Palmatine inhibits expression fat mass and obesity associated protein (FTO) and exhibits a curative effect in dextran sulfate sodium (DSS)-induced experimental colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory disease whose pathogenesis and mechanisms have not been fully described. The m6A methylation modification is a general mRNA modification in mammalian cells and is closely associated with the onset and progression of inflammatory bowel disease (IBD). Palmatine (PAL) is a biologically active alkaloid with anti-inflammatory and protective effects in animal models of colitis. Accordingly, we examined the role of PAL on colitis by regulating N6-methyladenosine (m6A) methylation. METHODS: A rat experimental colitis model was established by 5 % dextran sulfate sodium (DSS) in drinking water for seven days, then PAL treatment was administered for seven days. The colonic tissue pathology was assessed using hematoxylin-eosin (HE) and disease activity index (DAI). In in vitro studies, a human, spontaneously immortalized non-cancerous colon mucosal epithelial cell line (NCM460) was exposed to 2 % DSS and treated with PAL and cell viability was assayed using Cell Counting Kit-8 (CCK-8). The levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA) kits. The level of Zonula occludens-1 (ZO-1) was dectected by immunofluorescence. Transepithelial electrical resistance (TEER) of cells was also assessed. The methyltransferase-like 3 (METTL3), METTL14, AlkB homologate 5 (ALKBH5), and fat mass and obesity-associated protein (FTO) expression levels were assessed by western blotting. The localized expression of m6A was measured by immunofluorescence. RESULTS: PAL significantly prevented bodyweight loss and shortening of the colon in experimental colitis rats, as well as decreasing the DAI and histological damage scores. Furthermore, PAL inhibited the levels of inflammatory factors (TNF-α, IL-6, IL-8, and IL-1ß) in both DSS treated rats and NCM460 cells. In addition, PAL enhanced the expression level of ZO-1, and increased the transepithelial electrical resistance to repaire intestinal barrier dysfunction. Colitis occurred due to decreased m6A levels, and the increased FTO expression led to a colitis phenotype. PAL markedly enhanced the METTL3 and METTL14 expression levels while decreasing ALKBH5 and FTO expression levels. CONCLUSIONS: The findings demonstrated that PAL improved DSS-induced experimental colitis. This effect was associated with inhibiting FTO expression and regulating m6A methylation.

Inflammasome and pyroptosis in autoimmune liver diseases.

Autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and IgG4-related sclerosing cholangitis (IgG4-SC) are the four main forms of autoimmune liver diseases (AILDs), which are all defined by an aberrant immune system attack on the liver. Most previous studies have shown that apoptosis and necrosis are the two major modes of hepatocyte death in AILDs. Recent studies have reported that inflammasome-mediated pyroptosis is critical for the inflammatory response and severity of liver injury in AILDs. This review summarizes our present understanding of inflammasome activation and function, as well as the connections among inflammasomes, pyroptosis, and AILDs, thus highlighting the shared features across the four disease models and gaps in our knowledge. In addition, we summarize the correlation among NLRP3 inflammasome activation in the liver-gut axis, liver injury, and intestinal barrier disruption in PBC and PSC. We summarize the differences in microbial and metabolic characteristics between PSC and IgG4-SC, and highlight the uniqueness of IgG4-SC. We explore the different roles of NLRP3 in acute and chronic cholestatic liver injury, as well as the complex and controversial crosstalk between various types of cell death in AILDs. We also discuss the most up-to-date developments in inflammasome- and pyroptosis-targeted medicines for autoimmune liver disorders.

Banxia Xiexin decoction ameliorates dextran sulfate sodium (DSS)-induced ulcerative colitis via inhibiting serine-threonine protein kinase (Akt)/mitogen-activated protein kinase (MAPK) signaling pathway.

Banxia Xiexin decoction (BXD), a traditional Chinese medicine, was widely used in treating ulcerative colitis (UC). However, the active components of BXD and its mechanism in UC remain elusive. Therefore, we used network pharmacology in vivo experiments, molecular docking, and surface plasmon resonance strategy (SPR) to uncover BXD's potential mechanism. A UC rat model was established by orally administering 7% dextran sulfate sodium (DSS) in drinking water, BXD and palmatine were orally administered for 7 days. Network pharmacology was used to investigate the main bioactive components and crucial targets of BXD in treating UC. Molecular docking was used to investigate interactions between components and crucial targets, verifying the results by SPR. By network pharmacology predicting, 20 active components and 44 candidate anti-UC targets of BXD were identified, and the crucial proteins were screeded from PPI network, including extracellular regulated protein kinases (ERK), AKT1, and tumor necrosis factor-α (TNF). In addition, some key active components (palmatine, sexangularetin, and skullcapflavone II) were screened out from the active components-targets network. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and in vivo experiments showed that protein-serine-threonine kinase (Akt)/MAPK pathway was involved in BXD treatment for UC; BXD and palmatine significantly ameliorated the severity of DSS-induced UC in rats. Our study might assist in further investigation of the active components in Chinese medicine.

