mRNA Expression Levels Of Interleukin Research Articles

Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1β,IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1β concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

A Matched Case-Control Study of the Serum Level and Gene Expression of IL-6 and IL-12 in Patients with Schizophrenia Spectrum Disorder: An Azeri Acute Phase/Recent-Onset Psychosis Survey (ARAS) Study

Background: The causes of schizophrenia spectrum disorders are believed to be multifactorial, with both genetic and environmental factors, as well as gene-environment interactions, influencing the course of this disorder. Research suggests that immunological factors may play a role in the development of psychotic disorders. Objectives: The aim of this study is to evaluate and compare the serum levels of interleukin-6 (IL-6) and interleukin-12 (IL-12), as well as the expression levels of IL-6 and IL-12 genes, between an Iranian Azeri population of patients with first-episode psychosis and healthy controls. Methods: Forty patients who had recently experienced first-episode non-affective psychosis were allocated to the patient group. Forty healthy volunteers, matched for age and gender, were also recruited as controls. The study was conducted in 2020 in Tabriz, Iran. Peripheral blood samples were collected (using the Ficoll-Paque process) and assessed to determine the expression levels of IL-6 and IL-12 mRNA (using real-time Polymerase Chain Reaction-PCR- system), as well as the serum levels of IL-6 and IL-12 (with a sandwich ELISA kit). GraphPad Prism Version 6.0 was used for all statistical analyses. Results: Each group consisted of 40 participants in this study. Patients had significantly higher levels of IL-6 mRNA expression compared to the healthy controls (P < 0.0001), while no significant difference was found between the two groups in terms of IL-12 mRNA expression (P = 0.5697). A similar pattern was reported for the serum levels of IL-6 (P < 0.0001) and IL-12 (P = 0.0777). The area under the curve (AUC), which indicates the accuracy of the predictive model, was 0.9669 for the level of IL-6 mRNA expression (P < 0.0001). This Area (AUC) was 0.9584 for the serum concentration of IL-6 (P < 0.0001). The levels of IL-6 or IL-12 were not correlated with the severity of symptoms, gender, or age of the patients. Conclusions: The expression level of IL-6 mRNA and the serum concentration of IL-6 were altered in patients with first-episode non-affective psychosis. This finding supports the role of inflammatory pathways in the pathogenesis of psychosis. According to our results, measurement of the circulating level of IL-6 may be useful in distinguishing patients with first-episode psychosis from healthy individuals.

Study on the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogelin post-traumatic osteoarthritis

Objective: To explore the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogel (cHA-Dex) in inhibiting chondrocyte apoptosis and alleviating early post-traumatic osteoarthritis (PTOA). Methods: To generate PTOA model, anterior cruciate ligament transection (ACLT)was performed on SD rats (n=70), and the sham surgery group (n=70) was set as control. The changes in inflammatory indicators such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) in the joint lavage fluid were measured at different time points (1-14 days, 5 rats at each time point) after surgery. The cHA-Dex (0.5 mg/ml) hydrogel (experimental group, n=70) and ordinary low-molecular-weight hyaluronic acid (HA) hydrogel premixed with Dex, that was, HA-Dex (0.5 mg/ml) hydrogel (control group, n=70) were injected into the joint cavity of PTOA rats, and the release amount and cumulative release amount of Dex in the joint fluid of rats at each time point(1-14 days, 5 rats at each time point) were detected to reveal the release mechanism of cHA-Dex hydrogel. The cartilage of knee joint of patients with osteoarthritis (OA) who underwent knee arthroplasty in the Second Hospital of Shanxi Medical University from January 2020 to December 2022 was taken for in vitro tissue block culture (Outbridge score=1 or 2,n=18). After the cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1, 0.2, and 0.5 mg/ml Dex, the mRNA expression levels of IL-1β, IL-6, TNF-α, MMP-3, and MMP-13 in the articular cartilage tissue block were detected. OA chondrocytes were isolated from cartilage samples using enzymatic hydrolysis and cultured in vitro (n=18). Chondrocytes were divided into 4 groups: saline, cHA hydrogel, Dex (0.5 mg/ml), and cHA-Dex (0.5 mg/ml) hydrogel group. The effects of different interventions on chondrocyte proliferation and apoptosis were tested. Results: The Osteoarthritis Research Society International (OARSI) score of safranine O-solid green staining in PTOA group was 3.34±0.35, and it was 1.17±0.21 in Sham group(P=0.010). The Meachim score of knee joint osteophytes in PTOA rats was significantly higher than that in the Sham group (2.66±0.41 vs 0.22±0.17, P=0.010), indicating PTOA model in rat was established successfully. The cHA-Dex hydrogel, which corresponded to the peak changes of inflammatory factors in the joints of PTOA rats in the early stage, was also released in the early stage and sustained-released in the late stage. After the OA articular cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1, 0.2, and 0.5 mg/ml Dex, the mRNA expression levels of IL-1 β, IL-6, TNF-α, MMP-3, and MMP-13 in the tissue block were reduced significantly (all P<0.05) and in a dose-dependent manner. Compared with Dex (0.5 mg/ml) alone group, the apoptosis rate of cHA-Dex (0.5 mg/ml) hydrogel group was significantly reduced (0.60±0.07 vs 6.63±0.98, P=0.010).Compared with the normal saline or the cHA hydrogel alone group, the cHA-Dex (0.5 mg/ml) hydrogel group had significant cell proliferation, and the difference at each time point were all significant statistically (all P<0.05). Conclusion: For the early inflammation of PTOA, cHA-Dex hydrogel can not only inhibit cartilage inflammation, but also reverse the increased apoptosis and decreased proliferation rate of chondrocytes caused by Dex, and finally alleviate the progress of PTOA by releasing Dex.

