TNF-α Protein Expression Research Articles

Xuanbai Chengqi Decoction alleviates acute lung injury by inhibiting NLRP3 inflammasome

Ethnopharmacological relevanceAcute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a prevalent critical respiratory disorder caused mostly by infection and other factors. However, effective drug therapies are currently lacking. Xuanbai Chengqi Decoction (XCD), a traditional Chinese medicine (TCM) prescription, is commonly employed to treat lung diseases. It has been recommended by Chinese health authorities as one of the TCM prescriptions for COVID-19. Nonetheless, its underlying mechanism for the treatment of ALI has not been fully understood. Aim of the studyThe study aims to investigate the therapeutic effect of XCD on lipopolysaccharide (LPS) -induced ALI in mice and explore its anti-inflammatory mechanism involving pyroptosis. Materials and methodsUltra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was employed to identify the active compounds of XCD, and quantitative analysis of the main compounds was conducted. Male C57BL/6J mice were given different doses of XCD (4.5 and 9.0 g/kg/day) or dexamethasone (5 mg/kg/day) by oral gavage for 5 consecutive days. Subsequently, ALI was induced by injecting LPS (20 mg/kg) intraperitoneally 2 h after the last administration, and serum and lung tissues were collected 8 h later. J774A.1 cells were pretreated with different doses of XCD (100, 200, 400 μg/ml) for 12 h, then incubated with LPS (1 μg/ml) for 4 h and ATP (1 mM) for 2 h to induce pyroptosis. Supernatant and cells were collected. Moreover, J774A.1 cells were transfected with an NLRP3 overexpression plasmid for 24 h, followed by subsequent experiments with XCD (400 μg/ml). Lung histopathological changes were evaluated using hematoxylin and eosin (HE) staining. To assess the efficacy of XCD on ALI/ARDS, the levels of inflammatory factors, chemokines, and proteins associated with NLRP3 inflammasome signaling pathway were evaluated. ResultsXCD was found to ameliorate lung inflammation injury in ALI mice, and reduce the protein expression of TNF-α, IL-1β, and IL-6 in both mouse serum and J774A.1 cell supernatant. Meanwhile, XCD significantly decreased the mRNA levels of IL-1β, pro-IL-1β, CXCL1, CXCL10, TNF-α, NLRP3, NF-κB P65, and the protein expression of NLRP3, Cleaved-Caspase1, and GSDMD-N in the lung and J774A.1 cells. These effects were consistent with the NLRP3 inhibitor MCC950. Furthermore, overexpression of NLRP3 reversed the anti-inflammatory effect of XCD. ConclusionThe therapeutic mechanism of XCD in ALI treatment may involve alleviating inflammatory responses in lung tissues by inhibiting the activation of the NLRP3 inflammasome-mediated pyroptosis in macrophages.

Circ_0000396 suppresses the proliferation and inflammation of rheumatoid arthritis synovial fibroblasts by targeting miR-574-5p/RSPO1 axis

