protein expression of IL-6, IL-10, TLR4, iNOS, NF-κB, p-P38/P38, p- ERK/ERK and p-JNK/JNK. Results: 2-(3,5-Dichlorophenyl)phthalazin-2-ium bromide (2) with a concentration below 1 μg/mL showed no significant effect on the growth inhibition of RAW264.7 cells, so the concentrations of compound 2 used for experiments were set to 0, 0.25, 0.5 and 1 μg/mL. Compared with the blank control group, the model group showed increased release of NO, transcription levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS (p&lt; 0.05), and ratios of p-P38/P38, p-ERK/ERK, p-JNK/JNK (p&lt; 0.05). Compared with the model group, the sample groups displayed decreased NO release and reduced transcriptional levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4, and iNOS and reduced protein expression ratios of IL-6, IL-1ß, IL-10, TNF-α, NF-κB, TLR4, iNOS, p-P38/P38, p-ERK/ERK and p- JNK/JNK (p&lt; 0.05). Conclusion: This study showed that 2-phenylphthalazin-2-ium bromides partially protected macrophages from the LPS-induced inflammatory response by suppressing TLR4-NF-κB/MAPK signaling and reducing NO production.

Human inborn errors of long-chain fatty acid oxidation show impaired inflammatory responses to TLR4-ligand LPS.

Stimulation of mammalian cells with inflammatory inducers such as lipopolysaccharide (LPS) leads to alterations in activity of central cellular metabolic pathways. Interestingly, these metabolic changes seem to be important for subsequent release of pro-inflammatory cytokines. This has become particularly clear for enzymes of tricarboxylic acid (TCA) cycle such as succinate dehydrogenase (SDH). LPS leads to inhibition of SDH activity and accumulation of succinate to enhance the LPS-induced formation of IL-1β. If enzymes involved in beta-oxidation of fatty acids are important for sufficient responses to LPS is currently not clear. Using cells from various patients with inborn long-chain fatty acid oxidation disorders (lcFAOD), we report that disease-causing deleterious variants of Electron Transfer Flavoprotein Dehydrogenase (ETFDH) and of Very Long Chain Acyl-CoA Dehydrogenase (ACADVL), both cause insufficient inflammatory responses to stimulation with LPS. The insufficiencies included reduced TLR4 expression levels, impaired TLR4 signaling, and reduced or absent induction of pro-inflammatory cytokines such as IL-6. The insufficient responses to LPS were reproduced in cells from healthy controls by targeted loss-of-function of either ETFDH or ACADVL, supporting that the deleterious ETFDH and ACADVL variants cause the attenuated responses to LPS. ETFDH and ACADVL encode two distinct enzymes both involved in fatty acid beta-oxidation, and patients with these deficiencies cannot sufficiently metabolize long-chain fatty acids. We report that genes important for beta-oxidation of long-chain fatty acids are also important for inflammatory responses to an acute immunogen trigger like LPS, which may have important implications for understanding infection and other metabolic stress induced disease pathology in lcFAODs.

HIV1 gp120 activates microglia via TLR2-NF-κB signaling to up-regulate inflammatory cytokine expression and induce neuropathic pain

HIV associated neuropathic pain (HANP) is a common complication of AIDS. Intrathecal injection of recombinant HIV-1 gp120 in mice is a well-known model. Previous RNA sequencing revealed spinal TLR2 acts as a differentially expressed gene in HANP mice. The spinal TLR2 is involved in HANP, but its role and underlying mechanism remains unclear. In this study the transcription, expression and distribution characteristics of TLR2 in the spinal cord of HANP male mice have been analyzed by qRT-PCR, Western blotting, and immunofluorescent staining. We found that TLR2 expression was upregulated in the spinal dorsal horn and mainly distributed in microglial cells, and blocking TLR2 relieved pain of HANP mice. Following stimulation by gp120 microglial cells upregulate TLR2 expression and become activated. The activation stimulates their differentiation into the M1 type, increasing IL-1β and TNF-α expression while inhibiting IL-10 expression. Silencing the Tlr2 gene slows down the activation, polarization, and secretion of pro-inflammatory factors in microglial cells induced by gp120, and enhances the expression of anti-inflammatory factors. Further analysis of the impact of gp120 on downstream signaling pathways of TLR2 in microglial cells, including NF-κB, MAPK (p38MAPK, ERK, and JNK) and PI3K/AKT, revealed that TLR2-NF-κB signaling plays a crucial role in the activation and polarization of microglial cells by gp120. Activation of NF-κB signaling aggravates pain in HANP mice, while blocking it lightens pain. This data indicates that gp120, through the TLR2-NF-κB signaling, activates spinal microglial cells, promotes the secretion of inflammatory cytokines, leading to HANP. This provides new targets to develop drugs for HANP.