Effects of simvastatin on immunoreaction and inflammation in rats with asthma by regulating NOTCH signaling pathway.

To investigate the effect of simvastatin on the immunoreaction and inflammation in rats with asthma through the NOTCH signaling pathway, a total of 36 Sprague-Dawley (SD) rats were enrolled and randomly divided into the normal group (n=12), model group (n=12) and simvastatin group (n=12). The rats in the normal group were fed normally, those in the model group were prepared into models of asthma, and those in the simvastatin group were prepared into models of asthma and intervened with simvastatin. Next, the morphology of airway tissues was observed via hematoxylin-eosin (HE) staining assay. Besides, immunohistochemistry was employed to determine the expression of interferon-γ (INF-γ), and the relative protein expression levels of NOTCH2 and NOTCH3 were measured by Western blotting (WB). Additionally, enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR) assay were carried out to detect the content of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) and the relative mRNA expression levels of INF-γ, IL-6 and TNF-α, respectively. HE staining results uncovered that the airway tissues displayed normal morphology in the normal group and disordered morphology and obvious inflammatory infiltration in the model group. In comparison with the model group, the simvastatin group exhibited significantly improved morphology of airway tissues. Based on immunohistochemistry, the average optical density of INF-γ positive expression was increased in the model group and simvastatin group compared with that in the normal group (P<0.05), and it was distinctly lower in the simvastatin group than that in the model group (P<0.05). The results of WB showed that compared with those in the normal group, the relative protein expression levels of NOTCH2 and NOTCH3 were elevated in model group and simvastatin group (P<0.05), whereas they were overtly reduced in simvastatin group compared with those in model group (P<0.05). It was found through ELISA that the model group and simvastatin group had raised content of IL-6 and TNF-α in comparison with the normal group (P<0.05), while the simvastatin group exhibited markedly decreased content of IL-6 and TNF-α in comparison with the model group (P<0.05). The results of qPCR revealed that the relative mRNA expression levels of INF-γ, IL-6 and TNF-α were distinctly up-regulated in the model group and simvastatin group compared with those in the normal group, displaying statistically significant differences (P<0.05), whereas they were markedly lowered in simvastatin group compared with those in the model group, showing statistically significant differences (P<0.05). Simvastatin represses the immunoreaction and inflammation in rats with asthma by down-regulating the NOTCH signaling pathway.

Mechanism of action of Shaoyao-Gancao decoction in relieving chronic inflammatory pain via Sema3G protein regulation in the dorsal root ganglion

ObjectiveThe purpose of this study was to analyze the impact of Shaoyao-Gancao decoction (SGD) on proteins with significant changes in the dorsal root ganglion (DRG) in rats and to explore the role of the Semaphorin 3G (Sema3G) protein in the DRG and its downstream factors, interleukin-6 (IL-6) and CC-motif chemokine ligand 2(CCL2), in the treatment of chronic inflammatory pain (CIP). MethodsWe created a CIP rat model using 100 μL of complete Freund's adjuvant (CFA) that was injected into the left posterior plantar of rats. Then, we administered SGD intragastrically. We tested the animals for behavioral changes and protein expression levels in DRG pre- and post-drug intervention. ResultsRats in the SGD group showed significantly increased paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and relative expression levels of the Sema3G protein in the DRG (all P < 0.05), while the relative mRNA expression levels of IL-6 and CCL2 in the DRG of the rats were significantly decreased (P < 0.05) when compared with the model group. ConclusionIn this study, we found that Shaoyao-Gancao decoction was effective in improving the PWT and PWL of rats with CIP. It reduced CIP by upregulating the expression of Sema3G in the DRG and inhibiting the relative mRNA expression levels of IL-6 and CCL2.

