Content Of Inflammatory Factors Research Articles

USP7 alleviates neuronal inflammation and apoptosis in spinal cord injury via deubiquitinating NRF1/KLF7 axis

ABSTRACT Background Ubiquitin-specific protease 7 (USP7) has been found to be associated with motor function recovery after spinal cord injury (SCI). Therefore, its role and mechanism in SCI process need further exploration. Methods SCI rat models were established via performing laminectomy at the T9-T11 spinal vertebrae and cutting spinal cord tissues. SCI cell models were constructed by inducing PC12 cells with lipopolysaccharide (LPS). The protein levels of USP7, nuclear respiratory factor 1 (NRF1), Krüppel-like factor 7 (KLF7) and apoptosis-related markers were detected by western blot. Cell viability and apoptosis were tested by cell counting kit-8 assay and flow cytometry. The contents of inflammatory factors were examined using ELISA. The interaction between NRF1 and USP7 or KLF7 was analyzed by co-immunoprecipitation assay, chromatin immunoprecipitation assay and dual-luciferase reporter assay, respectively. Results USP7 was downregulated in SCI rat models and LPS-induced PC12 cells. Overexpressed USP7 promoted viability, while repressed apoptosis and inflammation in LPS-induced PC12 cells. USP7 could stabilize NRF1 protein expression via deubiquitination, and NRF1 knockdown reversed the protective effect of USP7 against LPS-induced PC12 cell injury. NRF1 is bound to KLF7 promoter to enhance its transcription. NRF1 overexpression inhibited LPS-induced PC12 cell inflammation and apoptosis via increasing KLF7 expression. Conclusion USP7 alleviated inflammation and apoptosis in LPS-induced PC12 cells via NRF1/KLF7 axis, indicating that targeting of USP7/NRF1/KLF7 axis might be a promising treatment strategy for SCI.

GOLPH3 knockdown alleviates the inflammation and apoptosis in lipopolysaccharide-induced acute lung injury by inhibiting Golgi stress mediated autophagy

Pneumonia, an acute inflammatory lesion of the lung, is the leading cause of death in children aged < 5 years. We aimed to study the function and mechanism of Golgi phosphoprotein 3 (GOLPH3) in infantile pneumonia. Lipopolysaccharide (LPS)-induced acute lung injury (ALI) mice and injury of MLE-12 cells were used as the pneumonia model in vitro. After GOLPH3 was knocked down, the histopathological changes of lung tissues were assessed by hematoxylin-eosin (H&E) staining. The Wet/Dry ratio of lung tissues was calculated. The enzyme-linked immunosorbent assay (ELISA) method was used to detecte the contents of inflammatory factors in bronchoalveolar lavage fluid (BALF). The damaged DNA in apoptotic cells in lung tissues was tested by Terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling (TUNEL) staining. Immunofluorescence staining analyzed LC3II and Golgi matrix protein 130 (GM130) expression in lung tissues and MLE-12 cells. The apoptosis of MLE-12 cells was measured by flow cytometry analysis. Additionally, the expression of proteins related to apoptosis, autophagy and Golgi stress was examined with immunoblotting. Results indicated that GOLPH3 knockdown alleviated lung tissue pathological changes in LPS-triggered ALI mice. LPS-induced inflammation and apoptosis in lung tissues and MLE-12 cells were remarkably alleviated by GOLPH3 deficiency. Besides, GOLPH3 depletion suppressed autophagy and Golgi stress in lung tissues and MLE-12 cells challenged with LPS. Moreover, Rapamycin (Rap), an autophagy inhibitor, counteracted inflammation and apoptosis inhibited by GOLPH3 silencing in LPS-induced MLE-12 cells. Furthermore, brefeldin A (BFA) pretreatment apparently abrogated the inhibitory effect of GOLPH3 knockdown on autophagy in MLE-12 cells exposed to LPS. To be concluded, GOLPH3 knockdown exerted lung protective effect against LPS-triggered inflammation and apoptosis by inhibiting Golgi stress mediated autophagy.

