Serum IL-6 Levels Research Articles

Cytokine and immunoglobulin profiles of Arbor Acres broiler chickens at different stages of physiological development.

Modern scientific research focuses on a detailed study of the immune system, the mechanisms of immunosuppression, and the search for an effective means to restore disturbed immune homeostasis in farm animals. The present study examined the cytokine and immunoglobulin (Ig) profiles of healthy broiler chickens during physiological development. Arbor Acres broilers (n = 28) were used in the study to achieve this objective. The immune status of broiler chickens was assessed on 7, 14, 28, and 42 days of age, including serum levels of cytokines, Igs, and lysozyme by enzyme immunoassay. We observed a decrease in the efficiency of immune system functioning of birds with increasing age. The most pronounced immunological deficiency in the body of broiler chickens is noted at the age of 7-14 days, which is associated with immaturity of the immune system and is characterized by the fact that non-specific humoral and specific cellular defense factors are at a rather low level. The levels of lysozyme, interleukin (IL)-2, IL-10, and IgA in blood serum at this age were minimal; starting from 28 days of age, there is a specific humoral immune deficiency, which is compensated by strengthening of cellular defense factors. The serum level of IgY intensively decreases against the background of an increase of lysozyme, IL-2, IL-4, and IL-10. During postnatal ontogenesis, the immune system of broiler chickens undergoes dynamic changes that have an age direction and phase character. Changes in the immune system may affect immunocompetence, disease susceptibility, and, consequently, productivity.

Use of Bacterial DNA Concentration in Ascites as a Marker for Spontaneous Bacterial Peritonitis

Background & AimsSpontaneous bacterial peritonitis (SBP) is a common and serious complication in patients with decompensated cirrhosis. Precise quantification of bacterial DNA (bactDNA) and the related inflammatory response might add further information on the course of disease. The aim of the study was to evaluate the association between bactDNA, cytokine levels and clinical outcome. MethodsAscites and serum samples of 98 patients with decompensated liver cirrhosis (42 with SBP and 56 without SBP) as well as serum samples of 21 healthy controls were collected. BactDNA in ascites and serum was detected and quantified by 16S rRNA PCR. Concentrations of IL-1β, TNF-α, IL-6, IL-8 and IL-10 were measured by a LEGENDplexTM multi-analyte flow assay. Clinical data were collected and analyzed retrospectively. ResultsBactDNA was detected more frequently in ascites of patients with SBP (n=24/42; 57.1%) than in ascites of patients without SBP (n=5/56; 8.9%; p < 0.001). Additionally, IL-6 levels in both ascites and serum were significantly higher in patients with SBP (ascites p < 0.001, serum p = 0.036). The quantity of bactDNA in ascites was strongly correlated with polymorphonuclear neutrophil count in ascites (r = 0.755; p < 0.001) as well as ascites IL-6 levels (r = 0.399; p < 0.001). Receiver operating characteristic (ROC) curve analysis to diagnose SBP provided an AUC of 0.764 (95% CI: 0.661 – 0.867) for serum IL-6 levels, an AUC of 0.810 (95% CI: 0.714 - 0.905) for ascites IL-6 levels, and an AUC of 0.755 (95% CI: 0.651 – 0.858) for bactDNA levels in ascites. ConclusionsThe correlation between the amount of bactDNA and IL-6 confirms the pathophysiological relevance of bactDNA and IL-6 as potential biomarkers for the diagnosis of SBP.

Mechanism of total saponins of Panax japonicus against liver injury induced by acetaminophen in mice based on ERK/NF-κB/COX-2 signaling pathway

This study investigated the effects and mechanisms of total saponins of Panax japonicus(TSPJ) against liver injury induced by acetaminophen(APAP). Male Kunming mice were randomly divided into a blank control group, TSPJ group(200 mg·kg~(-1), ig), model group, APAP+ TSPJ low-dose group(50 mg·kg~(-1), ig), APAP+ TSPJ medium-dose group(100 mg·kg~(-1), ig), APAP+ TSPJ high-dose group(200 mg·kg~(-1), ig), and APAP+ N-acetyl-L-cysteine group(200 mg·kg~(-1), ip). The administration group received the corresponding medications via ig or ip once a day for 14 consecutive days. After the last administration for one hour, except for the blank control group and TSPJ group, all groups of mice were given 500 mg·kg~(-1) APAP by gavage. After 24 hours, mouse serum and liver tissue were collected for serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), reactive oxygen species(ROS), tumor necrosis factor alpha(TNF-α), interleukin-1 beta(IL-1β), cyclooxygenase-2(COX-2), IL-6, IL-4, IL-10, as well as lactate dehydrogenase(LDH), glutathione(GSH), superoxide dismutase(SOD), catalase(CAT), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and myeloperoxidase(MPO) liver tissue. Hematoxylin-eosin staining was used to observe the morphological changes of liver tissue. The mRNA expression levels of lymphocyte antigen 6G(Ly6G), galectin 3(Mac-2), TNF-α, IL-1β, COX-2, IL-6, IL-4, and IL-10 in liver tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expression levels of Ly6G, Mac-2, extracellular regulated protein kinases(ERK), phosphorylated extracellular regulated protein kinases(p-ERK), COX-2, inhibitor of nuclear factor κB protein α(IκBα), phosphorylated inhibitor of nuclear factor κB protein α(p-IκBα), and nuclear factor-κB subunit p65(NF-κB p65) in cytosol and nucleus in liver tissue. The results manifested that TSPJ dramatically reduced liver coefficient, serum ALT, AST, ROS, TNF-α, IL-1β, IL-6, and COX-2 levels, LDH, MPO, and MDA contents in liver tissue, and mRNA expressions of TNF-α, IL-1β, and IL-6 in APAP-induced liver injury mice. It prominently elevated serum IL-4 and IL-10 levels, GSH, CAT, SOD, and T-AOC contents, and mRNA expressions of IL-4 and IL-10 in liver tissue, improved the degree of liver pathological damage, and suppressed neutrophil infiltration and macrophage recruitment in liver tissue. In addition, TSPJ lessened the mRNA and protein expressions of neutrophil marker Ly6G, macrophage marker Mac-2, and COX-2 in liver tissue, protein expressions of p-ERK, p-IκBα, and NF-κB p65 in nuclear, and p-ERK/ERK and p-IκBα/p-IκBα ratios and hoisted protein expression of NF-κB p65 in cytosol. These results suggest that TSPJ has a significant protective effect on APAP-induced liver injury in mice, and it can alleviate APAP-induced oxidative damage and inflammatory response. Its mechanism may be related to suppressing ERK/NF-κB/COX-2 signaling pathway activation, thus inhibiting inflammatory cell infiltration, cytokine production, and liver cell damage.