Ischemic stroke protected by ISO-1 inhibition of apoptosis via mitochondrial pathway.

Macrophage migration inhibitory factor (MIF) is an immune mediator associated with inflammation, which is upregulated after ischemia in brain tissue. ISO-1 is a potent inhibitor of MIF tautomerase and can protect neurons by reducing the permeability of blood brain barrier (BBB). In this study, we investigated the role of ISO-1 in cerebral ischemia/reperfusion injury by establishing a model of middle cerebral artery occlusion/reperfusion in rats. Rats were randomly divided into four groups: the sham operation group, the ISO-1group, the cerebral I/R group, and the ISO-1 + I/R group. We assessed the degree of neurological deficit in each group and measured the volume of cerebral infarction. We detected the expression of MIF in the core necrotic area and penumbra. We detected the expression of apoptosis-related proteins, apoptosis-inducing factor (AIF), endonuclease G (EndoG) and cytochrome c oxidase-IV (COX-IV) in the ischemic penumbra region. The results showed that MIF was expressed in the ischemic penumbra, while the injection of ISO-1 was able to alleviate neurological damage and reduce the infarction volume. In the cerebral ischemic penumbra region, ISO-1 could reduce the expression of Bax and Caspase3 and inhibit the displacement of AIF and EndoG to the nucleus simultaneously. Besides, ISO-1 also exhibited the ability to reduce apoptosis. In summary, ISO-1 may inhibit neuronal apoptosis through the endogenous mitochondrial pathway and reduce the injury of brain I/R after ischemic stroke.

Revealing therapeutic targets and mechanism of baicalin for anti-chronic gastritis using proteomic analysis of the gastric tissue.

BACKGROUND: Baicalin (BCL) is a natural compound associated with antioxidant, anti-inflammatory and immunomodulatory activities, among many others. To investigate the therapeutic effect of BCL treatment in ethanol-induced chronic gastritis, we investigated proteomic changes in the gastric tissue to elucidate the therapeutic targets of BCL in chronic gastritis using tandem mass tag (TMT)-based quantitative proteomics. METHODS: Sprague-Dawley (SD) rats received 8.7 ml/kg body weight of 56% ethanol intragastrically, which induced chronic gastritis model. Then, BCL (50 mg/kg) or omeprazole (20 mg/kg) was orally administered for 7 days to treat the induced chronic gastritis. After sacrifice, the total protein of the gastric antrum was determined. In addition, a tandem tag labelling for relative and absolute quantification (TMT)-based liquid chromatography with tandem mass spectrometry analysis was performed on the sample to identify differentially expressed proteins (DEPs) between therapy and model groups. Furthermore, the potential protein targets, signalling pathways and protein-protein interaction (PPI) network were analysed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, some potential targets were validated using Western blotting. RESULTS: A total of 4,452 proteins were identified and quantified in the gastric antrum tissue of SD rats using TMT-based quantitative proteomics. Of these, 107 DEPs, including 44 upregulated and 63 downregulated proteins, were discovered in the BCL-treated group compared with the untreated group with ethanol-induced gastritis, of these were 33 callback proteins. Biological information analysis demonstrated that the selected differential proteins were involved in the enriched pathways, including MAPK, PI3K-Akt and NF-κB. Furthermore, the expression of TPM2, GIMAP4, ICAM-1 and Mpc1 was validated using Western blotting, which was consistent with the proteomics results. The study results confirmed the reliability of the proteomic analysis. Additionally, BCL could decrease the production of interleukin (IL)-2, IL-8 and tumour necrosis factor-α while increasing the expression of epidermal growth factor and B-cell lymphoma-2. Notably, PPI network analysis revealed widespread interactions mediated by BCL. CONCLUSIONS: We investigated the effects and potential mechanism of BCL in chronic gastritis. Proteomic technology was used to explore BCL-affected proteins and some signalling pathways. The results may provide important insights into discovering potential target proteins for treating chronic gastritis.

Diagnostic and Prognostic Significance of serum miR-18a-5p in Patients with Atherosclerosis.