Protective effects of chlorogenic acid against cyclophosphamide induced liver injury in mice

ABSTRACT We investigated possible protective effects of chlorogenic acid (CGA) against cyclophosphamide (CP) induced hepatic injury in mice. We measured aminotransferase alanine transaminase (ALT) and aspartate transaminase (AST) levels in the serum. We assayed catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in hepatic tissue. We assessed expression of nuclear transcription factor 2 (Nrf2) and Kelch sample related protein-1 (keap1) proteins in hepatic tissues using immunohistochemistry. The relative mRNA expression levels of heme oxygenase-1 (HO-1), NADH quinone oxidoreductase 1 (NQO1), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Hematoxylin & eosin staining was used to assess liver histopathology. We found that administration of CGA prior to induction of injury by CP decreased serum ALT, AST and MDA expressions in hepatic tissue, while CAT, SOD, GSH and GSH-Px concentrations were increased. We found that hepatocytes of animals administered CGA gradually returned to normal morphology. CGA increased the protein expression of Nrf2 in murine hepatic tissue. Administration of CGA up-regulated mRNA expression levels of HO-1, NQO1, TNF-α and IL-6 in hepatic tissue. CGA exhibited a marked protective effect on CP induced liver injury in mice.

Phorbal-12-mysristate-13-acetate-induced inflammation is restored by protectin DX through PPARγ in human promonocytic U937 cells

AimsProtectin DX (PDX), a specialized pro-resolving mediator, is an important pharmaceutical compound with potential antioxidant and inflammation-resolving effects. However, the fundamental mechanism by which PDX's ameliorate chronic inflammatory diseases has not yet been elucidated. This study aims to evaluate the anti-inflammatory properties and PPARγ-mediated mechanisms of PDX in phorbal-12-mysristate-13-acetate (PMA)-stimulated human promonocytic U937 cells. Main methodsWe confirmed the effects of PDX on expressions of pro-inflammatory cytokines, mediators, and CD14 using conventional PCR, RT-qPCR, ELISA, and flow cytometry. Using western blotting, immunofluorescence, and reactive oxygen species (ROS) determination, we observed that PDX regulated PMA-induced signaling cascades. Molecular docking analysis and a cellular thermal shift assay were conducted to verify the interaction between PDX and the proliferator-activated receptor-γ (PPARγ) ligand binding domain. Western blotting was then employed to explore the alterations in PPARγ expression levels and validate PDX as a PPARγ full agonist. Key findingsPDX attenuated protein and mRNA expression levels of interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 in PMA-treated U937 cells. PDX acts as a PPARγ agonist, exerting a modulating effect on the ROS/JNK/c-Fos signaling pathways. Furthermore, PDX reduced human monocyte differentiation antigen CD14 expression levels. SignificancePPARγ exhibits pro-resolving effects to regulate the excessive inflammation. These results suggest that PDX demonstrates the resolution of inflammation, indicating the potential for therapeutic targeting of chronic inflammatory diseases.

Coptisine attenuates sepsis lung injury by suppressing LPS-induced lung epithelial cell inflammation and apoptosis

Objective: This study aimed to investigate the functioning and mechanism of coptisine in acute lung injury (ALI). Methods: Murine Lung Epithelial 12 (MLE-12) cells were stimulated with lipopolysaccharide (LPS) to construct an in vitro pulmonary injury model to study the functioning of coptisine in sepsis-induced ALI. The viability of MLE-12 cells was assessed by the cell counting kit-8 assay. The cytokine release of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and IL-1β was measured by enzyme-linked-immunosorbent serologic assay. The relative expression levels of TNF-α, IL-6, and IL-1β mRNA were examined by reverse transcription-quantitative polymerase chain reaction. The cell apoptosis of MLE-12 cells was determined by Annexin V/propidium iodide staining and analyzed by flow cytometry. The expressions of apoptosis-related proteins Bax and cleaved Caspase-3 were observed by Western blot analysis. The activation of nuclear factor kappa B (NF-κB) signaling pathway was discovered by the determination of phospho-p65, p65, phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and IκBα through Western blot analysis. Results: Coptisine treatment could significantly restore decrease in MLE-12 cell viability caused by LPS stimulation. The release of TNF-α, IL-6, and IL-1β was significantly inhibited by coptisine treatment. Coptisine treatment inhibited MLE-12 cell apoptosis induced by LPS, and also inhibited the expression levels of Bax and cleaved Caspase-3. Coptisine treatment along with LPS stimulation, significantly reduced the protein level of phospho-IκBα, increased the level of IκBα, and reduced phospho-p65–p65 ratio. Conclusion: These results indicated that coptisine attenuated sepsis lung injury by suppressing lung epithelial cell inflammation and apoptosis through NF-κB pathway. Therefore, coptisine may have potential to treat sepsis-induced ALI.