BackgroundCircular RNAs (circRNAs) are important regulators on the onset and progression of rheumatoid arthritis (RA). Our purpose is to explore the role and underpin mechanism of circ_0000396 in RA progression.MethodsRA patients (n = 39) and healthy volunteers (n = 33) were recruited from the Affiliated Hospital of Shaanxi University of Chinese Medicine for the present work. Circ_0000396, microRNA-574-5p (miR-574-5p) and R-spondin 1 (RSPO1) RNA levels were analyzed by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was analyzed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and 5-ethynyl-2′-deoxyuridine (EDU) assay. Cell apoptosis was assessed by flow cytometry. Protein expression levels of proliferating cell nuclear antigen (PCNA), Cyclin D1, Cyclin E1, BCL2-associated × protein (Bax), B-cell lymphoma-2 (Bcl2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and RSPO1 were detected by western blot assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the secretion of pro-inflammatory cytokines including IL-1β and TNF-α. The interaction between miR-574-5p and circ_0000396 or RSPO1 was confirmed by dual-luciferase reporter assay and RNA-pull down assay.ResultsCirc_0000396 expression was notably down-regulated in RA patients compared with healthy controls. Circ_0000396 overexpression suppressed the proliferation and inflammatory response and triggered the apoptosis of RA synovial fibroblasts (RASFs), accompanied by decreases in PCNA, Cyclin D1, Cyclin E1, Bcl2, IL-1β and TNF-α protein expression and an increase in Bax protein expression. Circ_0000396 acted as a molecular sponge for miR-574-5p, and circ_0000396 overexpression-mediated protective effects on RASFs dysfunction were largely reversed by the introduction of miR-574-5p mimics. miR-574-5p interacted with the 3’ untranslated region (3’UTR) of RSPO1, and miR-574-5p negatively regulated RSPO1 expression in RASFs. Circ_0000396 could up-regulate the expression of RSPO1 by sponging miR-574-5p in RASFs. RSPO1 interference largely overturned circ_0000396 overexpression-mediated effects in RASFs.ConclusionCirc_0000396 restrained the proliferation and inflammation and induced the apoptosis of RASFs by mediating miR-574-5p/RSPO1 axis, which provided novel potential targets for RA treatment.

Effect of comprehensive nursing based on evidence-based nursing on reducing the incidence of pressure ulcers in patients undergoing posterior orthopedic surgery.

To analyze the effect of comprehensive nursing based on evidence-based nursing during the perioperative period on reducing the incidence of pressure ulcers in patients undergoing posterior orthopedic surgery. Data on 120 patients who underwent orthopedic posterior surgery in our hospital from February 2021 to December 2022 were retrospectively analyzed. The patients were divided into an observation group (n = 60) and a control group (n = 60) based on different nursing methods. Patients in the control group received routine nursing, whereas those in the observation group received comprehensive nursing under the guidance of the concept of evidence-based nursing. The incidence of postoperative pressure ulcer was also recorded. Fasting venous blood (5 mL) was collected from patients before and after surgery and used to measure levels of myeloperoxidase (MPO) and superoxide dismutase (SOD) using enzyme-linked immunosorbent assay. Ulcer tissue samples of patients with pressure ulcers were collected and used to detect the expression of caspase-3 protein, vascular endothelial growth factor (VEGF) mRNA, tumor necrosis factor-α (TNF-α) mRNA, and interleukin-1β (IL-1β) mRNA. The incidence of postoperative pressure ulcers was 8% in the observation group and 23% in the control group (P = .024). The scores of sensory perceptions of the patients in the observation group were significantly lower than those in the control group (P < .001), as were the scores for moisture (P < .001), activity (P = .008), mobility (P < .001), nutrition (P = .003), friction, and shear (P < .001). After surgery, the serum MPO level in the observation group was significantly lower than that in the control group (P < .001), whereas the SOD level in the observation group was significantly higher than that in the control group (P < .001). The expression of TNF-α, IL-1β, VEGF mRNA, and caspase-3 protein in pressure ulcer tissues in the observation group was significantly lower than that in the control group. Comprehensive nursing based on the concept of evidence-based nursing can significantly reduce the incidence of postoperative pressure ulcers following posterior orthopedic surgery.

Mechanism of Yushenhuoxue prescription in treating endometriosis based on network pharmacology and the effect on the TNF pathway

Endometriosis is a common disease in the field of gynaecology, exhibiting clinical manifestations such as dysmenorrhoea, pelvic masses, and infertility, affecting 2–10% of women of reproductive age worldwide. Currently, the acceptance rate of hormonal drugs in patients is low and certain side effects exist. In this study, based on network pharmacology, it was found that the Yushenhuoxue (YSHX) formula could potentially affect endometriosis through the TNF signalling pathway. Clinical studies indicated that YSHX demonstrated the ability to reduce the vas score of dysmenorrhoea, resulting in a significant down-regulation of serum ca125 and inflammatory factors (IL-6, IL-1β, TNF-α). In vivo studies showed that stem cell mice in the YSHX group exhibited significantly reduced lesion volumes than those in the model group. Serum levels of IL-1β and IL-6 were significantly decreased. Moreover, the phosphorylation levels of NF-κB p65 and the expression of TNF-α protein were significantly decreased. In vitro studies have shown that YSHX inhibits the proliferation, invasion, and migration of endometriotic cells. This study partially verified that YSHX contributed to the treatment of endometriosis by regulating the TNF signalling pathway and improving the inflammatory state of endometriosis.