Neutrophils Disrupt B-1a Cell Homeostasis by Targeting Siglec-G to Exacerbate Sepsis

Abstract In sepsis, B-1a cells diminish in the peritoneal cavity (PerC) as they relocate to the spleen via CXCL12/CXCR4. In the spleen, these cells differentiate into plasma cells, skewing from innate to adaptive phenotype. The mechanism governing the PerC B-1a cell relocation in sepsis remains elusive. Sialic-acid-binding immunoglobulin-like lectin (Siglec)-G is a B-1a cell-specific receptor. We found that Siglec-G-/- PerC B-1a cells had a higher migration rate than WT B-1a cells. Siglec-G negatively regulated B-1a cell migration by interacting with CXCR4, as Siglec-G-/- B-1a cells showed increased activation of the CXCR4’s downstream pathway. LPS-activated neutrophils decreased Siglec-G expression on B-1a cells and promoted B-1a cell migration, while NE inhibitor reversed these. Recombinant NE treatment decreased Siglec-G expression by cleavage on B-1a cells. A small Siglec-G-derived decoy peptide (SDP) was developed to prevent Siglec-G cleavage based on the interaction site of NE at the Siglec-G protein in silico. Treatment of SDP significantly reduced the decrease in PerC B-1a cell numbers and Siglec-G expression on B-1a cells in septic mice. SDP treatment significantly reduced serum IL-6 and TNFα levels and improved survival rates in septic mice. In conclusion, we discover Siglec-G to be a new regulator of B-1a cell migration. Preventing NE-mediated Siglec-G cleavage upholds a promising strategy for maintaining peritoneal B-1a cell homeostasis to mitigate sepsis.

Regulation of Inflammation, Without Impacting Heart Rate, via Electrical Stimulation of the Dorsal Motor Nucleus of the Vagus

The vagus nerve plays an important role in neuroimmune interactions and in the regulation of inflammation. Recently, using optogenetics it has been demonstrated that the brainstem’s dorsal motor nucleus of the vagus (DMN) is a significant source of efferent vagus nerve fibers that control inflammation. In contrast to optogenetics, electrical neuronal stimulation is an approved therapeutic approach. However, the anti-inflammatory effectiveness of electrical stimulation of the DMN (eDMNS) and the possible heart rate (HR) alterations associated with this approach have not been investigated. Here, we examined the effects of eDMNS on the HR and cytokine levels in murine endotoxemia as well as the cecal ligation and puncture (CLP) model of sepsis. C57BL/6 mice (8-10 weeks old), under anesthesia and secured in a stereotaxic frame, received eDMNS via a concentric bipolar electrode in the left or right DMN, or sham treatment. eDMNS (50, 250 or 500 μA and 30 Hz, for 1 min) was performed and HR recorded. In endotoxemia experiments, mice underwent sham or 5-minute eDMNS (250 μA or 50 μA) followed by LPS (0.5 mg/kg) i.p. injection. eDMNS was also studied on mice with cervical unilateral vagotomy or sham surgery. In CLP experiments sham or left eDMNS was performed immediately post CLP. Cytokines and corticosterone were measured 90 mins after LPS administration or 24h after CLP. CLP survival was monitored for 14 days. Both left and right eDMNS at 250 μA and 500 μA significantly reduced HR, compared with pre- and post-stimulation, while 50 μA did not. Left-side eDMNS at 50 μA decreased serum and splenic TNF levels, and increased serum IL-10 during endotoxemia. The anti-inflammatory effect of eDMNS was abrogated in mice with ipsilateral unilateral vagotomy, which was also not associated with serum corticosterone alterations. Right-side eDMNS lowered serum TNF levels and increased IL-10 but had no impact on splenic cytokines. In CLP, left eDMNS decreased serum TNF and IL-6 levels, lowered splenic IL-6 and increased splenic IL-10, and improved survival in mice. For the first time, we demonstrate that an eDMNS regimen, which does not cause bradycardia, mitigates LPS-induced inflammation. These effects are dependent on an intact vagus nerve and are unrelated to corticosteroid alterations. Additionally, eDMNS reduces inflammation and enhances survival in a polymicrobial sepsis model. These findings hold promise for further research into bioelectronic anti-inflammatory strategies aimed at the brainstem DMN. This work was partially funded by NIGMS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Utilizing network pharmacology, molecular docking, and animal models to explore the therapeutic potential of the WenYang FuYuan recipe for cerebral ischemia-reperfusion injury through AGE-RAGE and NF-κB/p38MAPK signaling pathway modulation