TYROBP serve as potential immune-related signature genes in the acute phase of intracerebral hemorrhage

The development of intracerebral hemorrhage (ICH) is a dynamic process and intervention during the acute phase of ICH is critical for subsequent recovery. Therefore, it is crucial to screen potential signature genes and therapeutic target genes in the acute phase of ICH. In this study, based on the results of mRNA sequencing in mouse ICH and mRNA sequencing of human ICH from online databases, top five potential signature genes after ICH, Tyrobp, Itgb2, Tlr2, Ptprc and Itgam, were screened. Quantitative PCR results showed higher mRNA expression of Tyrobp, Itgb2, Tlr2, Ptprc, and Itgam in the 1-, 3- and 5-day mouse ICH groups compared to the sham-operated group. Immune infiltration correlation analysis shows that the top-ranked signature gene, Tyrobp, is negatively correlated with M2 macrophages and plasma cells, and Western blot analysis shows higher expression of the Tyrobp protein in the 1-, 3-, and 5-day mouse ICH groups compared to the sham-operated group. Furthermore, immunohistochemistry revealed that TYROBP protein expression was significantly higher in human ICH tissues than in normal brain tissues. Our results suggest that Tyrobp is a signature gene in the acute phase of ICH and may be a potential target for the treatment of the acute phase of ICH.

Dihydromyricetin Nanoparticles Alleviate Lipopolysaccharide-Induced Acute Kidney Injury by Decreasing Inflammation and Cell Apoptosis via the TLR4/NF-κB Pathway.

Acute kidney injury (AKI) is the most severe and fatal complication of sepsis resulting from infectious trauma. Currently, effective treatment options are still lacking. Dihydromyricetin is the main component extracted from Vine tea (Ampelopsis megalophylla Diels et Gilg). In our previous research, chitosan-tripolyphosphate-encapsulated nanoparticles of dihydromyricetin (CS-DMY-NPs) have been proven to have potential protective effects against cisplatin-induced AKI. Here, we investigated the protective effects and mechanisms of DMY and its nano-formulations against LPS-induced AKI by assessing pathological and inflammatory changes in mice. In mice with LPS-AKI treated with 300 mg/kg CS-DMY-NPs, the levels of creatinine (Cr), blood urea nitrogen (BUN), and KIM-1 were significantly reduced by 56%, 49%, and 88%, respectively. CS-DMY-NPs can upregulate the levels of GSH, SOD, and CAT by 47%, 7%, and 14%, respectively, to inhibit LPS-induced oxidative stress. Moreover, CS-DMY-NPs decreased the levels of IL-6, IL-1β, and MCP-1 by 31%, 49%, and 35%, respectively, to alleviate the inflammatory response. TUNEL and immunohistochemistry showed that CS-DMY-NPs reduced the number of apoptotic cells, increased the Bcl-2/Bax ratio by 30%, and attenuated renal cell apoptosis. Western blot analysis of renal tissue indicated that CS-DMY-NPs inhibited TLR4 expression and downregulated the phosphorylation of NF-κB p65 and IκBα. In summary, DMY prevented LPS-induced AKI by increasing antioxidant capacity, reducing inflammatory responses, and blocking apoptosis, and DMY nanoparticles were shown to have a better protective effect for future applications.