Atherosclerosis (AS) is a common vascular disease with great harm. The current study examined the expression pattern of miR-18a-5p in AS patients, and explored its clinical values. 110 AS patients and 68 healthy controls were collected clinically, and the expression pattern of miR-18a-5p in the serum of AS patients was detected using qRT-PCR. All AS patients were followed up for five years to record the adverse cardiovascular events. ROC and Kaplan-Meier (K-M) curve were plotted to assess the diagnostic ability. The multiple Cox regression analysis was performed for independent influencing factors analysis. MiR-18a-5p was at high expression in AS patients, and showed positive correlation with the CIMT value (r = 0.789, P < .001). ROC curve suggested the high diagnostic value of serum miR-18a-5p for AS, with the AUC of 0.894. The diagnostic specificity and sensitivity were 86.8% and 79.1%, respectively. K-M plot demonstrated that cases with high miR-18a-5p levels were more likely to suffer from cardiovascular events, and it is an independent influence factor for the poor clinical outcome. Serum miR-18a-5p serves as a promising biomarker for AS diagnosis, and is related to the occurrence of adverse cardiovascular events.

Identifying the active compounds and mechanism of action of Banxia Xiexin decoction for treating ethanol-induced chronic gastritis using network pharmacology combined with UPLC-LTQ-Orbitrap MS.

BACKGROUND: Banxia Xiexin decoction (BXD), a traditionally prescribed Chinese medicine, has been used to treat chronic gastritis for many years. However, the underlying mechanism and targets for its effects remain unknown. In the present study, we predicted the targets and active compounds of BXD in the treatment of chronic gastritis through network pharmacology and ultra-performance liquid chromatography coupled with linear trap quadrupole-Orbitrap mass spectrometry (UPLC-LTQ-Orbitrap MS). METHOD: A chronic gastritis model was established in rats by oral administration of 56 % ethanol. BXD was orally administered for 7 days. Stomach tissues were collected for histopathological analysis, and tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-8, and lactate dehydrogenase (LDH) levels were measured by enzyme-linked immunosorbent assay. UPLC-LTQ-Orbitrap MS was established to analyse compounds in rat plasma following oral BXD administration. The absorbed ingredients were selected as candidate active compounds. The chronic gastritis-related targets were screened using multiple databases. The potential targets for the treatment of chronic gastritis were used to construct a protein-protein interaction (PPI) network and were also analysed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Finally, molecular docking was used to uncover the interaction between multi-components and putative targets, and the results were verified by surface plasmon resonance (SPR). RESULTS: Intragastric administration of BXD ameliorated stomach injury resulting from chronic gastritis in rats and decreased the levels of TNF-α, IL-2, IL-8, and LDH. A comprehensive systematic strategy was used to successfully identify 38 candidate targets and 14 active compounds in BXD. Based on the network of compounds-targets and PPI, three hub genes that were associated with BXD therapy for chronic gastritis were selected and included intercellular adhesion molecule-1, peroxisome proliferator-activated receptor gamma and mitogen-activated protein kinase 14. The results of molecular docking and SPR demonstrated that the active compounds in BXD demonstrate affinity for these targets. Additionally, an enrichment analysis revealed that treatment of chronic gastritis with BXD primarily involves cytokine activation, the inflammatory response and nuclear factor-kappa B, hypoxia-inducible factor-1, phosphatidylinositol-3-kinase-protein-serine-threonine kinase and Janus kinase-signal transducer and activator of transcription signalling pathways, which may mediate the effects of BXD in the treatment of chronic gastritis. CONCLUSION: BXD exhibits a therapeutic effect in ethanol-induced gastritis through multi-compound, multi-target and multi-pathway mechanisms. A strategy of network pharmacology combined with SPR may provide a feasible approach to explore the targets of herbal medicine and uncover novel bioactive components.