BackgroundStroke is a debilitating condition with high morbidity, disability, and mortality that significantly affects the quality of life of patients. In China, the WenYang FuYuan recipe is widely used to treat ischemic stroke. However, the underlying mechanism remains unknown, so exploring the potential mechanism of action of this formula is of great practical significance for stroke treatment. ObjectiveThis study employed network pharmacology, molecular docking, and in vivo experiments to clarify the active ingredients, potential targets, and molecular mechanisms of the WenYang FuYuan recipe in cerebral ischemia-reperfusion injury, with a view to providing a solid scientific foundation for the subsequent study of this recipe. Materials and methodsActive ingredients of the WenYang FuYuan recipe were screened using the traditional Chinese medicine systems pharmacology database and analysis platform. Network pharmacology approaches were used to explore the potential targets and mechanisms of action of the WenYang FuYuan recipe for the treatment of cerebral ischemia-reperfusion injury. The Middle Cerebral Artery Occlusion/Reperfusion 2 h Sprague Dawley rat model was prepared, and TTC staining and modified neurological severity score were applied to examine the neurological deficits in rats. HE staining and Nissl staining were applied to examine the pathological changes in rats. Immunofluorescence labeling and Elisa assay were applied to examine the expression levels of certain proteins and associated factors, while qRT-PCR and Western blotting were applied to examine the expression levels of linked proteins and mRNAs in disease-related signaling pathways. ResultsWe identified 62 key active ingredients in the WenYang FuYuan recipe, with 222 highly significant I/R targets, forming 138 pairs of medication components and component-targets, with the top five being Quercetin, Kaempferol, Luteolin, β-sitosterol, and Stigmasterol. The key targets included TP53, RELA, TNF, STAT1, and MAPK14 (p38MAPK). Targets related to cerebral ischemia-reperfusion injury were enriched in chemical responses, enzyme binding, endomembrane system, while enriched pathways included lipid and atherosclerosis, fluid shear stress and atherosclerosis, AGE-RAGE signaling in diabetic complications. In addition, the main five active ingredients and targets in the WenYang FuYuan recipe showed high binding affinity (e.g. Stigmasterol and MAPK14, total energy <−10.5 Kcal/mol). In animal experiments, the WenYang FuYuan recipe reduced brain tissue damage, increased the number of surviving neurons, and down-regulated S100β and RAGE protein expression. Moreover, the relative expression levels of key targets such as TP53, RELA and p38MAPK mRNA were significantly down-regulated in the WenYang FuYuan recipe group, and serum IL-6 and TNF-a factor levels were reduced. After WenYang FuYuan recipe treatment, the AGE-RAGE signaling pathway and downstream NF-kB/p38MAPK signaling pathway-related proteins were significantly modulated. ConclusionThis study utilized network pharmacology, molecular docking, and animal experiments to identify the potential mechanism of the WenYang FuYuan recipe, which may be associated with the regulation of the AGE-RAGE signaling pathway and the inhibition of target proteins and mRNAs in the downstream NF-kB/p38MAPK pathway.

Distinct Neuronal Populations in the Bed Nuclei of the Stria Terminalis (BNST) Control IL1-β - mediated Stress Responses

The immune and nervous systems are intricately linked. However, mechanistic pathways linking cytokine-mediated stress networks in the brain to peripheral immune and cardiac functions remain poorly understood. Here, we identify a specific population of neurons in the bed nucleus of the stria terminalis (BNST), which shape diverse responses to interleukin-1β (IL-1β)-mediated stress phenotype. Using activity-dependent cell labeling in mice, we identify distinct neuronal ensembles in the BNST that are active in response to IL-1β. Interestingly, re-exposure of mice to acute restraint stress results in the labeling of same populations in the BNST that were responsive to IL-1β administration. Chemogenetic reactivation of these IL-1β-responsive neuronal subsets in the BNST is suffcient to broadly retrieve the stress-induced responses. Specifically, reactivation of these IL-1β -responsive neurons in the BNST recapitulates tachycardia and inflammation induced by acute restraint stress, whereas specific ablation of IL-1β-responsive neurons in the BNST attenuates stress-induced serum IL-6 levels and IL-1β-induced tachycardia. Our data define a neuronal population in the BNST that is both necessary and suffcient for mediating IL-1β-associated stress responses, revealing a role for BNST coordination of inflammatory responses. Supported in part by grants from NIH, NIGMS, 1R35 GM118182 to KT and 1R01AR083159-01 to SC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Doxycycline decelerates aging in progeria mice.