Chemical structure and immunomodulatory activity of a polysaccharide from Saposhnikoviae Radix

A new polysaccharide, named SP40015A01, was obtained from Saposhnikoviae Radix by water extraction, isolation and purification. SP40015A01 (9.7 × 105 Da) is composed of Rhamnose (Rha), Galacturonic acid (GalA), Galactose (Gal), and Arabinose (Ara) with the proportion of 1.6:85.6:5.8:7.6. The backbone of SP40015A01 is composed of 3-α-GalAp, 2-α-GalAp, 2,3-β-GalAp and 2,3-β-Galp, and branched at C3 of 2,3-β-GalAp, C3 of 2,3-β-Galp. Zebrafish experiments were used to explore the immunomodulatory activity of SP40015A01. Results showed that SP40015A01 could significantly improve the neutrophils density of immunocompromised zebrafish and reduce the content of nitric oxide (NO) and interleukin-1β (IL-1β). This study demonstrated that SP40015A01 has significant immunomodulatory activity, which can improve the neutrophils density and reduce inflammatory factor content, suggesting SP40015A01 may be a potential immunomodulator in Saposhnikoviae Radix (SR) for treatment of hypoimmunity related disease. This study supplemented the research on the polysaccharide components in traditional Chinese medicine and provided a scientific explanation for the development and clinical applications of SR.

Silencing LINC00663 inhibits inflammation and angiogenesis through downregulation of NR2F1 via EBF1 in bladder cancer

ABSTRACT This study is to elucidate the effect of the LINC00663/EBF1/NR2F1 axis on inflammation and angiogenesis in bladder cancer (BC) and related molecular mechanisms. After transfection, functional experiments were conducted to test cell proliferation and invasion, tube formation ability, and content of inflammatory factors, Snail, E-cadherin, and VEGFA. Meanwhile, the relationships among LINC00663, EBF1, and NR2F1 were predicted and verified. In addition, xenograft experiments in nude mice were performed to observe the oncogenicity of 5637 BC cells in vivo. In BC tissues and cells, LINC00663 and NR2F1 were upregulated. Silencing NR2F1 or LINC00663 repressed cell proliferation and invasion, weakened vascular mimicry in vitro, decreased inflammatory factor, Snail, and VEGFA levels, and increased expression of E-cadherin. LINC00663 positively regulated NR2F1 expression through EBF1. Additionally, in vivo experiments showed that NR2F1 upregulation reversed the suppression effects of LINC00663 silencing on tumour growth, inflammation, and angiogenesis. Silencing LINC00663 decreased NR2F1 expression by mediating EBF1, thereby inhibiting BC inflammation and angiogenesis.

Mechanism of Mailuo Shutong Pills on posterior limb muscle swelling caused by femoral fracture in rats based on network pharmacology and experimental verification

This study aims to investigate the mechanism of Mailuo Shutong Pills(MLST) on posterior limb muscle swelling caused by femoral fracture(SCFF) through network pharmacology and animal experiments. The plasma components of MLST were analyzed by LC-MS, and the target and signal pathway of SCFF were predicted by network pharmacology and verified by molecular docking. SCFF model rats were established through animal experiments, and different doses of MLST were administered to detect the degree of limb swelling. Hematoxylin-eosin(HE) staining was used to observe pathological changes in muscle tissue, and interleukin-6(IL-6), interleukin-1β(interleukin-1β), and tumor necrosis factor-α(TNF-α) in peripheral blood were determined by enzyme-linked immunosorbent assay(ELISA). The expression of relevant signaling pathways was measured by Western blot. Network pharmacological results showed that MLST and SCFF had a total of 153 disease targets, and the key targets were IL-6, TNF, etc., involving mitogen-activated protein kinase(MAPK) signaling pathway, phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT) signaling pathway, etc. The binding energies of the main components and key targets were lower than-7.0 kcal·mol~(-1), indicating that the network analysis results were reliable. The results of animal experiments showed that MLST could reduce the swelling degree and pathological damage of the posterior limb muscles of SCFF rats compared with the model group. ELISA results showed that MLST could reduce the levels of IL-6, IL-1β, and TNF-α in the serum of SCFF rats. Western blot results showed that MLST can reduce the expression of p-AKT, p-PI3K, p-NF-κB, p-p38 MAPK, and p-ERK in SCFF rats. MLST may reduce the content of inflammatory factors in serum by regulating the expression of PI3K/AKT and MAPK-related signaling pathway protein and improving posterior limb muscle SCFF in rats.

Overexpression of CISD2 alleviates septic acute kidney injury via activating Sonic Hedgehog signaling pathway.

Patients with sepsis are often complicated by acute kidney injury (AKI), which greatly increases mortality. In this study, our purpose was to explore the expression and function of CDGSH iron sulfur domain 2 (CISD2) in septic AKI, and the underlying molecular mechanism. Western blot and quantitative real-time polymerase chain reaction (RT-PCR) were employed to detect protein and mRNA levels in cells. The inflammation level of cells was evaluated by detecting the content of inflammatory factors (TNF-α, IL-1β, IL-6). Apoptosis of cells was evaluated by Caspase-3 activity assay, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) staining. CISD2 was down-regulated in HK-2 cells treated with lipopolysaccharide (LPS). LPS treatment increased the level of inflammatory factors, the activity of Caspase-3, and the rate of apoptosis in HK-2 cells. However, overexpression of CISD2 significantly suppressed these effects. Moreover, overexpression of CISD2 activated the Sonic Hedgehog (SHH) signaling pathway. The use of cyclopamine (Cyc), a SHH signaling pathway inhibitor, eliminated the effect of overexpressing CISD2, that is, inhibiting LPS-induced inflammation and apoptosis of HK-2 cells. LPS treatment down-regulated CISD2 in HK-2 cells, and overexpression of CISD2 could inhibit LPS-induced inflammation and apoptosis of HK-2 cells by activating the SHH signaling pathway.