Role and mechanism of IL-6/STAT3/Th17 signaling pathway in the intervention of forsythia in acute lung injury of septic rats

Objective: To investigate the mechanism of interleukin-6 (IL-6) /signal transducers and activators of transcriptional 3 (STAT3) /helper T cell 17 (Th17) signaling pathway in lung injury of rats with sepsis intervened by forsythia, with a view to provide experimental basis for the role and mechanism of forsythia in the treatment of sepsis. Methods: In July 2021, 30 healthy Wistar male rats were selected and randomly divided into sham operation group, model group and treatment group, with 10 rats in each group. The rat model of sepsis was established by cecal ligation and puncture. 2 h after recovery, the rats were given traditional Chinese medicine forsythia orally, twice a day at an interval of 12 h. The wet/dry mass ratio (W/D) of lung tissue was detected 24 h after surgery. The morphological changes of lung tissue were detected by HE staining. Flow cytometry was used to detect Th17 population in peripheral blood. The expression levels of IL-6 and interleukin-17A (IL-17A) in serum were determined by enzyme-linked immunosorbent assay. The mRNA expression levels of IL-17A and IL-6 in lung tissues were detected by reverse transcription-polymerase chain reaction. The expression levels of STAT3 and IL-17A in lung tissue were determined by Western blotting. Results: Compared with model group, the W/D of lung tissue in treatment group was decreased, and the difference was statistically significant (P<0.05). HE staining of lung tissue showed that compared with the model group, the degree of lung lesion and injury was reduced in the treatment group. Compared with sham operation group, the proportion of Th17 cells in CD4 lymphocytes in peripheral blood of rats in model group was significantly increased (P<0.05). Compared with model group, the proportion of Th17 cells in CD4 lymphocytes in peripheral blood of rats in treatment group was significantly decreased (P<0.05). Compared with sham operation group, peripheral blood serum IL-6 and IL-17 of rats in model group were significantly increased (P<0.05). Compared with model group, IL-6 and IL-17 in peripheral blood serum of rats in treatment group were decreased (P<0.05). Compared with sham operation group, the expressions of IL-17A and IL-6 mRNA in lung tissue of model group were significantly increased (P<0.05). Compared with model group, the expressions of IL-17A and IL-6 mRNA in lung tissue of rats in treatment group were significantly decreased (P<0.05). Compared with sham operation group, the protein expressions of STAT3 and IL-17A in lung tissue in model group were significantly increased (P<0.05). Compared with model group, the pritein expressions of STAT3 and IL-17A in lung tissue in treatment group were significantly decreased (P<0.05) . Conclusion: Forsythia plays a role in alleviating lung injury by down-regulating the expressions of the signaling pathway IL-6/STAT3/Th17, which providing a new target for the treatment of sepsis induced lung injury.

Effect of Formononetin on Lipopolysaccharide-Induced Depressive-Like Behaviors and Neuroinflammation in Mice

Abstract Objective The objective of this article is to explore the effect of formononetin (FMN) on depressive-like behaviors and neuroinflammation in lipopolysaccharide (LPS)-induced mice.Methods After acclimatization, male Institute of Cancer Research mice were randomly divided into normal group, LPS group, paroxetine group (20 mg/kg), FMN low-dose group (20 mg/kg, FMN20), and FMN high-dose group (40 mg/kg, FMN40), with eight mice in each group. The depressive-like behaviors were observed by sucrose preference test, tail suspension test (TST), and open field test. The protein and mRNA levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the hippocampus were determined by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction. The expression level of ionized calcium-binding adapter molecule 1 (Iba-1) in the hippocampus was observed by immunofluorescence staining to evaluate the activation level of microglia. Results Compared with the control group, the sucrose preference rate, the activity time of the central area, the distance of the central area, and the number of times of entering the central area were significantly decreased in the LPS group (p &lt; 0.01), and the immobility time of TST was significantly prolonged (p &lt; 0.05), the expression levels of IL-6, IL-1β, and TNF-α protein and mRNA in hippocampus were significantly increased (p &lt; 0.01), and the fluorescence intensity of Iba-1 in CA1, CA3, and DG regions of hippocampus was significantly increased (p &lt; 0.01). Compared with the LPS group, the sucrose preference rate, central area activity time, central area activity distance, and the number of times of entering the central area were significantly increased (p &lt; 0.05 or p &lt;0.01) in the FMN group, and TST immobility time was significantly shortened (p &lt; 0.01), the expression levels of IL-6, IL-1β, and TNF-α protein and mRNA in the hippocampus were significantly decreased (p &lt; 0.05 or p &lt; 0.01), and the fluorescence intensity of Iba-1 in CA1, CA3, and DG regions of hippocampus was significantly decreased (p &lt; 0.01). Conclusion FMN could inhibit LPS-induced activation of microglia, reduce hippocampal neuroinflammation, and improve depressive-like behaviors in mice.