Protective effect of Acanthus ilicifolius extracts against acute alcoholic liver injury via suppressing TLR4/NF-κB signal pathway and modulating intestinal microbiota in mice

Excessive alcohol consumption is leading to increased rates of liver injury and disease. A new research strategy focuses on manipulating gut microbiota to lessen alcohol-induced harm. This study examined the hepatoprotective effects of extracts from Acanthus ilicifolius (EAI) on acute alcoholic liver injury by inhibiting the TLR4/NF-κB signalling pathway and modulating intestinal microbiota in mice. The results showed that EAI dose-dependently reduced alcohol-induced elevations of AST, ALT, and ALP levels. EAI showed significant inhibitory effects on the expressions of TLR4, NF-κB, and pNF-κB proteins. Furthermore, EAI caused a notable reduction in hepatic levels of IL-1β, IL-6, and TNF-α. Supplementation with EAI could ameliorate alcohol-induced dysbiosis of intestinal bacteria. The levels of ALT, AST, and ALP levels were negatively correlated with Ligilactobacillus, Lactobacillus, and Alistipes, but positively correlated with Helicobacter and Bacteroides. Overall, EAI alleviated alcoholic liver injury in mice by inhibiting the TLR4/NF-κB signalling pathway and modulating intestinal bacteria.

Proton-activated chloride channel increases endplate porosity and pain in a mouse spine degeneration model.

Chronic low back pain (LBP) can severely affect daily physical activity. Aberrant osteoclast-mediated resorption leads to porous endplates, which allow the sensory innervation that causes LBP. Here, we report that expression of the proton-activated chloride (PAC) channel was induced during osteoclast differentiation in the porous endplates via a RANKL/NFATc1 signaling pathway. Extracellular acidosis evoked robust PAC currents in osteoclasts. An acidic environment of porous endplates and elevated PAC activation-enhanced osteoclast fusion provoked LBP. Furthermore, we found that genetic knockout of the PAC gene Pacc1 significantly reduced endplate porosity and spinal pain in a mouse LBP model, but it did not affect bone development or homeostasis of bone mass in adult mice. Moreover, both the osteoclast bone-resorptive compartment environment and PAC traffic from the plasma membrane to endosomes to form an intracellular organelle Cl channel had a low pH of approximately 5.0. The low pH environment activated the PAC channel to increase sialyltransferase St3gal1 expression and sialylation of TLR2 in the initiation of osteoclast fusion. Aberrant osteoclast-mediated resorption is also found in most skeletal disorders, including osteoarthritis, ankylosing spondylitis, rheumatoid arthritis, heterotopic ossification, and enthesopathy. Thus, elevated Pacc1 expression and PAC activity could be a potential therapeutic target for the treatment of LBP and osteoclast-associated pain.

The hijacking of HBV by small extracellular vesicles inhibits M1 macrophages to facilitate immune evasion

Small extracellular vesicles (sEVs) have the ability to transfer genetic material between cells, but their role in mediating HBV infection and regulating M1 macrophages to promote immune evasion remains unclear. In this study, we utilized PMA + LPS + IFN-γ to induce THP-1 into M1 macrophages. We then extracted sEVs from HepG2.2.15 cell and treated the M1 macrophages with these sEVs. QPCR detection revealed the presence of HBV-DNA in the M1 macrophages. Additionally, RT-qPCR and WB analysis demonstrated a significantly decreased in the expression of TLR4, NLRP3, pro-caspase-1, caspase-1p20, IL-1β and IL-18 in the M1 macrophages (P < 0.05). Furthermore, RT-qPCR results displayed high expression levels of that miR-146a and FEN-1 in the sEVs derived from HepG2.2.15 cells (P < 0.01). RT -qPCR and WB analysis showed that these sEVs enhanced the expression of FEN-1 or miR-146a in the M1 macrophages through miR-146a or FEN-1 (P < 0.05), while simultaneously reducing the expression of TLR4, NLRP3, caspase-1p20, IL-1β and IL-18 in the M1 macrophages (P < 0.05). In summary, our findings indicate that sEVs loaded with HBV inhibit the inflammatory function of M1 macrophages and promote immune escape. Additionally, miR-146a and FEN-1 present in the sEVs play a crucial role in this process.