Effects of PM2.5 on mucus hypersecretion in airway through miR-133b-5p/EGFR/Claudin1/MUC5AC axis.

To investigate the role of the EGFR/MAPK signaling pathway in PM2.5 in promoting the MUC5AC hypersecretion in airway and exacerbating airway inflammation. By establishing rat model exposed to PM2.5, overexpressing miR-133b-5p and Claudin1, the content of IL-1 and TNF-α in serum were detected by ELISA, the pathology of lung tissue was observed by HE staining, p-EGFR, Claudin1, MUC5AC, p-ERK1/2, p-JNK, p-p38 in rats lung tissue were detected by immunohistochemical and WB, the expression level of miR-133b-5p in rats lung tissue were detected by qPCR. After the rats were exposed to PM2.5, the content of inflammatory factors in serum increased, the inflammatory damage of lung tissues occurred, the expression of miR-133b-5p was down-regulated, and the expression of MUC5AC protein was increased. The ELISA test results showed that the expression of IL-1 and TNF-α in the model group was significantly higher than that in the control group, and the model +AG1478 treatment group was down-regulated compared with the model group, and the +miR-133b-5p agomir treatment group was significantly lower than that in the control group, the model group and the model +Claudin1 overexpression blank load group, and the model +Claudin1 overexpression group was down-regulated compared with the model group and the model +Claudin1 overexpression blank load group. The protein detection results showed that the expression of p-EGFR, MUC5AC, p-ERK1/2, p-JNK and p-p38 proteins was increased and the expression of Claudin1 protein was decreased in the model group compared with the control group. In the model + AG1478 treatment group, model + miR-133b-5p agomir treatment group and model + Claudin1 overexpression group, compared with the model group, p-EGFR, MUC5AC, p-ERK1/2, p-JNK, p-p38 protein expression was down-regulated, and Claudin1 protein expression was up-regulated. PM2.5 inhibited the expression of miR-133b-5p to activate the EGFR/MAPK signal pathway, induce the hypersecretion of MUC5AC, thus aggravating PM2.5-related airway inflammation in rats.

Banxia Xiexin Decoction inhibits colitis-associated colorectal cancer development by modulating STAT3 signaling and gut microbiota

ObjectiveTo investigate the therapeutic effects of Banxia Xiexin Decoction (BXD), a herbal medicine formula, on inflammation and the imbalance of the gut microbiota in a rat model of colorectal cancer (CRC) induced by azoxymethane (AOM) /dextran sulfate sodium (DSS). MethodsA total of 75 male C57BL/6 mice were randomly divided into five groups: normal control group (NC), model group (MODEL), low-dose BXD treatment group (L-BXD), high-dose BXD treatment (H-BXD) group and MS treatment group (MS). BXD and MS were used in CRC mice at the doses of 3.915 g/kg, 15.66 g/kg, 0.6 g/kg for 3 weeks consecutively.Histopathological changes in the colon were observed using hematoxylin-eosin (HE) staining. The content of inflammatory factors in serum was detected by an enzyme-linked immunosorbent assay (ELISA), and the expression of mRNA and protein of genes related to immunity, apoptosis, inflammation, and inflammatory factors was evaluated. Changes in the intestinal flora of mouse fecal were determined based on high-throughput sequencing of the 16S rRNA microbial gene. ResultsCompared to the model group, the low-dose BXD and high-dose BXD groups decreased the number of colon tumors, reversed weight loss, and shortened colon length of mice. The pathological examination showed that BXD alleviated the malignancy of intestinal tumors. It also suppressed signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase-9 (MMP-9), and transforming growth factor beta 1 (TGF-β1) expression, while increasing the expression of the tight junction protein ZO-1 in colon tissues. Additionally, the levels of key pathway proteins involved in inflammation (phosphorylated-STAT3, Bcl-2, COX-2) and cell cycle regulatory molecules (c-Myc and PCNA) were reduced. According to 16S rRNA sequence analysis, BXD enhanced the relative abundance of potentially beneficial bacteria, while that of cancer-related bacteria decreased. ConclusionBXD plays a preventive role in developing colorectal cancer; its mechanisms are related to the inhibition of inflammation and tumor proliferation, as well as maintenance of intestinal homeostasis.