Expression profile of circular RNA in inflammatory response in human bronchial epithelial cells induced by carbon black nanoparticles

Objective: To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells, and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing, so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology. Methods: In June 2020, 16 HBE cells were treated with carbon black nanoparticles at concentrations of 20, 40 and 80 μg/ml, and 16 HBE cells without any intervention were used as the control group. The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments. Real-time quantitative fluorescent PCR (qRT-PCR) and ELISA were used to detect the changes of interleukin-6 (IL-6) and interleukin-8 (IL-6, IL-8) mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure. Western blot analysis was conducted to detect the expression levels of toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (P-NF-κB), apoptosis-related speckled protein (ASC) and Caspase-1 associated with nuclear factor-κB. According to high-throughput sequencing results, differentially expressed Circrnas were screened and identified by qRT-PCR, and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification. Results: After being exposed to carbon black nanoparticles for 72 h, the activity of 16HBE cells decreased significantly (P<0.05), and the release of lactate dehydrogenase increased significantly (P<0.05). Compared with control group, mRNA expression levels of IL-6 and IL-8, protein levels of IL-6 and IL-8 were increased, and protein levels of TLR4, p-NF-κB, ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations, with statistical significance (P<0.05). Compared with the control group, a total of 492 differentially expressed circular Rnas (|log2 FC|>1) were detected. Among the 5 differentially expressed (P<0.05) circular Rnas, circ_002642 was selected as the object of subsequent research on circular Rnas, affter 72 hours of exposure to 80 μg/ml CBNPs, 16HBE cells showed signlficantly higher expression of circ_002642 (P<0.05) . Conclusion: Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.

Effect and mechanism of Bovis Calculus on ulcerative colitis by inhibiting IL-17/IL-17RA/Act1 signaling pathway

This study aimed to elucidate the effect and underlying mechanism of Bovis Calculus in the treatment of ulcerative colitis(UC) through network pharmacological prediction and animal experimental verification. Databases such as BATMAN-TCM were used to mine the potential targets of Bovis Calculus against UC, and the pathway enrichment analysis was conducted. Seventy healthy C57BL/6J mice were randomly divided into a blank group, a model group, a solvent model(2% polysorbate 80) group, a salazosulfapyridine(SASP, 0.40 g·kg~(-1)) group, and high-, medium-, and low-dose Bovis Calculus Sativus(BCS, 0.20, 0.10, and 0.05 g·kg~(-1)) groups according to the body weight. The UC model was established in mice by drinking 3% dextran sulfate sodium(DSS) solution for 7 days. The mice in the groups with drug intervention received corresponding drugs for 3 days before modeling by gavage, and continued to take drugs for 7 days while modeling(continuous administration for 10 days). During the experiment, the body weight of mice was observed, and the disease activity index(DAI) score was recorded. After 7 days of modeling, the colon length was mea-sured, and the pathological changes in colon tissues were observed by hematoxylin-eosin(HE) staining. The levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), and interleukin-17(IL-17) in colon tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA expression of IL-17, IL-17RA, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1β, CXCL1, CXCL2, and CXCL10 was evaluated by real-time polymerase chain reaction(RT-PCR). The protein expression of IL-17, IL-17RA, Act1, p-p38 MAPK, and p-ERK1/2 was investigated by Western blot. The results of network pharmacological prediction showed that Bovis Calculus might play a therapeutic role through the IL-17 signaling pathway and the TNF signaling pathway. As revealed by the results of animal experiments, on the 10th day of drug administration, compared with the solvent model group, all the BCS groups showed significantly increased body weight, decreased DAI score, increased colon length, improved pathological damage of colon mucosa, and significantly inhibited expression of TNF-α,IL-6,IL-1β, and IL-17 in colon tissues. The high-dose BCS(0.20 g·kg~(-1)) could significantly reduce the mRNA expression levels of IL-17, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1β, CXCL1, and CXCL2 in colon tissues of UC model mice, tend to down-regulate mRNA expression levels of IL-17RA and CXCL10, significantly inhibit the protein expression of IL-17RA,Act1,and p-ERK1/2, and tend to decrease the protein expression of IL-17 and p-p38 MAPK. This study, for the first time from the whole-organ-tissue-molecular level, reveals that BCS may reduce the expression of pro-inflammatory cytokines and chemokines by inhibiting the IL-17/IL-17RA/Act1 signaling pathway, thereby improving the inflammatory injury of colon tissues in DSS-induced UC mice and exerting the effect of clearing heat and removing toxins.