Polysaccharide of Atractylodes macrocephala Koidz alleviates NAFLD-induced hepatic inflammation in mice by modulating the TLR4/MyD88/NF-κB pathway

Non-alcoholic fatty liver disease (NAFLD) not only could cause abnormal lipid metabolism in the liver, but also could cause liver inflammation. Previous studies have shown that Polysaccharide of Atractylodes macrocephala Koidz (PAMK) could alleviate animal liver inflammatory damage and alleviate NAFLD in mice caused by high-fat diet(HFD), but regulation of liver inflammation caused by NAFLD has rarely been reported. In this study, an animal model of non-alcoholic fatty liver inflammation in the liver of mice was established to explore the protective effect of PAMK on the liver of mice. The results showed that PAMK could alleviate the abnormal increase of body weight and liver weight of mice caused by HFD, alleviate the abnormal liver structure of mice, reduce the level of oxidative stress and cytokine secretion in the liver of mice, and downregulate the mRNA expression of TLR4, MyD88, NF-κB and protein expression of P-IκB, P-NF-κB-P65, TLR4, MyD88, NF-κB in the liver. These results indicate that PAMK could alleviate hepatocyte fatty degeneration and damage, oxidative stress and inflammatory response of the liver caused by NAFLD in mice.

The Expression of Toll-like Receptors in Cartilage Endplate Cells: A Role of Toll-like Receptor 2 in Pro-Inflammatory and Pro-Catabolic Gene Expression.

The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), it is unclear if CEP cells (CEPCs) share this trait. The CEP has a higher cell density than the disc, making CEPCs an important contributor. This study aimed to identify TLRs on CEPCs and their role in pro-inflammatory and catabolic gene expression. Gene expression of TLR1-10 was measured in human CEPs and expanded CEPCs using quantitative polymerase chain reaction. Additionally, surface TLR expression was measured in CEPs grouped into non-MC and MC. CEPCs were stimulated with tumor necrosis factor alpha, interleukin 1 beta, small-molecule TLR agonists, or the 30 kDa N-terminal fibronectin fragment. TLR2 signaling was inhibited with TL2-C29, and TLR2 protein expression was measured with flow cytometry. Ex vivo analysis found all 10 TLRs expressed, while cultured CEPCs lost TLR8 and TLR9 expression. TLR2 expression was significantly increased in MC1 CEPCs, and its expression increased significantly after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer upregulated TLR2 protein expression. The TLR2/1 and TLR2/6 ligands upregulated pro-inflammatory genes and matrix metalloproteases (MMP1, MMP3, and MMP13), and TLR2 inhibition inhibited their upregulation. Endplate resorptive capacity of TLR2 activation was confirmed in a CEP explant model. The expression of TLR1-10 in CEPCs suggests that the CEP is susceptible to PAMP and DAMP stimulation. Enhanced TLR2 expression in MC1, and generally in CEPCs under inflammatory conditions, has pro-inflammatory and pro-catabolic effects, suggesting a potential role in disc degeneration and MC.

Molecular mechanism of Rolupram reducing neuroinflammation in noise induced tinnitus mice through TLR4/NF kB/NLRP3 protein/caspase-1/IL-1 β signaling pathway

Noisy tinnitus is a common auditory system disease characterized by persistent tinnitus symptoms. The TLR4/NF - κ B/NLRP3 signaling pathway plays an important role in neuroinflammatory response. Select 6 control and 6 noise exposed mice for transcriptome sequencing analysis in the hippocampus, conduct high-throughput data analysis, identify differentially expressed genes, and screen for pathways. Auditory brainstem response (ABR) detection was performed to understand the hearing changes, and the modeling effect was evaluated using the GPIAS% inhibition experiment of auditory startle reflex. Morphological observation of the basement membrane was performed to determine whether the inner hair cells were damaged. Immunohistochemistry and immunofluorescence were used to determine the activation of microglia in the hippocampus of noise induced tinnitus mice. Finally, qPCR and Western Blot were used to detect the expression of TLR4, NF kB, NLRP3, caspase-1, and IL-1 β in the hippocampus of each group of mice. Through high-throughput data analysis, it was found that there was no significant difference in the auditory threshold of the three groups of mice; After 2 h of exposure to 100 dB SPL noise, the GPIAS% of mice decreased significantly compared to before exposure, and membrane construction was successful. After 7 days, the GPIAS% of the drug intervention group increased. After noise exposure, mice developed tinnitus, and hippocampus neuroinflammation. Roflupram can inhibit neuroinflammation and improve tinnitus through the TLR4/NF kB/NLRP3/caspase-1/IL-1 β signaling pathway.