Potential value of Interleukin-6 as a diagnostic biomarker in human MDD and the antidepressant effect of its receptor antagonist tocilizumab in lipopolysaccharide-challenged rats

Depression is a common mental disease with disastrous effect on the health and wealth globally. Focusing on the role for inflammation and immune activation in the pathogenesis of depression, many tries have been taken into effect targeting at the blockage of inflammatory cytokines, among which interleukin- 6 (IL-6) and its receptor antagonist tocilizumab attracts more attention, with inconsistent findings. Moderate to severe depressive disorder (MSDD) patients were enrolled and the serum concentrations of IL-6 and tumor necrosis factor-α (TNF-α) measured, their correlation with the Hamilton Depression Rating Scale-24 (HAMD-24) scores was analyzed, and their role in discriminating MSDD patients from the health controls were evaluated. Meanwhile, a depression rat model was established by intraperitoneal injection of LPS, and tocilizumab was administrated doing 50 mg/kg via intravenous injection. The behavioral performance was observed, the serum concentration of IL-6, TNF-α, and C-reactive protein (CRP) was measured, and the protein expression of IL-6 and TNF-α in the hippocampus were also detected. The activity of the Hypothalamic-pituitary-adrenal (HPA) axis was observed, and the protein expression levels in the hippocampus were detected via western blot. Moreover, the immunofluorescence staining (IF) technique was used to investigate the co-location of IL-6 and neuron (MAP2), astrocyte (GFAP), or microglial (IBA-1). The results showed that the serum IL-6 level was significantly increased in the MSDD patients and lipopolysaccharide (LPS)-challenged rats, with a significant correlation with the HAMD-24 scores or struggling time in the FST and corticosterone (CORT) abundance. Results of ROC analysis showed a significant diagnosis value of IL-6 in discriminating MSDD patients or depression rats from the controls in the present study. Tocilizumab could relieve the depression-like behaviors induced by LPS, together with a normal abundance of serum CORT and hypothalamic CRH expression. Moreover, tocilizumab could alleviate the “inflammatory storm” and impaired hippocampal synaptic plasticity in LPS-challenged depression rats, inhibiting the hyperactivation of astrocyte and microglia, decreasing the peripheral and central abundance of IL-6, CRP, and TNF-α, and balancing the hippocampal expression levels of synaptic plasticity-associated proteins and key molecular in Wnt/β-catenin signaling pathway. These results indicated a predictive role of IL-6 in discriminating depression from controls, and demonstrated an antidepressant effect of tocilizumab in LPS-challenged rats, targeting at the inflammatory storm and the subsequent impairments of hippocampal synaptic plasticity.

Pro-inflammatory regulatory mechanism of C5a/C5aR pathway in GCRV-infected grass carp (Ctenopharyngodon idella)