Oncostatin M stimulates prostaglandin D2-induced osteoprotegerin and interleukin-6 synthesis in osteoblasts

Oncostatin M produced by osteal macrophages plays a significant role in fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in bone resorption suppression. Interleukin-6 (IL-6) is a pro-inflammatory cytokine and generally regulates bone resorption. However, accumulating evidence suggests that IL-6 plays pivotal roles in bone formation. We previously showed that prostaglandin D2 (PGD2) induces OPG synthesis by activating p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we demonstrated that PGD2 stimulates IL-6 synthesis by activating p38 MAP kinase and p44/p42 MAP kinase in MC3T3-E1 cells. In the present study, we investigated whether oncostatin M affects PGD2-stimulated OPG and IL-6 synthesis in MC3T3-E1 cells through MAP kinase activation. The osteoblast-like MC3T3-E1 cells and normal human osteoblasts were treated with oncostatin M and subsequently stimulated with PGD2. Consequently, oncostatin M significantly increased the PGD2-stimulated OPG and IL-6 release in both cells. Oncostatin M significantly enhanced mRNA expression levels of OPG and IL-6 induced by PGD2 similarly in both cells. Regarding the signaling mechanism, oncostatin M did not affect the phosphorylation of p38 MAP kinase, SAPK/JNK, and p44/p42 MAP kinase. Our results suggest that oncostatin M upregulates the PGD2-stimulated OPG and IL-6 synthesis in osteoblasts and therefore affects bone remodeling. However, OPG and IL-6 synthesis are not mediated through p38 MAP kinase, p44/p42 MAP kinase, or SAPK/JNK pathways.

TXLNG improves insulin resistance in obese subjects in vitro and in vivo by inhibiting ATF4 transcriptional activity

Lipotoxicity contributes to insulin resistance and dysfunction of pancreatic β-cells. Insulin promotes 3T3-L1 preadipocyte differentiation and facilitates glucose entry into muscle, adipose, and other tissues. In this study, differential gene expression was analyzed using four datasets, and taxilin gamma (TXLNG) was the only shared downregulated gene in all four datasets. TXLNG expression was significantly reduced in obese subjects according to online datasets and in high-fat diet (HFD)-induced insulin-resistant (IR) mice according to experimental investigations. TXLNG overexpression significantly improved IR induced by HFD in mouse models by reducing body weight and epididymal adipose weight, decreasing mRNA expression of pro-inflammatory factors interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), and reducing adipocyte size. High-glucose/high-insulin-stimulated adipocytes exhibited decreased TXLNG and increased signal transducer and activator of transcription 3 (STAT3) and activating transcription factor 4 (ATF4). IR significantly decreased glucose uptake, cell surface glucose transporter type 4 (GLUT4) levels, and Akt phosphorylation, while increasing the mRNA expression levels of IL-6 and TNF-α in adipocytes. However, these changes were significantly reversed by TXLNG overexpression, while they were exacerbated by TXLNG knockdown. TXLNG overexpression had no effect on ATF4 protein levels, while ATF4 overexpression increased ATF4 protein levels. Furthermore, ATF4 overexpression notably abolished the improvements in IR adipocyte dysfunction caused by TXLNG overexpression. In conclusion, TXLNG improves IR in obese subjects in vitro and in vivo by inhibiting ATF4 transcriptional activity.

Isoliquiritigenin alleviates the development of alcoholic liver fibrosis by inhibiting ANXA2

The study aimed to investigate the effect of isoliquiritigenin (ISL) on model of alcoholic liver fibrosis (ALF). C57BL/6 mice were used to establish animal model of ALF, HSC-T6 cells were used to establish alcohol-activated cell model, and tandem mass tag (TMT) assays were used to analyze the proteome. The results showed that ISL obviously alleviated hepatic fibrosis in model mice. ISL visually improved the area of liver pathological stasis and deposition of fibrillar collagen (Sirius Red staining, Masson staining), inhibited the mRNA expression levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in liver tissues. ISL down-regulated the mRNA expression levels of IL-6 and transforming growth factor-β1(TGF-β1) in activated hepatic stellate cells (HSCs). And ISL significantly reduced annexin A2 (ANXA2) in vitro detected by TMT proteomics technology. Interestingly, it was found for the first time that ISL could inhibit ANXA2 expression both in vivo and in vitro, block the sphingosine kinases (SPHKs)/sphingosine-1-phosphate (S1P)/interleukin 17 (IL-17) signaling pathway and regulate the expression of α-smooth muscle actin (α-SMA) by inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at the downstream signal to finally reverse HSCs activation and hepatic fibrosis. Thus, we demonstrated that ISL is a drug monomer with notable anti-hepatic fibrosis activity.