THE IMPACT OF ELECTRO-ACUPUNCTURE ON “BAIHUAN SHU” (BL 30) AND “HUIYANG” (BL 35) POINTS ON THE TLR4/NF-κB PATHWAY IN RATS WITH CHRONIC PROSTATITIS

The objective of this research is to investigate the potential mechanisms underlying electro-acupuncture intervention in chronic prostatitis (CP). Rats were randomly divided into five groups: a control group, a model group (suffering from CP), an electro-acupuncture group, an inhibitor group, and an activator group. One day post-modeling, electro-acupuncture intervention was conducted on the electro-acupuncture group, inhibitor group, and activator group at the acupoints “Baihuan Shu” (BL 30) and “Huiyang” (BL 35). Relevant indicators were used to evaluate the efficacy. Compared with models, the electro-acupuncture group showed significantly increased horizontal and vertical movement scores and sugar water intake, and significantly decreased prostate wet weight and prostate index. Compared with the electro-acupuncture group, the inhibitor group showed significantly increased horizontal and vertical movement scores and sugar water consumption. In contrast, for the activator group, these scores and consumption levels significantly decreased, while their prostate wet weight and prostate index increased significantly. Compared with models, the expression of TLR4 and p-NF-[Formula: see text]B/NF-[Formula: see text]B proteins in the prostate tissue of electro-acupuncture group was significantly lower. In terms of the inhibitor group, they also showed a decrease in TLR4 and p-NF-[Formula: see text]B/NF-[Formula: see text]B protein expression, whilst the activator group showed an increase. Therefore, electro-acupuncture at “Baihuan Shu” (BL 30) and “Huiyang” (BL 35) can significantly enhance movement scores, vertical movement scores, and sugar water consumption in rats with CP. Moreover, it can decrease the wet weight and index of the prostate, possibly by regulating the TLR4/NF-[Formula: see text]B pathway.

Immunomodulatory effects of live and UV-killed Bacillus subtilis natto on inflammatory response in human colorectal adenocarcinoma cell line in vitro.

Colorectal cancer (CRC) is a heterogeneous disease of the colon or rectum arising from adenoma precursors and serrated polyps. Recently, probiotics have been proposed as an effective and potential therapeutic approach for CRC prevention and treatment. Probiotics have been shown to alleviate inflammation by restoring the integrity of the mucosal barrier and impeding cancer progression. In this study, we aimed to investigate the immunomodulatory effects of live and UV-killed Bacillus subtilis natto on the inflammatory response in CRC. Caco-2 cells were exposed to various concentrations of live and UV- killed B. subtilis natto, and cell viability was assessed using MTT assay. Gene expression analysis of IL-10, TGF-β, TLR2 and TLR4 was performed using RT-qPCR. Our findings showed that both live and UV-killed B. subtilis natto caused significant reduction in inflammatory response by decreasing the gene expression of TLR2 and TLR4, and enhancing the gene expression of IL-10 and TGF-β in Caco-2 cells as compared to control group. The results of this study suggest that live and UV-killed B. subtilis natto may hold potential as a therapeutic supplement for modulating inflammation in CRC.

Neuroinflammation and Neurometabolomic Profiling in Fentanyl Overdose Mouse Model Treated with Novel β-Lactam, MC-100093, and Ceftriaxone.