Beyond the antimicrobial activity, doxycycline (DOX) exhibits longevity-promoting effect in nematodes, while its effect on mammals is unclear. Here, we applied a mouse model of Hutchinson-Gilford progeria syndrome (HGPS), Zmpste24 knockout (KO) mice, and analyzed the antiaging effect of DOX. We found that the DOX treatment prolongs lifespan and ameliorates progeroid features of Zmpste24 KO mice, including the decline of body and tissue weight, exercise capacity and cortical bone density, and the shortened colon length. DOX treatment alleviates the abnormal nuclear envelope in multiple tissues, and attenuates cellular senescence and cell death of Zmpste24 KO and HGPS fibroblasts. DOX downregulates the level of proinflammatory IL6 in both serum and tissues. Moreover, the elevated α-tubulin (K40) acetylation mediated by NAT10 in progeria, is rescued by DOX treatment in the aorta tissues in Zmpste24 KO mice and fibroblasts. Collectively, our study uncovers that DOX can decelerate aging in progeria mice via counteracting IL6 expression and NAT10-mediated acetylation of α-tubulin.

Exploring the efficacy and mechanism of Bailing capsule to improve polycystic ovary syndrome in mice based on intestinal-derived LPS-TLR4 pathway

Ethnopharmacological relevancePolycystic ovary syndrome (PCOS) is a common endocrine disorder associated with reproductive dysfunction and metabolic abnormalities, particularly characterized by insulin resistance and chronic low-grade inflammation. Multiple clinical studies have clearly demonstrated the significant efficacy and safety of the combination of Bailing capsules (BL) in the treatment of PCOS, but its pharmacological effects and mechanisms still require further study. Aim of the studyTo evaluate the effect of BL on improving PCOS in mice and explore the mechanism. MethodsIn this study, Dehydroepiandrosterone (DHEA) injection was administered alone and in combination with a high-fat and high-sugar diet to induce PCOS-like mouse. They were randomly divided into five groups: normal group (N), PCOS group (P), Bailing capsule low-dose group (BL-L), Bailing capsule high-dose group (BL-H) and Metformin + Daine-35 group (M + D). Firstly, the effects of BL on ovarian lesions, serum hormone levels, HOMA-IR, intestinal barrier function, inflammation levels, along with the expression of IRS1, PI3K, AKT, TLR4, Myd88, NF-κB p65, TNF-α, IL-6, and Occludin of the ovary, liver and colon were investigated. Finally, the composition of the gut microbiome of fecal was tested. ResultsThe administration of BL significantly reduced body weight, improved hormone levels, improved IR, and attenuated pathological damage to ovarian tissues, up-regulated the expression of IRS1, PI3K, and AKT in liver. It also decreased serum LPS, TNF-α, and IL-6 levels, while downregulating the expression of Myd88, TLR4, and NF-κB p65. Additionally, BL improved intestinal barrier damage and upregulated the expression of Occludin. Interestingly, the abundance of norank_f__Muribaculacea and Lactobacillus was down-regulated, while the abundance of Akkermansia was significantly up-regulated. ConclusionThe results of the study showed that BL exerts a treatment PCOS effect, which may be related to the modulation of the gut microbiota, the improvement of insulin resistance and the intestinal-derived LPS-TLR4 inflammatory pathway. Our research will provide a theoretical basis for the clinical treatment of PCOS.

Long-term clinicopathological characteristics of TAFRO syndrome and its relapse: a case series study.

This study aimed to analyze the clinical course of TAFRO syndrome in patients through extended follow-up, focusing on recurrent cases and long-term remission. This was a retrospective case series study. We assessed the clinical course of patients diagnosed with TAFRO syndrome between January 2012 and September 2022 at Toranomon Hospital or Toranomon Hospital Kajigaya, excluding those patients who died during the initial hospitalization. Twelve patients were included. Baseline characteristics, laboratory findings, treatment modalities, and outcomes were assessed. During the median follow-up period of 1474 days, two patients experienced recurrence following a reduction in tocilizumab (TCZ) dose, whereas two achieved remission for >400 days without TCZ treatment. The remaining eight patients maintained remission under the continued TCZ therapy. Recurrence diagnosis was complicated by the non-simultaneous presentation of the five manifestations of TAFRO syndrome. The patients who experienced recurrence showed milder manifestations and faster recovery than the initial onset. Glomerular endotheliopathy was evident in kidney biopsies during recurrence, which was similar to the initial presentation. In a case where only inflammation preceded other manifestation, a kidney biopsy was pivotal in distinguishing TAFRO syndrome relapse from other inflammatory conditions such as infection. Pretreatment serum IL-6 levels were within the reference range only in patients who experienced long-term remission without TCZ treatment. This is the first study to perform kidney biopsies on recurrent TAFRO cases, highlighting recurrence after TCZ dosage reduction, non-simultaneous manifestation of symptoms, the utility of kidney biopsies in recurrence diagnosis, and potential non-IL-6 pathogenesis factors. Pretreatment serum IL-6 levels may help identify patients suitable for maintenance therapy without TCZ. Further investigation is warranted to identify stratified treatment approaches based on individual etiologic factors.

Therapeutic effect of Yinhuapinggan granules mediated through the intestinal flora in mice infected with the H1N1 influenza virus.