Death of grass carp (Ctenopharyngodon idella) caused by grass carp reovirus (GCRV) infection severely restricts the development of the grass carp industry. The interaction of C5a and C5a receptor (C5aR) in the complement system promotes inflammation and aggravates bleeding in grass carps. To reveal the regulatory mechanism of the C5a/C5aR pro-inflammatory response after GCRV infection, the expression of C5 in the liver and muscle tissues of grass carp after GCRV infection was analyzed using immunofluorescence. The effects of over expression of C5a and the C5aR antagonist PMX205 in grass carp kidney cell line (CIK) on the phosphorylation of p38 and ERK and the gene and protein expression of IL-6 and TNF-α were also analyzed using RT-qPCR and western blot. The results showed that the expression of the C5a protein in the liver and muscle of grass carp infected with GCRV first increased and then decreased, and the C5a protein level peaked on the 5th day after infection. In CIK cells, over expression of C5a significantly increased C5aR expression, phosphorylation of p38 and ERK, and overexpression of IL-6 and TNF-α. Treatment of CIK cells with PMX205 significantly decreased the expression of C5a and C5aR, phosphorylation of p38 and ERK proteins, and expression of IL-6 and TNF-α after GCRV infection. These results implied that the interaction of C5a and C5aR in grass carp resulted in the phosphorylation of p38 and ERK proteins, a significant increase in the expression of IL-6 and TNF-α, and ultimately induction of an inflammatory response of grass carp infected with GCRV. These results provide a theoretical basis for relieving grass carp hemorrhagic diseases by regulating the interaction of C5a and C5aR.

Azilsartan inhibits inflammation-triggered bone resorption and osteoclastogenesis in vivo via suppression of TNF-α expression in macrophages.

Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.

DDAH1 Protects against Cardiotoxin-Induced Muscle Injury and Regeneration.

Nitric oxide (NO) is an important biological signaling molecule affecting muscle regeneration. The activity of NO synthase (NOS) is regulated by dimethylarginine dimethylaminohydrolase 1 (DDAH1) through degradation of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). To investigate the role of DDAH1 in muscle injury and regeneration, muscle-specific Ddah1-knockout mice (Ddah1MKO) and their littermates (Ddah1f/f) were used to examine the progress of cardiotoxin (CTX)-induced muscle injury and subsequent muscle regeneration. After CTX injection, Ddah1MKO mice developed more severe muscle injury than Ddah1f/f mice. Muscle regeneration was also delayed in Ddah1MKO mice on Day 5 after CTX injection. These phenomena were associated with higher serum ADMA and LDH levels as well as a great induction of inflammatory response, oxidative stress and cell apoptosis in the gastrocnemius (GA) muscle of Ddah1MKO mice. In the GA muscle of CTX-treated mice, Ddah1 deficiency decreased the protein expression of M-cadherin, myogenin, Bcl-2, peroxiredoxin 3 (PRDX3) and PRDX5, and increased the protein expression of MyoD, TNFα, Il-6, iNOS and Bax. In summary, our data suggest that DDAH1 exerts a protective role in muscle injury and regeneration.

Effect of spirocyclopiperazinium salt compound LXM-15 on spinal nerve injury in rats.

Currently available therapies for neuropathic pain show limited efficacy. This study aimed to investigate the anti-nociceptive effect of the spirocyclopiperazinium salt compound LXM-15 in spinal nerve ligation (SNL) rats and to explore the potential mechanisms. Mechanical allodynia and thermal hyperalgesia tests were used to evaluate the effects of LXM-15 in SNL rats. The expression of CaMKIIα, CREB, JAK2, STAT3, c-fos and TNF-α was detected by western blotting, ELISA or qRT-PCR analysis. Receptor blocking test was performed to explore possible target. Administration of LXM-15 (1, 0.5, 0.25 mg/kg, i.g.) dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in rats subjected to SNL (p < 0.01, p < 0.05), and the effects were completely blocked by peripheral α7 nicotinic or M4 muscarinic receptor antagonist (p > 0.05). LXM-15 significantly decreased the overexpression of phosphorylated CaMKIIα, CREB, JAK2 and STAT3 proteins and the mRNA levels of TNF-α and c-fos (p < 0.01, p < 05). All of the effects could be blocked by α7 or M4 receptor antagonist. Furthermore, LXM-15 reduced the protein expression of TNF-α and c-fos (p < 0.01, p < 0.05). No significant acute toxicity or abnormal hepatorenal function was observed. This is the first study to report that LXM-15 exerts significant anti-nociceptive effect on SNL rats. This effect may occur by activating peripheral α7 nicotinic and M4 muscarinic receptors, further inhibiting the CaMKIIα/CREB and JAK2/STAT3 signalling pathways, and finally inhibiting the expression of TNF-α and c-fos. Existing treatments for neuropathic pain show limited efficacy with severe adverse reactions. This paper is the first to report that LXM-15, a new spirocyclopiperazinium salt compound, exerts a significant anti-nociception in SNL rats without obvious toxicity. The underlying mechanisms include activating peripheral α7 nicotinic and M4 muscarinic receptors, then inhibiting the signalling pathways of CaMKIIα/CREB and JAK2/STAT3 and the expressions of TNF-α and c-fos. This study sheds new light on the development of novel analgesic drugs with fewer side effects.

Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

Elucidating the chemical interaction effects of herb pair Danshen-Chuanxiong and its anti-ischemic stroke activities evaluation

Ethnopharmacological relevanceSalvia miltiorrhiza Bunge (Danshen) and Ligusticum chuanxiong Hort. (Chuanxiong) is the core herb pair in traditional Chinese medicines (TCMs) formulae for treating ischemic stroke. However, the synergistic effect of Danshen-Chuanxiong against anti-ischemic stroke and its compatibility mechanism remains unclear. Aim of the studyThis study aimed to uncover the compatibility mechanism of Danshen-Chuanxiong against ischemic stroke through chemical profiling, pharmacodynamics evaluation, network pharmacology and experimental validation. Materials and methodsUltra-high performance liquid chromatography (UHPLC) combined with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS) and UHPLC connected with tandem triple quadrupole mass spectrometry (QQQ-MS) were utilized to conduct the chemical interaction analysis. Then the synergistic effects of Danshen-Chuanxiong against ischemic stroke were comprehensively evaluated by the middle cerebral artery occlusion reperfusion (MCAO/R) mice model, zebrafish ischemic stroke model and glutamic acid-induced PC12 cells injury model. Afterwards, network pharmacology and molecular docking were applied to dissect the significant active compounds and potential mechanisms. Finally, the key target proteins were experimentally validated by Western blot. Results83 compounds were characterized in Danshen-Chuanxiong by UHPLC-QTOF-MS analysis, and 4 compounds were tentatively identified for the first time. The quantification results (24 accurately identified compounds) in 13 proportions of Danshen-Chuanxiong revealed that Danshen significantly increased the dissolution of most phthalides (from Chuanxiong), while Chuanxiong facilitated the dissolution of most phenolic acids (from Danshen) in solution. The anti-ischemic stroke effects of Danshen-Chuanxiong were significantly better than Danshen or Chuanxiong in attenuating infarct size, reducing brain edema and neurological scores in MCAO/R mice. Also, compared with single herbs, this herb pair exerted better effects of suppressing the incidence of cerebral thrombosis in zebrafish, and increasing the cell viability of glutamic acid-induced PC12 cells. In network pharmacology, 7 effective compounds (rosmarinic acid, chlorogenic acid, salvianolic acid B, (Z)-ligustilide, ferulic acid, caffeic acid, tanshinone IIA) and 5 hub targets (AKT, TNF−α, IL-1β, CASP3 and BCL2) as well as 4 key pathways were predicted. Western blot results showed that Danshen-Chuanxiong exert therapeutic effects mainly through decreasing the protein expressions of TNF-α, IL-1β and Cleaved-CASP3, elevating the levels of p-AKT and BCL2. ConclusionsThis work provided an integration strategy for uncovering the synergistic effects and compatibility mechanism of Danshen-Chuanxiong herb pair for treating ischemic stroke, and laid foundation for the further development and utilization of this herb pair.