Coptisine attenuates sepsis lung injury by suppressing LPS-induced lung epithelial cell inflammation and apoptosis.

This study aimed to investigate the functioning and mechanism of coptisine in acute lung injury (ALI). Murine Lung Epithelial 12 (MLE-12) cells were stimulated with lipopolysaccharide (LPS) to construct an in vitro pulmonary injury model to study the functioning of coptisine in sepsis-induced ALI. The viability of MLE-12 cells was assessed by the cell counting kit-8 assay. The cytokine release of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and IL-1β was measured by enzyme-linked-immunosorbent serologic assay. The relative expression levels of TNF-α, IL-6, and IL-1β mRNA were examined by reverse transcription-quantitative polymerase chain reaction. The cell apoptosis of MLE-12 cells was determined by Annexin V/propidium iodide staining and analyzed by flow cytometry. The expressions of apoptosis-related proteins Bax and cleaved Caspase-3 were observed by Western blot analysis. The activation of nuclear factor kappa B (NF-κB) signaling pathway was discovered by the determination of phospho-p65, p65, phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and IκBα through Western blot analysis. Coptisine treatment could significantly restore decrease in MLE-12 cell viability caused by LPS stimulation. The release of TNF-α, IL-6, and IL-1β was significantly inhibited by coptisine treatment. Coptisine treatment inhibited MLE-12 cell apoptosis induced by LPS, and also inhibited the expression levels of Bax and cleaved Caspase-3. Coptisine treatment along with LPS stimulation, significantly reduced the protein level of phospho-IκBα, increased the level of IκBα, and reduced phospho-p65-p65 ratio. These results indicated that coptisine attenuated sepsis lung injury by suppressing lung epithelial cell inflammation and apoptosis through NF-κB pathway. Therefore, coptisine may have potential to treat sepsis-induced ALI.

Effect of crocin on experimental gastrocnemius muscle ischemia/reperfusion injury in rat.

Ischemia-reperfusion (IR) injury to the lower extremities causes damage to various tissues, notably the limbs. Because research in recent years have demonstrated that saffron and its components are useful in ischemic strokes, the goal of this study was to see whether Crocin (Cr), one of the active constituents in saffron, could protect the gastrocnemius muscle from IR injury. A total number of 32 Sprague-Dawley rats were randomized into four groups randomly: control, Cr, IR, and IR + Cr. Xylazine and ketamine were used to anesthetize all of the rats. The left lower limbs of the other two groups were subjected to 2 hr of ischemia and 2 hr of reperfusion with tourniquet, with the exception of the control and Cr groups. Tumor necrosis factor alfa (TNF-α), interleukin 6 (IL-6), IL-1β, total antioxidant status (TAS) and total oxidant status (TOS) levels were assessed in the blood as well as muscle IL-6, IL1β, SOD1-2, catalase (CAT) and glutathione peroxidase (GPx) expression. According to the IR group, increases in TAS levels and decreases in TNF-α, IL-6, and IL-1β levels were substantial in the Cr therapy group. Cr significantly reduced IL-6 and IL-1β mRNA expression levels in the muscle of the IR group and increased superoxide dismutases 1 (SOD1), SOD2, catalase (CAT), and GPx. Our data showed that Cr protected the gastrocnemius muscle from IR injury in rats and reduced inflammatory markers significantly. These effects of Cr might have been mediated by improved antioxidant enzyme activity, suppression of free radical generation and reduction of oxidative stress.

Correlations of Myeloperoxidase (MPO), Adenosine deaminase (ADA), C–C motif chemokine 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in the nasopharyngeal specimens with the diagnosis and severity of SARS-CoV-2 infections

ABSTRACT Cytokine dynamics in patients with coronavirus disease 2019 (COVID-19) have been studied in blood but seldomly in respiratory specimens. We studied different cell markers and cytokines in fresh nasopharyngeal swab specimens for the diagnosis and for stratifying the severity of COVID-19. This was a retrospective case-control study comparing Myeloperoxidase (MPO), Adenosine deaminase (ADA), C–C motif chemokine ligand 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in 490 (327 patients and 163 control) nasopharyngeal specimens from 317 (154 COVID-19 and 163 control) hospitalized patients. Of the 154 COVID-19 cases, 46 died. Both total and normalized MPO, ADA, CCL22, TNFα, and IL-6 mRNA expression levels were significantly higher in the nasopharyngeal specimens of infected patients when compared with controls, with ADA showing better performance (OR 5.703, 95% CI 3.424–9.500, p < 0.001). Receiver operating characteristics (ROC) curve showed that the cut-off value of normalized ADA mRNA level at 2.37 × 10–3 had a sensitivity of 81.8% and specificity of 83.4%. While patients with severe COVID-19 had more respiratory symptoms, and elevated lactate dehydrogenase, multivariate analysis showed that severe COVID-19 patients had lower CCL22 mRNA (OR 0.211, 95% CI 0.060–0.746, p = 0.016) in nasopharyngeal specimens, while lymphocyte count, C-reactive protein, and viral load in nasopharyngeal specimens did not correlate with disease severity. In summary, ADA appears to be a better biomarker to differentiate between infected and uninfected patients, while CCL22 has the potential in stratifying the severity of COVID-19.