In this study, we examined the therapeutic effects of Yinhuapinggan granules (YHPGs) in influenza-infected mice. We also examined how YHPGs affect the composition of the intestinal flora and associated metabolites. We used the nasal drip method to administer the influenza A virus (IAV) H1N1 to ICR mice. Following successful model construction, the mice were injected with 0.9% sterile saline and low (5.5 g/kg), medium (11 g/kg), and high (22 g/kg) doses of YHPGs. The pathological changes in the lungs and intestines were evaluated by gavage for 5 consecutive days. Detection of sIgA, IL-6, TNF-α, INF-γ, and TGF-β cytokine levels in serum by enzyme-linked immunosorbent assay. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to measure the mRNA and protein expression of the tight junction proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) in the colon. To assess the influence of YHPGs on the intestinal microbiota, feces were obtained from the mice for 16s rRNA sequencing, and short-chain fatty acids (SCFAs) were measured in the feces. By reducing the production of pro-inflammatory cytokines and increasing the relative expression of claudin-1, occludin, and ZO-1 in colon tissues, YHPGs had a protective effect in tissues from the lungs and colon. When YHPGs were administered to mice with IAV infection, the relative abundance of Lactobacillus, Coprobacillus, Akkermansia, Prevotella, Oscillospira, and Ruminococcus increased, whereas the relative abundance of Desulfovibrio decreased. The therapeutic mechanism of YHPGs against IAV infection in mice may be underpinned by modulation of the structural composition of colonic bacteria and regulation of SCFA production.

Quantitative and qualitative analysis of stability for 16 serum immunoregulators over 50 freeze-thaw cycles.

To evaluate the reliability of data from the assay of bio-archived specimens, a 50-freeze-thaw-cycle (FTC) degradation study of fresh sera was conducted to test the stability of 16 immunoregulators. Twenty de-identified serum specimens were obtained from volunteers at United Health Services-Wilson Memorial Hospital. Specimens were stored at -20°C and underwent daily 1 h thawing and subsequent freezing for each FTC over 50 consecutive days. Immunoregulator concentrations were assessed via enzyme-linked immunosorbent assay (ELISA) in participant samples at 2 FTC (baseline), 25 FTC, and 50 FTC. Specific immunoregulators observed in the study were C-reactive protein (CRP), interleukin (IL)-1α, 4, 6, 8, 10, monocyte chemoattractant protein-1 (MCP-1, CCL2), monocyte chemoattractant protein-2 (MCP-2, CCL8), eotaxin-1, thymus-and-activation-regulated chemokine (TARC, CCL17), regulated on activation normal T-cell expressed and secreted (RANTES, CCL5), growth-regulated oncogene-alpha (GRO-α, CXCL1), small inducible cytokine A1 (I-309, CCL1), interferon-gamma (IFN-γ), interferon-gamma inducible protein-10 (IP-10, CXCL10), and tumor necrosis factor-alpha (TNF-α). Quantitative stability of serum immunoregulators: Serum CRP, IL-8, IL-10, IFN-γ, IP-10, and eotaxin-1 levels appear to be statistically equivalent from baseline to 50 FTC (p ≤ .05). Retention of patterns in serum immunoregulators: patterns across FTC were retained for TARC (age) and CRP, IFN-γ, and MCP-2 (sex). While the effect of multiple FTC on serum immunoregulator levels may not replicate prolonged freezer storage, the results of this study provide valuable information on the robustness of immunoregulators for research using bio-archived sera.

Revealing the mechanism of Dahuang Huanglian Xiexin Decoction attenuates dysbiosis via IL-17 signaling pathway based on network pharmacology and experimental validation

Ethnopharmacological relevanceDahuang Huanglian Xiexin Decoction (XXD), derived from Zhang Zhongjing's Treatise on Typhoid Fever, has a long history of medicinal use and is widely used for digestive system diseases. It is mainly composed of three natural medicines, including Dahuang (Rheum palmatum L.), Huanglian (Coptis chinensis Franch.), and Huangqin (Scutellaria baicalensis Georgi). Modern pharmacological research shows that the active ingredients of XXD can have a positive effect on intestinal flora regulatory effect, but its mechanism of action is unclear. Aims of this studyClarify the effect of XXD on regulating dysbiosis, and elucidate the mechanism of XXD in alleviating dysbiosis based on network pharmacology, molecular docking and experimental verification. MethodsHistopathological observation and intestinal high-throughput sequencing were used to observe the effect. Preliminary prediction of the mechanism of action of XXD in treating dysbiosis through network pharmacology and molecular docking. Finally, the effect of XXD on the IL-17 signaling pathway was verified through in vivo experiments. ResultsHistopathology and high-throughput sequencing of intestinal flora indicated that XXD has a good regulatory effect on bacterial dysbiosis. At the same time, network pharmacology identified a total of 40 active compounds, 14 of which may be key compounds for XXD to treat dysbiosis. In addition, the study also revealed 14 potential key targets as well as the top 5 therapeutic targets: IL-6, TNF-α, IL-1β, TP53 and PTGS2. GO and KEGG predicted the key pathway for IL-17 signaling pathway to play a role in XXD. In the verification of the prediction results, it was found that the above targets and the IL-17 target showed strong activity in molecular docking. Furthermore, it was found that XXD can reduce the levels of IL-17, IL-6, TNF-α, IL-1β, p53 and COX-2 in serum, while inhibiting the expression of IL-17, IL-17RA, Act-1 and NF-κB protein and the mRNA expression of IL-17, IL-17RA and Act-1 in colon tissue. ConclusionsThis study found that XXD has a good regulatory effect on dysbiosis and its induced symptoms. Network pharmacology was used to predict the key compounds and therapeutic targets of XXD, and preliminary experiments confirmed that XXD may regulate bacterial dysbiosis by inhibiting the IL-17 signaling pathway.