Remote ischemic conditioning attenuates oxidative stress and inflammation via the Nrf2/HO-1 pathway in MCAO mice

The protective effects of remote ischemic conditioning (RIC) on acute ischemic stroke have been reported. However, the protective mechanisms of RIC have not been fully elucidated. This study aimed to investigate whether RIC could reduce oxidative stress and inflammatory responses in middle cerebral artery occlusion (MCAO)-reperfusion mice via the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. C57BL/6 mice were subjected to MCAO and underwent RIC twice daily at 1, 3, and 7 days after MCAO. ML385 was used to specifically inhibit Nrf2 in MCAO mice. Neurological deficit scores, infarct volume, and hematoxylin-eosin (HE) staining were assessed. Oxidative stress levels were assessed based on total antioxidant capacity (TAC), malonaldehyde (MDA), superoxide dismutase (SOD), and glutathione/glutathione disulfide (GSH/GSSG). mRNA levels were detected using real-time polymerase chain reaction (PCR), and protein levels were detected using western blotting and enzyme-linked immunosorbent assay (ELISA). Protein localization was investigated using immunofluorescence staining. RIC significantly reduced infarct volume and improved neurological function and histological changes after MCAO. RIC significantly increased TAC, SOD, and GSH/GSSG levels and decreased MDA levels. RIC significantly increased Nrf2 and HO-1 mRNA levels and decreased Keap1, NLRP3, and Cleaved Caspase-1 mRNA levels. RIC significantly increased Nrf2, HO-1, and NQO1 protein expression and decreased Keap1, NLRP3, Cleaved Caspase-1, Cleaved IL-1β, IL-6, and TNF-α protein expression. RIC promoted the activation and translocation of Nrf2 into the nucleus. The protective effects of RIC were abolished by ML385 treatment. In conclusion, our findings suggest that RIC alleviates oxidative stress and inflammatory responses via the Nrf2/HO-1 pathway, which in turn improves neurobehavioral function. RIC may provide novel therapeutic options for acute ischemic stroke.

Effects of Pera Orange Juice and Moro Orange Juice in Healthy Rats: A Metabolomic Approach.

Sixty male Wistar rats were allocated into 3 groups: control (C), Pera orange juice (PO), and Moro orange juice (MO). PO and MO groups received Pera orange juice or Moro orange juice, respectively, and C received water with maltodextrin (100 g/L). Echocardiogram and euthanasia were performed after 4 weeks. Plasma metabolomic analysis was performed by high-resolution mass spectrometry. Type I collagen was evaluated in picrosirius red-stained slides and matrix metalloproteinase (MMP)-2 activity by zymography. MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-4, type I collagen, and TNF-α protein expression were evaluated by Western blotting. We differentially identified three metabolites in PO (N-docosahexaenoyl-phenylalanine, diglyceride, and phosphatidylethanolamine) and six in MO (N-formylmaleamic acid, N2-acetyl-L-ornithine, casegravol isovalerate, abscisic alcohol 11-glucoside, cyclic phosphatidic acid, and torvoside C), compared to controls, which are recognized for their possible roles in cardiac remodeling, such as extracellular matrix regulation, inflammation, oxidative stress, and membrane integrity. Cardiac function, collagen level, MMP-2 activity, and MMP-9, TIMP-2, TIMP-4, type I collagen, and TNF-α protein expression did not differ between groups. Ingestion of Pera and Moro orange juice induces changes in plasma metabolites related to the regulation of extracellular matrix, inflammation, oxidative stress, and membrane integrity in healthy rats. Moro orange juice induces a larger number of differentially expressed metabolites than Pera orange juice. Alterations in plasma metabolomics induced by both orange juice are not associated with modifications in cardiac extracellular matrix components. Our results allow us to postulate that orange juice may have beneficial effects on pathological cardiac remodeling.

Heritability of Immunity Traits and Resistance of Atlantic Salmon against the Sea Louse Caligus rogercresseyi.

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h2 skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h2 skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h2 skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon.

Proinflammatory and Immunomodulatory Gene and Protein Expression Patterns in Spinal Cord and Spleen Following Acute and Chronic High Thoracic Injury.