Trans-anethole exerts protective effects on lipopolysaccharide-induced acute jejunal inflammation of broilers via repressing NF-κB signaling pathway

This study aimed to explore the effects of trans-anethole (TA) on lipopolysaccharide (LPS)-induced acute jejunal inflammation model of broilers. A total of 160 one-day-old broilers (male; Arbor Acres) were randomly allocated into four treatment groups with 8 replicates of 5 birds each. On d 20, the dose of 5 mg/kg body weight LPS solution and the equal amount of sterile saline were intraperitoneally injected into LPS-challenged and unchallenged broilers, respectively. Compared with the control group, LPS decreased (P < 0.05) the villus height (VH) and the ratio of villus height to crypt depth (VCR) but increased (P < 0.05) the crypt depth (CD), meanwhile, enhanced (P < 0.01) the levels of interleukin-6 (IL-6), interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) but decreased (P < 0.01) the level of interleukin-10 (IL-10). The group supplemented with 600 mg/kg of TA had lower (P < 0.01) CD and higher (P < 0.01) VCR than the LPS group. TA increased (P < 0.01) the level of IL-10 and decreased (P < 0.01) the level of IL-1β. The mRNA expression levels of IL-6, nuclear factor kappa B (NF-κB), TNF-α were up-regulated (P < 0.05) and the levels of IL-10 and inhibitor of NF-κB alpha (IκBα) were down-regulated (P < 0.05) by LPS as compared with the control group. TA down-regulated (P < 0.05) the increased mRNA expression levels of genes caused by LPS, as well as up-regulated (P < 0.05) the levels of IL-10 and IκBα. Furthermore, LPS down-regulated (P < 0.05) and up-regulated (P < 0.05) the protein expression levels of IκBα and NF-κB p65, respectively. TA up-regulated (P < 0.05) the level of IκBα and down-regulated (P < 0.05) the level of NF-κB p65. The conclusion of this study is that TA could exert protective effect on the LPS-induced acute jejunal inflammation of broilers via repressing the activation of NF-κB and the 600 mg/kg is the optimal dose against LPS-induced acute jejunal inflammation of broilers.

The effects of Ibuprofen and 1, 8- cineol on anxiety and spatial memory in hyperammonemic rats.

In hepatic encephalopathy, hyperammonemia (HA) causes cognitive impairment and anxiety by causing neuroinflammation. Ibuprofen and 1,8- cineol have anti-inflammatory and antioxidant properties, respectively. The aim of this study was to evaluate the effects of ibuprofen alone and in combination with 1,8- cineol on anxiety and oxidative stress in a HA rat animal model. For this purpose, 36 rats were divided into six groups (n = 6) including the HA (received intraperitoneally (IP) ammonium acetate 2.5mg/kg for four week), ibuprofen (induced HA rats that received 15mg/kg, IP), cineol (induced HA rats that received 5 and 10mg/kg, IP), Ib + cineol (induced HA rats that received 15 and 10mg/kg, respectively, IP), and the control groups (received normal saline, IP). Except the HA group, all other groups received the aforementioned treatment for two weeks.. The Morris water maze and elevated plus maze were used to assess cognitive function and anxiety in the animals, respectively. Superoxide dismutase (SOD) activity was measured to evaluate oxidative stress. The mRNA expression levels of interleukin (IL)-6 and IL-1β was assessed by real-time PCR in the animal's brain. The results showed a significant improvement in spatial memory and anxiety of the Ib group compared to the HA group (P < 0.01), but no significant change was observed in SOD activity (P > 0.05). There was a significant improvement in spatial memory and anxiety as well as a significant increase in SOD activity in the Ib + cineol group (P < 0.01) compared to the HA group. These results indicate that the Ib + cineol, not only improve cognitive function and reduce anxiety, also reduce oxidative stress, therefore, the simultaneous use of these two compounds may be useful in improving HA-induced cognitive disorders and anxiety.