Knockdown of IRF8 alleviates neuroinflammation through regulating microglial activation in Parkinson’s disease

Neuroinflammation associated with microglial activation plays a role in the development of Parkinson's disease (PD). The upregulation of interferon regulatory factor 8 (IRF8) in microglia following peripheral nerve injury has been observed to induce microglial activation. This suggests the potential therapeutic significance of IRF8 in PD. This research aims to explore the effects of IRF8 on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and lipopolysaccharide (LPS)-induced neuroinflammation, along with its underlying mechanisms. The study examines the differential expression of IRF8 and its effects on neuropathological changes using a PD mouse model and a PD model established from BV2 cells in vitro. IRF8 was found to be prominently expressed in the substantia nigra pars compacta (SNpc) region of PD mice and LPS-stimulated BV2 cells, while the expression of tyrosine hydroxylase (TH) and dopamine (DA) content in the SNpc region of PD mice was notably reduced. MPTP treatment and LPS stimulation intensified microglial activation, inflammation, and activation of the AMPK/mTOR signaling pathway in vivo and in vitro, respectively. Upon IRF8 silencing in the PD mouse and cell models, the knockdown of IRF8 ameliorated MPTP-induced behavioral deficits, increased the counts of TH and Nissl-positive neurons and DA content, reduced the number of Iba-1-positive microglia, and reduced the content of inflammatory factors, possibly by inhibiting the AMPK/mTOR signaling pathway. Similar outcomes were observed in the PD cell model. In conclusion, the suppression of IRF8 alleviates neuroinflammation through regulating microglial activation in PD models in vivo and in vitro by the AMPK/mTOR signaling pathway.

Sijunzi decoction alleviates inflammation and intestinal epithelial barrier damage and modulates the gut microbiota in ulcerative colitis mice.

As a representative classical prescription, Sijunzi decoction has powerful therapeutic effects on spleen-stomach qi insufficiency. Ulcerative colitis (UC) is a chronic, diffuse, and non-specifically inflammatory disorder, the etiology of which still remains unclear. In the traditional Chinese medicine (TCM) perspective, splenic asthenia is the primary cause of UC. Based on this, Sijunzi decoction has been extensively used in TCM clinical practice to alleviate UC in recent years. However, the pharmacological mechanism of Sijunzi decoction in modern medicine is still not completely clear, which limits its clinical application. The purpose of this study was to investigate the Sijunzi decoction's curative effect on acute UC mice and probe into its potential pharmacological mechanism. The UC mouse model was set up by freely ingesting a 3% dextran sulfate sodium (DSS) solution. The relieving role of Sijunzi decoction on UC in mice was analyzed by evaluating the changes in clinical parameters, colon morphology, histopathology, inflammatory factor content, intestinal epithelial barrier protein expression level, and gut microbiota balance state. Finally, multivariate statistical analysis was conducted to elucidate the relationship between inflammatory factors, intestinal epithelial barrier proteins, and gut microbiota. First, the research findings revealed that Sijunzi decoction could visibly ease the clinical manifestation of UC, lower the DAI score, and attenuate colonic damage. Moreover, Sijunzi decoction could also significantly inhibit IL-6, IL-1β, and TNF-α while increasing occludin and ZO-1 expression levels. Subsequently, further studies showed that Sijunzi decoction could remodel gut microbiota homeostasis. Sijunzi decoction was beneficial in regulating the levels of Alistipes, Akkermansia, Lachnospiraceae_NK4A136_group, and other bacteria. Finally, multivariate statistical analysis demonstrated that key gut microbes were closely associated with inflammatory factors and intestinal epithelial barrier proteins. Sijunzi decoction can significantly prevent and treat UC. Its mechanism is strongly associated with the improvement of inflammation and intestinal epithelial barrier damage by regulating the gut microbiota.

L-AP Alleviates Liver Injury in Septic Mice by Inhibiting Macrophage Activation via Suppressing NF-κB and NLRP3 Inflammasome/Caspase-1 Signal Pathways.