Opioid-related deaths are attributed to overdoses, and fentanyl overdose has been on the rise in many parts of the world, including the USA. Glutamate transporter 1 (GLT-1) has been identified as a therapeutic target in several preclinical models of substance use disorders, and β-lactams effectively enhance its expression and function. In the current study, we characterized the metabolomic profile of the nucleus accumbens (NAc) in fentanyl-overdose mouse models, and we evaluated the protective effects of the functional enhancement of GLT-1 using β-lactams, ceftriaxone, and MC-100093. BALB/c mice were divided into four groups: control, fentanyl, fentanyl/ceftriaxone, and fentanyl/MC-100093. While the control group was intraperitoneally (i.p.) injected with normal saline simultaneously with other groups, all fentanyl groups were i.p. injected with 1 mg/kg of fentanyl as an overdose after habituation with four repetitive non-consecutive moderate doses (0.05 mg/kg) of fentanyl for a period of seven days. MC-100093 (50 mg/kg) and ceftriaxone (200 mg/kg) were i.p. injected from days 5 to 9. Gas chromatography-mass spectrometry (GC-MS) was used for metabolomics, and Western blotting was performed to determine the expression of target proteins. Y-maze spontaneous alternation performance and the open field activity monitoring system were used to measure behavioral manifestations. Fentanyl overdose altered the abundance of about 30 metabolites, reduced the expression of GLT-1, and induced the expression of inflammatory mediators IL-6 and TLR-4 in the NAc. MC-100093 and ceftriaxone attenuated the effects of fentanyl-induced downregulation of GLT-1 and upregulation of IL-6; however, only ceftriaxone attenuated fentanyl-induced upregulation of TRL4 expression. Both of the β-lactams attenuated the effects of fentanyl overdose on locomotor activities but did not induce significant changes in the overall metabolomic profile. Our findings revealed that the exposure to a high dose of fentanyl causes alterations in key metabolic pathways in the NAc. Pretreatment with ceftriaxone and MC-100093 normalized fentanyl-induced downregulation of GLT-1 expression with subsequent attenuation of neuroinflammation as well as the hyperactivity, indicating that β-lactams may be promising drugs for treating fentanyl use disorder.

Protective effect of menthol against thioacetamide-induced hepatic encephalopathy by suppressing oxidative stress and inflammation, augmenting expression of BDNF and α7-nACh receptor, and improving spatial memory

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that can occur in people with acute or chronic liver disease. Here, we investigated the effects of menthol, a natural monoterpene, on HE induced by thioacetamide (TA) in male Wistar rats. The rats received 200 mg/kg of TA twice a week for four weeks and were administered 10 mg/kg of menthol intraperitoneally daily for the same period. The results showed that menthol treatment reduced oxidative stress and inflammation in the livers and hippocampi of the rats that received TA. It also lowered the levels of ammonium and liver enzymes AST, ALT, ALP, and GGT in the serum of these animals and prevented liver histopathological damage. In addition, the expression and activity of acetylcholinesterase in the hippocampus of HE model rats were decreased by menthol. Likewise, this monoterpene reduced the expression of TLR4, MyD88, and NF-κB in the hippocampus while increasing the expression of BDNF and α7-nACh receptor. Menthol also reduced neuronal death in the hippocampal cornu ammonis-1 and dentate gyrus regions and reduced astrocyte swelling, which led to improved learning and spatial memory in rats with HE. In conclusion, the study suggests that menthol may have strong protective effects on the liver and brain, making it a potential treatment for HE and neurodegenerative diseases.

Immunobiological effects of lipopolysaccharide derived from Helicobacter pylori and influence of a proton pump inhibitor lansoprazole on human polymorphonuclear leukocytes.

Helicobacter pylori colonizes the human gastric mucosa of more than half of the human population and has a unique lipopolysaccharide (LPS) structure. LPS is the most dominant and suitable pathogen-associated molecular pattern that is detected via pattern recognition receptors. Although the priming effect of H. pylori LPS on reactive oxygen species (ROS) production of PMNs is lower than that of Escherichia coli O111:B4 LPS, LPS released from H. pylori associated with antibiotics eradication therapy may activate PMNs and increase ROS production. In addition, we describe the effects of H. pylori and E. coli O111:B4 LPSs on gene expression and the anti-inflammatory effect of lansoprazole (LPZ) in human polymorphonuclear leukocytes. LPS isolated from H. pylori and E. coli O111:B4 alters toll-like receptor 2 (TLR) and TLR4 expressions similarly. However, LPS from E. coli O111:B4 and H. pylori caused a 1.8-fold and 1.5-fold increase, respectively, in CD14 expression. All LPS subtypes upregulated TNFα and IL6 expression in a concentration-dependent manner. Although E. coli O111:B4 LPS upregulated IL8R mRNA levels, H. pylori LPS did not (≦ 100ng/mL). Gene expression levels of ITGAM demonstrated no significant change on using both LPSs. These different effects on the gene expression in PMNs may depend on variations in LPS structural modifications related to the acquired immunomodulatory properties of H. pylori LPS. Proton pump inhibitors, i.e., LPZ, are used in combination with antibiotics for the eradication therapy of H. pylori. LPZ and its acid-activated sulphenamide form AG-2000 suppress ROS production of PMNs in a dose-dependent manner. These results suggest that LPZ combination with antibiotics for H. pylori eradication reduces gastric inflammation by suppressing ROS release from PMNs.