In addition to paralysis and loss of sensation, high-level spinal cord injury (SCI) causes sympathetic dysfunction that can lead to autonomic dysreflexia (AD) and chronic immune suppression involving splenic leukopenia. Evidence has shown that treatment with either gabapentin or blockade of TNFα mitigates maladaptive plasticity and the underlying hemodynamic dysfunction, spleen atrophy, and immune dysfunction associated with AD. Because significant improvements long term was noted following treatments only during acute stages of recovery, we sought to systematically examine changes in proinflammatory and immunomodulatory cytokines to ascertain the reason. Adult female Wistar rats underwent complete T4 spinal transection before euthanasia at systematic intervals from 3 days to 8 weeks after injury. Using qRT-PCR and meso scale discovery (MSD) assays, the gene and protein expression of TNFα and IFNγ in the spleen, upper thoracic (T4-9) and lumbosacral (L5-S6) spinal cords were analyzed. We found that spleen atrophy occurs in a biphasic manner compared to naïve controls, with significant decreases in the spleen mass noted at 3 days and 8 weeks after injury. Splenic TNFα mRNA and protein levels did not change significantly over time, while IFNγ gene expression dipped acutely with trends for increased protein levels at more chronic time points. TNFα protein increased significantly only in thoracic spinal cord segments from 3 to 14 days post-injury. IFNγ mRNA and protein levels remained unelevated in injured spinal cords over time, with trends for increased protein levels at 2 and 8 weeks in the lumbosacral segments. Novel temporal-spatial cytokine expression profiles reveal that TNFα protein levels are increased solely in upper thoracic segments after high thoracic SCI, while IFNγ remains unaltered. Splenic leukopenia and latent systemic immunosuppression are not associated with altered TNFα or IFNγ expression in the spleen or spinal cord.

Effects and mechanisms of total flavones of Abelmoschus manihot in inhibiting podocyte necroptosis and renal fibrosis in diabetic kidney disease

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-β1(TGF-β1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.

Thymoquinone exposure on BV2 microglial cell line: an in vitro study on cell viability, lactate dehydrogenase activity, microglial morphological changes, and TNF-α protein expression

Thymoquinone exposure on BV2 microglial cell line: an in vitro study on cell viability, lactate dehydrogenase activity, microglial morphological changes, and TNF-α protein expression

The Effect of Training Type on The Signaling P athway of Ceramide-Dependent Insulin Resistance in The Flexor Hallucis Longus Muscle of Streptozotocin-Induced Diabetic Rats.

This study aimed to compare the effect of different physical training on the mechanism of ceramidedependent insulin resistance in the flexor hallucis longus (FHL) muscle of diabetic rats. In this experimental study, 7 healthy as a healthy control (HC) group, and 21 diabetics (55 mg/ kg Streptozotocin) Wistar rats (200-220 g; 8-10 weeks old) divided into the diabetic control (DC), moderate continuous training (MCT), and moderate intensity interval training (MIIT) groups. Both MCT (55-70% of maximal oxygen uptake (VO2max), and MIIT (85% VO2max) groups trained for 10-25 minutes at a speed of 10-20 m/minutes. The changes in the expression of blood glucose, insulin, insulin resistance, lipid profile and total ceramide were measured as well as ceramide synthase-1, Glucose transporter type 4 (GLUT4), Protein kinase B known as Akt, phosphorylated protein kinase B known as pAkt, protein kinase C (PKC), and tumour necrosis factor α (TNFα). Blood glucose, triglyceride (TG) and ceramide synthase-1 (CS1) expression levels in the MCT group decreased in comparison with the DC group. FHL protein expression of GLUT4 in the MCT group was higher than the DC group. FHL expression of GLUT4, pAKT, AKT/pAKT, PKC, CS1 and total ceramide in the MIIT group were higher than the DC group. Cholesterol, low-density lipoprotein (LDL), TG, and TNF-α protein expression in the MIIT group were lower than the DC group. GLUT4, PKC, pAKT, AKT/pAKT in the MIIT group were higher, and total ceramide and TNF-α were lower in the MIIT group than the MCT group. It seems that both training plan MIIT and MCT have favorable effects on the metabolism of glucose, insulin, lipids, and the decrease of TNFα level in the diabetes, but in connection with the improvement of the ceramides mechanism, it seems that the MIIT training plan is more optimal than MCT training plan.