Liver injury and progressive liver failure are severe life-threatening complications in sepsis, further worsening the disease and leading to death. Macrophages and their mediated inflammatory cytokine storm are critical regulators in the occurrence and progression of liver injury in sepsis, for which effective treatments are still lacking. l-Ascorbic acid 6-palmitate (L-AP), a food additive, can inhibit neuroinflammation by modulating the phenotype of the microglia, but its pharmacological action in septic liver damage has not been fully explored. We aimed to investigate L-AP's antisepticemia action and the possible pharmacological mechanisms in attenuating septic liver damage by modulating macrophage function. We observed that L-AP treatment significantly increased survival in cecal ligation and puncture-induced WT mice and attenuated hepatic inflammatory injury, including the histopathology of the liver tissues, hepatocyte apoptosis, and the liver enzyme levels in plasma, which were comparable to NLRP3-deficiency in septic mice. L-AP supplementation significantly attenuated the excessive inflammatory response in hepatic tissues of septic mice in vivo and in cultured macrophages challenged by both LPS and ATP in vitro, by reducing the levels of NLRP3, pro-IL-1β, and pro-IL-18 mRNA expression, as well as the levels of proteins for p-I-κB-α, p-NF-κB-p65, NLRP3, cleaved-caspase-1, IL-1β, and IL-18. Additionally, it impaired the inflammasome ASC spot activation and reduced the inflammatory factor contents, including IL-1β and IL-18 in plasma/cultured superannuants. It also prevented the infiltration/migration of macrophages and their M1-like inflammatory polarization while improving their M2-like polarization. Overall, our findings revealed that L-AP protected against sepsis by reducing macrophage activation and inflammatory cytokine production by suppressing their activation in NF-κB and NLRP3 inflammasome signal pathways in septic liver.

CircABCB10 Promotes the Apoptosis and Inflammatory Response of 16HBE Cells by Cigarette Smoke Extract by Targeting miR-130a/PTEN Axis.

Chronic obstructive pulmonary disease (COPD) has become a global public health problem due to its high mortality. So there is an urgent need to find an effective treatment. The targeting relationship among circABCB10, miR-130a and PTEN was predicted by the targetscan database (TargetScanHuman 8.0, https://www.targetscan.org/vert_80/). A total of 60 patients which were from the second affiliated hospital of Qiqihar Medical University from 2022 to 2023 were enrolled. The lung condition was detected by CT (Computed Tomography). The expression levels of circABCB10, miR-130a and PTEN in lung tissues were determined by qRT-PCR. The COPD model was established by stimulating normal and silenced 16HBE cells in circABCB10 genes with cigarette smoke extract (CSE) at different concentrations. qRT-PCR was conducted for the expression levels of circABCB10, miR-130a and PTEN, WB for the expression levels of apoptotic proteins, ELISA for the content of inflammatory factors, and CCK8 for the effect of CSE on the proliferation of cells. CircABCB10 expression increased in lung tissues from patients with COPD and in 16HBE cells treated with CSE. The stimulation on cells with CSE increased the expression of inflammatory factors, while knocking down circABCB10 could reverse this response. The inflammatory response to the knockdown of circABCB10 was reversed by miR-130a inhibitor, which increased the expression of c-caspase 3. The targetscan database predicted the target factor downstream miR-130a was PTEN. Transfecting OE-PTEN reversed the inflammation of knocking down circABCB10, and increased the apoptosis and inflammation. CircABCB10 can cause the inflammatory response by targeting miR-130a/PTEN axis, which is a mechanism that may lead to the occurrence and development of COPD.

The therapeutic effect of ultrasound targeted destruction of schisandrin A contrast microbubbles on liver cancer and its mechanism.

The aim of the study was to explore the therapeutic effect of ultrasound targeted destruction of schisandrin A contrast microbubbles on liver cancer and its related mechanism. The Span-PEG microbubbles loaded with schisandrin A were prepared using Span60, NaCl, PEG-1500, and schisandrin A. The loading rate of schisandrin A in Span-PEG composite microbubbles was determined by ultraviolet spectrophotometry method. The Walker-256 cell survival rate of schisandrin A was determined by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay. The content of schisandrin A in the cells was determined by high performance liquid chromatography. Ultrasound imaging was used to evaluate the therapeutic effect in situ. Enzyme linked immunosorbent assay (ELISA) was used to measure the content of inflammatory factors in serum. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of experimental animals in each group. Immunohistochemistry was used to detect the expression of hypoxia inducible factor-1α (HIF-1α), vascular endothlial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR-2) in tumor tissues, and western blot was used to detect the protein expression of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in tumor tissues. The composite microbubbles were uniform in size, and the particle size distribution was unimodal and stable, which met the requirements of ultrasound contrast agents. The loading rate of schisandrin A in Span-PEG microbubbles was 8.84 ± 0.14%, the encapsulation efficiency was 82.24±1.21%. The IC50 value of schisandrin A was 2.87 μg/mL. The drug + microbubbles + ultrasound (D+M+U) group had the most obvious inhibitory effect on Walker-256 cancer cells, the highest intracellular drug concentration, the largest reduction in tumor volume, the most obvious reduction in serum inflammatory factors, and the most obvious improvement in pathological results. The results of immunohistochemistry showed that HIF-1α, VEGF and VEGFR-2 protein decreased most significantly in D+M+U group (P < 0.01). WB results showed that D+M+U group inhibited the PI3K/AKT/mTOR signaling pathway most significantly (P < 0.01). Schisandrin A had an anti-tumor effect, and its mechanism might be related to the inhibition of the PI3K/AKT/mTOR signaling pathway. The schisandrin A microbubbles could promote the intake of schisandrin A in tumor cells after being destroyed at the site of tumor under ultrasound irradiation, thus playing the best anti-tumor effect.