Single and Mixed Strains of Probiotics Reduced Hepatic Fat Accumulation and Inflammation and Altered Gut Microbiome in a Nonalcoholic Steatohepatitis Rat Model.

As gut dysbiosis has been implicated in the pathogenesis of nonalcoholic steatohepatitis (NASH), probiotic supplementation might be a potential treatment for this condition. The aim of this study was to evaluate the effects of single- and mixed-strain probiotics on the severity of NASH induced by a high-fat, high-fructose (HFHF) diet and their mechanisms of action. Male Sprague-Dawley rats were divided into four groups (n = 7 per group): control group, NASH group, NASH + single-strain group, and NASH + mixed-strain group. In the single-strain and mixed-strain groups, rats received Lactobacillus plantarum B7 and Lactobacillus rhamnosus L34 + Lactobacillus paracasei B13 by oral gavage once daily, respectively. The duration of the study was 6 weeks. Liver tissue was used for histopathology, hepatic fat content was assessed by Oil Red O staining and hepatic free fatty acid (FFA), and hepatic TLR4 and CD14 expression were assessed by immunohistochemistry. Fresh feces was collected for gut microbiota analysis. Liver histology revealed a higher degree of fat accumulation, hepatocyte ballooning, and lobular inflammation in the NASH group, which improved in probiotics-treated groups. The amounts of hepatic fat droplets and hepatic FFA levels were more pronounced in the NASH group than in the control and treatment groups. Serum TNF- α levels were significantly higher in the NASH group than in control and probiotic groups. The expression of CD14 and TLR4 increased in the NASH group as compared with the control and probiotics-treated groups. Alpha diversity was reduced in the NASH group, but increased in both treatment groups. The relative abundance of Lactobacillus significantly decreased in the NASH group, but increased in both treatment groups. The relative abundance of Akkermansia significantly increased in the NASH group, but decreased in both treatment groups. In conclusion, both single-strain and mixed-strain probiotics could improve NASH histology by suppressing inflammatory responses in the liver, with this improvement potentially being associated with changes in the gut microbiota.

Alterations in DNA 5-hydroxymethylation patterns in the hippocampus of an experimental model of chronic epilepsy

Temporal lobe epilepsy (TLE) is a type of focal epilepsy characterized by spontaneous recurrent seizures originating from the hippocampus. The epigenetic reprogramming hypothesis of epileptogenesis suggests that the development of TLE is associated with alterations in gene transcription changes resulting in a hyperexcitable network in TLE. DNA 5-methylcytosine (5-mC) is an epigenetic mechanism that has been associated with chronic epilepsy. However, the contribution of 5-hydroxymethylcytosine (5-hmC), a product of 5-mC demethylation by the Ten-Eleven Translocation (TET) family proteins in chronic TLE is poorly understood. 5-hmC is abundant in the brain and acts as a stable epigenetic mark altering gene expression through several mechanisms. Here, we found that the levels of bulk DNA 5-hmC but not 5-mC were significantly reduced in the hippocampus of human TLE patients and in the kainic acid (KA) TLE rat model. Using 5-hmC hMeDIP-sequencing, we characterized 5-hmC distribution across the genome and found bidirectional regulation of 5-hmC at intergenic regions within gene bodies. We found that hypohydroxymethylated 5-hmC intergenic regions were associated with several epilepsy-related genes, including Gal, SV2, and Kcnj11 and hyperdroxymethylation 5-hmC intergenic regions were associated with Gad65, TLR4, and Bdnf gene expression. Mechanistically, Tet1 knockdown in the hippocampus was sufficient to decrease 5-hmC levels and increase seizure susceptibility following KA administration. In contrast, Tet1 overexpression in the hippocampus resulted in increased 5-hmC levels associated with improved seizure resiliency in response to KA. These findings suggest an important role for 5-hmC as an epigenetic regulator of epilepsy that can be manipulated to influence seizure outcomes.