Effects of miR-129-5p on inflammation and nucleus pulposus cell apoptosis in rats with intervertebral disc degeneration through JNK signaling pathway.

This study aimed to explore the effects of miR-129-5p on inflammation and nucleus pulposus (NP) cell apoptosis in rats with intervertebral disc degeneration (IVDD) through the c-Jun N-terminal kinase (JNK) signaling pathway. A total of 20 rats were randomly divided into control group (n=10) or IVDD group (n=10). The mRNA expressions of miR-129-5p and apoptosis index Fas in IVDD tissues were determined using RT-PCR. NP cell apoptosis rate was detected via TUNEL assay. NP cells were extracted from IVDD tissues for primary culture. Subsequently, the cells were transfected with miR-129-5p inhibitor or mimic to inhibit or overexpress miR-129-5p, respectively. Furthermore, the changes in the JNK pathway indexes and apoptosis indexes were detected using Western blotting. In IVDD group, the expression of miR-129-5p was significantly down-regulated, while the transcriptional level of Fas was up-regulated compared with those in control group. Pearson correlation analysis revealed a negative correlation between the expressions of miR-129-5p and Fas mRNA (r=-0.75, P<0.05). IVDD group exhibited significantly higher levels of serum TNF-α, IL-6 and IL-1 than control group. Subsequent TUNEL assay indicated that the apoptosis rate was evidently higher in IVDD group (60.6%) than control group (2.5%). The results of Western blotting showed that the protein expressions of JNK1, JNK2 and Fas remarkably rose in IVDD group compared with those in control group. However, they declined remarkably in miR-129-5p mimic group compared with those in control group. Furthermore, such trends were significantly reversed in miR-129-5p inhibitor group. MiR-129-5p was significantly down-regulated in IVDD, whose overexpression has anti-inflammatory and anti-apoptotic effects.

Chang-Kang-Fang alleviates diarrhea predominant irritable bowel syndrome (IBS-D) through inhibiting TLR4/NF-κB/NLRP3 pathway

Ethnopharmacological relevanceChang-Kang-Fang (CKF), originated from traditional Chinese medicine (TCM) formulas, has been utilized to treat diarrhea predominant irritable bowel syndrome (IBS-D) based on clinical experience. However, the underlying mechanism of CKF for treating IBS-D remains unclear and need further clarification. Aim of the studyThe objective of this present investigation was to validate the efficacy of CKF on IBS-D model rats and to uncover its potential mechanism for the treatment of IBS-D. Materials and methodsWe first established the IBS-D rat model through neonatal maternal separation (NMS) in combination with restraint stress (RS) and the administration of senna decoction via gavage. To confirm the therapeutic effect of CKF on treating IBS-D, abdominal withdrawal reflex (AWR) scores, the quantity of fecal pellets, and the fecal water content (FWC) were measured to evaluate the influence of CKF on visceral hypersensitivity and the severity of diarrhea symptom after the intragastric administration of CKF for 14 days. Subsequently, enzyme linked immunosorbent assay (ELISA) was applied to assess the effect of CKF on neuropeptides substance P (SP) and 5-hydroxytryptamine (5-HT), as well as inflammatory cytokines in serum and in intestinal tissues. Further, colonic pathological changes, the amount of colonic mast cells, and the expression level of occludin in rat colon tissues, were investigated by hematoxylin-eosin (HE) staining, toluidine blue staining, and immunohistochemistry, respectively. To explore the underlying mechanisms, alterations in colonic RNA transcriptomics for the normal, model, and CKF treatment groups were assessed using RNA sequencing (RNA-Seq). Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), and immunofluorescence (IF) assays were applied to validate the effect of CKF on predicted pathways in vivo and in vitro. In addition, to elucidate the potential active compounds in CKF, 11 representative components found in CKF were selected, and their anti-inflammation potentials were evaluated using LPS-treated RAW264.7 cell models. ResultsCKF treatment significantly reduced the number of fecal pellets, attenuated visceral hypersensitivity, and decreased 5-HT and SP concentrations in serum and colon tissues, along with a reduction in colonic mast cell counts, correlating with improved symptoms in IBS-D rats. Meanwhile, CKF treatment reduced the colonic inflammatory cell infiltration, lowered the levels of IL-6, TNF-α, and IL-1β in serum and colon tissues, and increased the occludin protein expression in colon tissues to improve inflammatory response and colonic barrier function. RNA-Seq, in conjugation with our previous network pharmacology analysis, indicated that CKF might mitigate the symptoms of IBS-D rats by inhibiting the Toll like receptor 4/Nuclear factor kappa-B/NLR family pyrin domain-containing protein 3 (TLR4/NF-κB/NLRP3) pathway, which was confirmed by WB, IF, and qRT-PCR experiments in vivo and in vitro. Furthermore, coptisine, berberine, hyperoside, epicatechin, and gallic acid present in CKF emerged as potential active components for treating IBS-D, as they demonstrated in vitro anti-inflammatory effects. ConclusionOur findings demonstrate that CKF effectively improves the symptoms of IBS-D rats, potentially through the inhibition of the TLR4/NF-κB/NLRP3 pathway. Moreover, this study unveils the potential bioactive components in CKF that could be applied in the treatment of IBS-D.