Itaconate inhibits corticosterone-induced necroptosis and neuroinflammation via up-regulating menin in HT22 cells.

Corticosterone (CORT) damages hippocampal neurons as well as induces neuroinflammation. The tricarboxylic acid cycle metabolite itaconate has an anti-inflammatory role. Necroptosis is a form of programmed cell death, also known as inflammatory cell death. Menin is a multifunctional scaffold protein, which deficiency aggravates neuroinflammation. In this study, we explored whether itaconate inhibits CORT-induced neuroinflammation as well as necroptosis and further investigated the mediatory role of Menin in this protective effect of itaconate by using an exposure of CORT to HT22 cells (a hippocampal neuronal cell line). The viability of HT22 cells was examined by the cell counting kit 8 (CCK-8). The morphology of HT22 cells was observed by transmission electron microscope (TEM). The expressions of necroptosis-related proteins (p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL) were evaluated by western blotting. The contents of inflammatory factors were detected by an enzyme-linked immunosorbent assay (ELISA) kit. Our results showed that CORT increases the contents of pro-inflammatory factors (IL-1β, TNF-α) as well as decreases the contents of anti-inflammatory factors (IL-4, IL-10) in HT22 cells. We also found that CORT increases the expressions of necroptosis-related proteins (p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL) and decreases the cell viability in HT22 cells, indicating that CORT induces necroptosis in HT22 cells. Itaconate improves CORT-induced neuroinflammation and necroptosis. Furthermore, itaconate upregulates the expression of Menin in CORT-exposed HT22 cells. Importantly, silencing Menin abolishes the antagonistic effect of itaconate on CORT-induced necroptosis and neuroinflammation. In brief, these results indicated that itaconate protects HT22 cells against CORT-induced neuroinflammation and necroptosis via upregulating Menin.

Effect of butylphthalide on prevention and treatment of high altitude cerebral edema in rats

3-n-butylphthalide (NBP) contains one of the main active ingredients of celery seed. It has a series of pharmacological mechanisms, including reconstitution of microcirculation, protection of mitochondrial function, inhibition of oxidative stress, and inhibition of neuronal apoptosis. Based on the complex multi-targeting of NBP pharmacological mechanisms, the clinical applications of NBP are increasing, and more and more clinical studies and animal experiments have focused on NBP. In this study, we used male Sprague Dawley rats as an animal model to elucidate the intervention effect of butylphthalide on high altitude cerebral edema (HACE), and also compared the effect of butylphthalide and rhodiola rosea on HACE. Firstly, we measured the changes of body weight and brain water content and observed the pathological changes of brain tissues. In addition, the contents of inflammatory factors, oxidative stress and brain neurotransmitters were assessed by enzyme-linked immunoassay kits, and finally, the expression of apoptotic proteins in brain tissues was determined by western blotting. The results showed that NBP reduced brain water content, attenuated brain tissue damage, altered inflammatory factors, oxidative stress indicators, and brain neurotransmitter levels, and in addition NBP inhibited the expression of Caspase-related apoptotic proteins. Therefore, NBP has the potential to treat and prevent HACE.

Bone mesenchymal stem cell-derived exosomal miR-26a-3p promotes autophagy to attenuate LPS-induced apoptosis and inflammation in pulmonary microvascular endothelial cells.