Ascorbic acid and IFNγ preconditioning enhance the potency of human mesenchymal stem cells to ameliorate LPS induced cytokine storm

The mesenchymal Stem Cells (MSCs) is one of the leading contender in therapeutic management of cytokine storm implicated in the COVID-19 and other inflammatory conditions. This study was aimed to investigate the effect of Interferon gamma (IFN-γ) and Ascorbic Acid (AA) preconditioning on the secretome of the human Umbilical Cord Derived MSCs (UCMSCs) and their potential to ameliorate the lipopolysaccharide (LPS) induced cytokine storm in the human peripheral blood mononuclear cells (PBMCs). UCMSCs were preconditioned with IFN-γ, AA and secretome (UCMSCs-S, IFNγ-UCMSCs-S and AA-UCSMCs-S) was analysed for the levels of growth factors and cytokines by flow cytometry. The potential of secretome to ameliorate cytokine storm and augment angiogenesis was assessed in the LPS induced PBMCs and yolk sac membrane (YSM) assay respectively. The mRNA transcript and protein levels of IL-6, IL-1β and TNF-α was analysed by RT-PCR and flow cytometry respectively. IFNγ-UCMSCs-S and AA-UCSMCs-S ameliorated the LPS induced cytokine storm as revealed by the decreased mRNA and protein expression of IL-6, IL-1β and TNF-α as compared to the UCMSCs-S. IFNγ-UCMSCs-S and AA-UCSMCs-S augmented angiogenesis in YSM assay. Furthermore, IFNγ and AA preconditioning of UCMSCs exhibited distinct growth factors and cytokine profile in the secretome. Our results unequivocally show that IFNγ and AA preconditioning of MSCs could give better therapeutic outcomes in the cell mediated therapies for COVID-19 and other inflammatory conditions.

Intravesical BCG in bladder cancer induces innate immune responses against SARS-CoV-2.

BCG is the most efficient adjuvant therapy for high-risk, non-muscle-invasive bladder cancer (NMIBC). Both innate and adaptive immune responses have been implicated in BCG-mediated effects. BCG vaccination can boost innate immune responses via trained immunity (TI), resulting in an increased resistance to respiratory viral infections. Here we evaluated for the first time whether intravesical application of BCG triggers increased immunity against SARS-CoV-2 in patients with high-risk NMIBC. Serum and peripheral blood mononuclear cells (PBMCs) from heparinized whole blood samples of 11 unvaccinated SARS-CoV-2-naïve high-risk NMIBC patients were collected at baseline and during BCG treatment in a pre-COVID-19 era. To examine B-cell or T cell-dependent adaptive immunity against SARS-CoV-2, sera were tested for the presence of SARS-CoV-2 neutralizing antibodies. Using a SARS-CoV-2 peptide pool, virus-specific T cells were quantified via IFNγ ELISpot assays. To analyze innate immune responses, mRNA and protein expression levels of pro- and anti-inflammatory cytokines were measured after a 24-hour stimulation of PBMCs with either BCG or SARS-CoV-2 wildtype. ATAC- sequencing was performed to identify a potential epigenetic reprogramming in immune cells. We neither identified SARS-CoV-2 neutralizing antibodies nor SARS-CoV-2- reactive T cells, indicating that intravesical BCG did not induce adaptive immunity against SARS-CoV-2. However, a significant increase in mRNA as well as protein expression of IL-1β, IL-6 and TNFα, which are key cytokines of trained immunity, could be observed after at least four intravesical BCG instillations. Genomic regions in the proximity of TI genes (TLR2, IGF1R, AKT1, MTOR, MAPK14, HSP90AA1) were more accessible during BCG compared to baseline. Although intravesical BCG did not induce adaptive immune responses, repetitive intravesical instillations of BCG induced circulating innate immune cells that produce TI cytokines also in response to SARS-CoV-2.