The Role of Cannabinoid-1 Receptor Ligands in the Ovalbumin-Induced Mouse Model of Allergic Asthma: Is It Related to Transient Receptor Potential Vanilloid-1 Channels?

Objectives: The aim of this study was to investigate the role of cannabinoid (CB1) receptors on airway inflammation and hypersensitivity in allergic asthma and the potential interactions with TRPV1 channels. Materials and Methods: BALB/c mice were sensitized and provoked with ovalbumin to create a model of allergic asthma. CB1 selective agonist arachidonoyl 2'-chloroethylamide (ACEA) was administered intraperitoneally at doses of 0.5, 3, and 5 mg/kg. Receptor antagonism studies were performed utilizing selective CB1 antagonists AM251 at a dose of 3 mg/kg. TRPV1 channel was selectively blocked by capsazepine at a dose of 2.5 mg/kg. Penh values were recorded in vivo by a whole-body plethysmograph under methacholine challenge. Inflammatory cell count was performed in bronchoalveolar lavage fluid (BALF). Serum levels of proinflammatory cytokines were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). Inflammation in the lung tissue was scored histopathologically. Statistical significance was determined using one-way analysis of variance or Kruskal-Wallis test and expressed as p<0.05. Results: In sensitized animals, provocation with inhaled ovalbumin increased Penh values, serum interleukin (IL)-4, IL-5, IL-13 levels, eosinophil, neutrophil, lymphocyte, macrophage counts in BALF, and inflammation in the lung tissue. ACEA applications did not significantly alter Penh values, BALF inflammatory cell levels, and histological changes related to inflammation in the lung tissue according to the disease group; however, only at a dose of 5 mg/kg, it reduced the levels of the inflammatory cytokine IL-4. AM251 decreased Penh values, eosinophil and neutrophil migration in BALF, and inflammation score of lung tissue compared with the disease group. Although BALF inflammatory cell levels and Penh values were higher in the AM251+ACEA group than in the AM251 group, the differences were insignificant. In the CPZ+ACEA group, Penh values were significantly higher, and serum IL-4 and IL-13 levels and BALF eosinophil counts were lower than that in the CPZ group. Conclusions: This study demonstrated an important role of the CB1 receptors in allergic asthma. CB1 antagonism reduced airway hyperresponsiveness and inflammation and showed immunomodulatory effects. The effect of the CB1 agonist ACEA on asthma does not appear to be related to TRPV1 channels.

Electrical stimulation of the dorsal motor nucleus of the vagus in male mice can regulate inflammation without affecting the heart rate

BackgroundThe vagus nerve plays an important role in neuroimmune interactions and in the regulation of inflammation. A major source of efferent vagus nerve fibers that contribute to the regulation of inflammation is the brainstem dorsal motor nucleus of the vagus (DMN), as recently shown using optogenetics. In contrast to optogenetics, electrical neuromodulation has broad therapeutic implications. However, the anti-inflammatory effectiveness of electrical stimulation of the DMN (eDMNS) and the possible heart rate (HR) alterations associated with this approach have not been investigated. Here, we examined the effects of eDMNS on HR and cytokine levels in mice administered with lipopolysaccharide (LPS, endotoxin) and in mice subjected to cecal ligation and puncture (CLP) sepsis. MethodsAnesthetized male 8–10-week-old C57BL/6 mice on a stereotaxic frame were subjected to eDMNS using a concentric bipolar electrode inserted into the left or right DMN or sham stimulation. eDMNS (500, 250 or 50 μA at 30 Hz, for 1 min) was performed and HR recorded. In endotoxemia experiments, sham or eDMNS utilizing 250 μA or 50 μA was performed for 5 mins and was followed by LPS (0.5 mg/kg) i.p. administration. eDMNS was also applied in mice with cervical unilateral vagotomy or sham operation. In CLP experiments sham or left eDMNS was performed immediately post CLP. Cytokines and corticosterone were analyzed 90 mins after LPS administration or 24 h after CLP. CLP survival was monitored for 14 days. ResultsEither left or right eDMNS at 500 μA and 250 μA decreased HR, compared with baseline pre-stimulation. This effect was not observed at 50 μA. Left side eDMNS at 50 μA, compared with sham stimulation, significantly decreased serum and splenic levels of the pro-inflammatory cytokine TNF and increased serum levels of the anti-inflammatory cytokine IL-10 during endotoxemia. The anti-inflammatory effect of eDMNS was abrogated in mice with unilateral vagotomy and was not associated with serum corticosterone alterations. Right side eDMNS in endotoxemic mice suppressed serum TNF and increased serum IL-10 levels but had no effects on splenic cytokines. In mice with CLP, left side eDMNS suppressed serum IL-6, as well as splenic IL-6 and increased splenic IL-10 and significantly improved the survival rate of CLP mice. ConclusionsFor the first time we show that a regimen of eDMNS which does not cause bradycardia alleviates LPS-induced inflammation. These eDMNS anti-inflammatory effects require an intact vagus nerve and are not associated with corticosteroid alterations. eDMNS also decreases inflammation and improves survival in a model of polymicrobial sepsis. These findings are of interest for further studies exploring bioelectronic anti-inflammatory approaches targeting the brainstem DMN.