Acute lung injury (ALI) is a serious lung disease. The apoptosis and inflammation of pulmonary microvascular endothelial cells (PMVECs) are the primary reasons for ALI. This study aimed to explore the treatment effect and regulatory mechanism of bone mesenchymal stem cell-derived exosomes (BMSC-expos) on ALI. PMVECs were stimulated by Lipopolysaccharide (LPS) to imitate ALI environment. Cell viability was determined by CCK-8 assay. Cell apoptosis was evaluated by TUNEL and flow cytometry. ELISA was utilized for testing the contents of TNF-α, IL-1β, IL-6, and IL-17. Western blot was applied for testing the levels of autophagy-related proteins LC3, p62, and Beclin-1. RNA interaction was determined by luciferase reporter assay. The ALI rat model was established by intratracheal injection of LPS. Evans blue staining was utilized for detecting pulmonary vascular permeability. Our results showed that LPS stimulation notably reduced cell viability, increased cell apoptosis rate, and enhanced the contents of inflammatory factors in PMVECs. However, BMSC-exo treatment significantly abolished the promoting effects of LPS on cell injury. In addition, we discovered that BMSC-exo treatment notably activated autophagy in LPS-induced PMVECs. Furthermore, BMSC-expos upregulated miR-26a-3p expression and downregulated PTEN in PMVECs. MiR-26a-3p was directly bound to PTEN. MiR-26a-3p overexpression reduced cell apoptosis, and inflammation and promoted autophagy by silencing PTEN. Animal experiments proved that miR-26a-3p overexpression effectively improved LPS-induced lung injury in rats. The results proved that BMSC-expos promotes autophagy to attenuate LPS-induced apoptosis and inflammation in pulmonary microvascular endothelial cells via miR-26a-3p/PTEN axis.

Saikosaponin D alleviates inflammatory response of osteoarthritis and mediates autophagy via elevating microRNA-199-3p to target transcription Factor-4

ObjectiveThis study was to investigate the underlying mechanism by which Saikosaponin D (SSD) mitigates the inflammatory response associated with osteoarthritis (OA) and regulates autophagy through upregulation of microRNA (miR)-199-3p and downregulation of transcription Factor-4 (TCF4).MethodsA mouse OA model was established. Mice were intragastrically administered with SSD (0, 5, 10 μmol/L) or injected with miR-199-3p antagomir into the knee. Then, pathological changes in cartilage tissues were observed. Normal chondrocytes and OA chondrocytes were isolated and identified. Chondrocytes were treated with SSD and/or transfected with oligonucleotides or plasmid vectors targeting miR-199-3p and TCF4. Cell viability, apoptosis, inflammation, and autophagy were assessed. miR-199-3p and TCF4 expressions were measured, and their targeting relationship was analyzed.ResultsIn in vivo experiments, SSD ameliorated cartilage histopathological damage, decreased inflammatory factor content and promoted autophagy in OA mice. miR-199-3p expression was downregulated and TCF4 expression was upregulated in cartilage tissues of OA mice. miR-199-3p expression was upregulated and TCF4 expression was downregulated after SSD treatment. Downregulation of miR-199-3p attenuated the effect of SSD on OA mice. In in vitro experiments, SSD inhibited the inflammatory response and promoted autophagy in OA chondrocytes. Downregulation of miR-199-3p attenuated the effect of SSD on OA chondrocytes. In addition, upregulation of miR-199-3p alone inhibited inflammatory responses and promoted autophagy in OA chondrocytes. miR-199-3p targeted TCF4. Upregulation of TCF4 attenuated the effects of miR-199-3p upregulation on OA chondrocytes.ConclusionsSSD alleviates inflammatory response and mediates autophagy in OA via elevating miR-199-3p to target TCF4.

MiR129-5p-loaded exosomes suppress seizure-associated neurodegeneration in status epilepticus model mice by inhibiting HMGB1/TLR4-mediated neuroinflammation

BackgroundNeuroinflammation contributes to both epileptogenesis and the associated neurodegeneration, so regulation of inflammatory signaling is a potential strategy for suppressing epilepsy development and pathological progression. Exosomes are enriched in microRNAs (miRNAs), considered as vital communication tools between cells, which have been proven as potential therapeutic method for neurological disease. Here, we investigated the role of miR129-5p-loaded mesenchymal stem cell (MSC)-derived exosomes in status epilepticus (SE) mice model.MethodsMice were divided into four groups: untreated control (CON group), kainic acid (KA)-induced SE groups (KA group), control exosome injection (KA + Exo-con group), miR129-5p-loaded exosome injection (KA + Exo-miR129-5p group). Hippocampal expression levels of miR129-5p, HMGB1, and TLR4 were compared among groups. Nissl and Fluoro-jade B staining were conducted to evaluate neuronal damage. In addition, immunofluorescence staining for IBA-1 and GFAP was performed to assess glial cell activation, and inflammatory factor content was determined by ELISA. Hippocampal neurogenesis was assessed by BrdU staining.ResultsThe expression of HMGB1 was increased after KA-induced SE and peaking at 48 h, while hippocampal miR129-5p expression decreased in SE mice. Exo-miR129-5p injection reversed KA-induced upregulation of hippocampal HMGB1 and TLR4, alleviated neuronal damage in the hippocampal CA3, reduced IBA-1 + and GFAP + staining intensity, suppressed SE-associated increases in inflammatory factors, and decreased BrdU + cell number in dentate gyrus.ConclusionsExosomes loaded with miR129-5p can protect neurons against SE-mediated degeneration by inhibiting the pro-inflammatory HMGB1/TLR4 signaling axis.Graphical

Study on anti-inflammatory effect of Shangkehuangshui in vitro and in vivo based on TLR4/TLR2-NF-κB signaling pathway

Ethnopharmacological relevanceShangkehuangshui (SK) has been traditionally used to treat traumatic injury, soft tissue and bone injury in Foshan hospital of traditional Chinese medicine for more than 60 years, which composed of many Chinese herbs such as Coptis chinensis Franch., Gardenia jasminoides Ellis, Phellodendron chinense Schneid. and etc. SK exhibits heat-clearing and detoxifying, enhancing blood circulation to eliminate blood stasis properties, and demonstrates noteworthy clinical efficacy. Nevertheless, the underlying mechanism remains uncertain. Aim of the studyThe early study found that SK had good anti-inflammatory effects in acute soft tissue injury model. This research is to verify the anti-inflammatory properties of SK both in vitro and in vivo via TLR4/TLR2-NF-κB signaling pathway, to clarify the underlying mechanisms responsible for the curative effect of SK. MethodsThe RAW264.7 cells inflammatory model was established with lipopolysaccharide (LPS) in vitro. NO and TNF-α, IL-6, IL-1β were determined with Griess method and ELISA method respectively. The mRNA and protein expression levels of TLR4/TLR2-NF-κB pathway were evaluated by qPCR and Western blot method. In vivo experiment, chronic soft tissue injury rat models were established by tracking gastrocnemius muscle with electrical stimulation, then local appearance and pathological changes were observed and recorded, the contents of inflammatory factors in serum and tissue were performed. Moreover, we also measured and contrasted the expression of TLR4/TLR2-NF-κB related factors. ResultsSK effectively inhibited the LPS-induced generation of inflammatory cytokines, including NO, TNF-α, IL-6 and IL-1β in RAW264.7 cells, and significantly suppressed the expression of TLR4, TLR2, MyD88, IκB, and NF-κB. In vivo, SK remarkably decreased the damage appearance scores after 4 and 14 days of administration and inhibit the quantity of NO and leukocytes present in the serum. Additionally, the inflammatory infiltration in the pathological section was alleviated, myofibrillar hyperplasia and blood stasis were reduced. SK markedly downregulated NO, TNF-α, IL-6 and IL-1β in injured tissues of rats, also declined the expression of TLR4, TLR2, MyD88, IκB, NF-κB, IL-6, TNF-α and IL-1β. ConclusionThis study revealed that SK had obvious effects of anti-inflammatory actions in vivo and vitro, effectively reduced acute and chronic soft tissue injury in clinical, this might be attributed to inhibit the TLR4/TLR2-NF-κB pathway, further inhibit the expression of downstream relevant pro-inflammatory cytokines.

Rosuvastatin: A Potential Therapeutic Agent for Inhibition of Mechanical Pressure-Induced Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) underlies the pathogenesis of degenerative diseases of the spine; however, its exact molecular mechanism is unclear. To explore the molecular mechanism of mechanical pressure (MP)-induced IDD and to assess the role and mechanism of Rosuvastatin (RSV) inhibits MP-induced IDD. SD rat nucleus pulposus cells (NPCs) were cultured in vitro and an apoptosis model of NPCs was constructed using MP. Proliferative activity, reactive oxygen species content, apoptosis, and wound healing were detected in each group of NPCs, respectively. The expression of relevant proteins was detected by qPCR and Western Blot techniques. 18 SD rats were randomly divided into control, pressure and RSV groups. Elisa, qPCR, Western Blot and immunohistochemical staining techniques were used to detect changes in the content of related proteins in the intervertebral discs of each group. HE staining and Modified Saffron-O and Fast Green Stain Kit were used to assess IDD in each group. MP treatment at 1.0 MPa could significantly induce apoptosis of NPCs after 24 h. MP could significantly inhibit the proliferative activity and wound healing ability of NPCs, and increase the intracellular reactive oxygen species content and apoptosis rate; pretreatment with RSV could significantly activate the Nrf2/HO-1 signaling pathway and reverse the cellular damage caused by MP; when inhibit the Nrf2/HO-1 signaling pathway activation, the protective effect of RSV was reversed. In vivo MP could significantly increase the content of inflammatory factors within the IVD and promote the degradation of extracellular matrix, leading to IDD. When the intervention of RSV was employed, it could significantly activate the Nrf2/HO-1 signaling pathway and improve the above results. RSV may inhibit MP-induced NPCs damage and IDD by activating the Nrf2/HO-1 signaling pathway.