IL-10 and IL-1β Serum Levels, Genetic Variants, and Metabolic Syndrome: Insights into Older Adults' Clinical Characteristics.

Populational aging is marked by chronic noncommunicable diseases, such as metabolic syndrome (MetS). IL-10 and IL-1β are pleiotropic cytokines with multiple biological effects linked to metabolic disorders. This cross-sectional study assessed 193 participants' IL-10 and IL-1β serum levels regarding their role in developing MetS, clinical characteristics, and their IL1B rs1143627 and IL10 rs1800890 variants' genotype frequencies in a population over 60. IL-10 levels correlated weakly with HDL levels and fat mass and inversely with triglycerides, glucose, glycated hemoglobin, and estimated average blood glucose levels. IL-10 levels were also indirectly influenced by the patient's T2DM duration, lean mass amount, and bone mineral content. Participants with altered HDL, elevated serum glucose, raised HbA1c levels, or those over 80 had reduced serum IL-10 levels compared to those with normal levels or other age groups, respectively. Women also had higher serum IL-10 levels than men. Dissimilarly, IL-1β levels correlated directly only with the number of total leukocytes and segmented neutrophils, showing only significant variations with self-reported alcohol consumption. Our study also found that those with the IL10 AA genotype (lower IL-10 levels) had a significantly higher risk of developing MetS. These findings may help direct future research and more targeted therapeutic approaches in older adults.

Ciplukan (Physalis angulata Linn) Bioactivity against Bleomycin-Induced Pulmonary Fibrosis in Mice by reducing Subpleural Fibrosis, KL-6 Level and Anti-Inflammatory properties

Pulmonary fibrosis is a fatal lung disease with unknown pathogenesis and limited treatment options. Herbal medicine began to be developed as an antifibrosis drug for this disease. Ciplukan plant (Physalis angulata Linn.), is a wild plant that has been widely used for generations as traditional Indonesian medicine for various diseases; but has never been studied as an antifibrosis. This study aimed to determine Ciplukan herb ethanol extract (CPL) bioactivity as antifibrosis in pulmonary fibrosis disorders in experimental mice model induced by bleomycin. A total of 35 male mice and 35 female mice of the ddy strain was divided into 7 groups respectively with 1 normal control group and 6 experimental animal models of pulmonary fibrosis induced by bleomycin groups. For the pulmonary fibrosis model, bleomycin (BLM) was injected subcutaneously 8 times with a frequency of twice a week for 4 weeks. Furthermore, the mice were given CPL orally starting at week 6 of treatment with 2 different doses, 1.95mg (CPL-1) and 3.9mg (CPL) every day for 4 weeks. Pulmonary fibrosis histopathology was analyzed using HE and MT staining methods. Serum IL-6, KL-6, and TGF-β1 levels determination was carried out using the ELISA method. The administration of CPL significantly reduced the fibrosis score from 2.80±1.095 to 1.67±0.577µm (p=0.026). The CPL also showed anti-inflammatory activity by reducing IL-6 levels from 1916.20±594.27 to 16.81±17.07pg/mL (p=0.003); TGF-β1 levels from 51.25±2.25 to 22.48±0.93ng/mL (p=0.021); and KL-6 levels from 28.09±2.25 to 13.99±0.93ng/mL (p=0.000). CPL was proven to have pulmonary antifibrotic activity in experimental mice model. The pulmonary antifibrotic effect was evidenced by a decrease in pulmonary fibrosis scores also a decrease in KL-6 levels, IL-6 levels, and TGF-β1. The administration of CPL-1 and CPL-2 can provide recovery of pulmonary fibrosis induced by bleomycin.

Serum Interleukin 6 (IL -6) as Prognostic Marker in Egyptian CLL patients.

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Currently, several biomarkers are being used as CLL prognosticators, including elevated protein levels, elevated RNA levels, gene mutations, and epigenetic changes. Materials and Methods: This study is a prospective study conducted on 55 patients newly diagnosed with CLL, serum IL-6 level was measured initially and after a 6-month treatment course. Correlation with the course of the disease and the known CLL prognostic parameters was done initially and after 6 months. Results: The initial serum IL-6 level in the patient group (pre-treatment) ranges from 36-91 pg/mL (median 57), and in the patient group (post-treatment) ranges from 1-32 pg/mL (median 2). Serum IL-6 level was positively correlated with WBC count, β2 microglobulin, LDH, ESR, B symptoms, Uric Acid, BM Aspirate (% of lymphocytes), and Binet and Rai staging systems. Conclusion: Serum IL-6 is a useful poor prognostic marker in newly diagnosed CLL patients; its prognostic value goes with the other known prognostic markers such as the BM lymphocyte count, ESR, and LDH.