Qinzhuliangxue mixture alleviates psoriasis-like skin lesions via inhibiting the IL6/STAT3 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoriasis is a chronic inflammatory skin disease mediated by immunity. Our pre-clinical studies have proved that QZLX mixture can improve patients' clinical symptoms with psoriasis without noticeable adverse reactions. In a psoriasis-like mouse model induced by imiquimod, QZLX mixture has been shown to alleviate epidermal inflammation and inhibit the hyperproliferation of keratinocytes. However, its related molecular mechanism remains to be elucidated. AIM OF THE STUDY: To assess the mechanism of QZLX mixture against psoriasis. MATERIALS AND METHODS: This study combines network pharmacology and experiments to study the mechanism of QZLX against psoriasis. First, construct the active compound-target network and PPI network. Secondly, determine possible drug targets through Molecular docking and KEGG. Thirdly, high-performance liquid chromatography (HPLC) was used for the quality control of QZLX. Finally, use a mouse model of psoriasis to further confirm the role of QZLX. RESULTS: (1) Network pharmacology analysis shows that QZLX alleviates psoriasis's epidermal inflammation, and neovascularization may be achieved by inhibiting the IL6/STAT3 signaling pathway. (2) QZLX improves the pathological characteristics of IMQ-induced skin damage in psoriasis-like mice. (3) QZLX inhibits the IL6/STAT3 signaling pathway and reduces the expression of IL-17, IL-23, and TNF-α related to inflammation in peripheral blood, as well as the expression of S100A7 in the lesion area. QZLX is better than MTX in inhibiting neovascularization by down-regulating the expression of HIF-1 and CD31 in the lesion area. Finally, inhibition of Ki67 alleviates the excessive proliferation of keratinocytes. CONCLUSION: In sum, this study clarifies the mechanism of QZLX against psoriasis and provides evidence to support its clinical use.

[Effect of Gegen Qinlian Decoction and it's different compatibility groups on gut microbiota in rats with acute enteritis based on high-throughput sequencing].

This study was designed to investigate the effect of Gegen Qinlian(GGQL) Decoction and its different compatibility groups on gut microbiota in rats with acute enteritis, and to explore the efficacy of GGQL Decoction in improving acute enteritis and gut microbiota. Male SD rats were randomly divided into control group, model group, positive control group(SASP), GGQL decoction group, Glycyrrhizae-free group(QGC), Puerariae-free group(QGG), Qinlian-free group(QQL), and Qinlian group(QL). The pathological sections and detection indexes of the rats were observed before and after modeling and administration. After 7 days of administration, fecal samples from 24 rats were collected and Illumina Miseq platform was used for high-throughput sequencing. From the anti-inflammatory and pharmacodynamic indicators, the effect was the most obvious in GGQL Decoction group, QGC group, QGG group and QL group(P<0.05). The alpha diversity and beta diversity showed that there were significant differences in the composition of intestinal flora in each group. As compared with the model group, the increased abundance and diversity of the flora caused by acute inflammation could be down-regulated in all groups except QQL group(P<0.05). The differential bacteria were explored by using LEfSe analysis, and the results showed that Bifidobacterium and other beneficial bacteria only appeared in the normal group. As compared with the normal group, Lactobacillus was significantly reduced(P<0.01), and Bacteroides, Flavonifractor and Clostridium_sensu_stricto_1 were up-regulated in model group(P<0.05, P<0.01). As compared with the model group, the number of Akkermansia was significantly increased(P<0.05), and the number of Clostridium_sensu_stricto_1 associated with intestinal inflammatory diseases was decreased in the GGQL Decoction group, QGC group and QL group. QGC group and QQL group caused the up-regulation of Ruminococcaceae and induced enrichment of Desulfovibrio which could lead to colon cell toxicity; QGG group caused the up-regulation of Proteobacteria and Burkhonderiales. The study suggests that the GGQL Decoction may play a role in the treatment of acute enteritis partially through improving the intestinal barrier, regulating the immune response and the structure of gut microbiota.

Baicalin protects against ethanol-induced chronic gastritis in rats by inhibiting Akt/NF-κB pathway.

AIMS: Currently, chronic gastritis is a high incidence of digestive diseases, along with loss of appetite, abdominal pain and diarrhea. Baicalin belongs to the major bioactive flavonoids compounds from Scutellariae Radix, it exhibited anti-inflammatory and anti-bacteria activities. Nonetheless, the protective effects of baicalin on ethanol-induced gastritis have not been completely clarified. Our study was designed to evaluate the protective activity of baicalin on ethanol-induced chronic gastritis. MAIN METHODS: Rat with chronic gastritis model was induced by the administration of 56% ethanol for four weeks. Baicalin (50 and 100 mg/kg) were orally administered for seven days to evaluate its curative effect, respectively. The production of TNF-α, interleukin (IL)-8, IL-1ß, NO, ET-1, PGE2, LDH and COX-2 were determined by ELISA. The activities of Akt, p-Akt, IκBα, p-IκBα, NF-κBp65 and NF-κBp-p65 were tested by western blot. Immunofluorescence staining was employed to assess the location of NF-κBp65. KEY FINDINGS: The changes of the histopathological analysis and the levels of NO, ET-1, PGE2, LDH and COX-2 demonstrated that baicalin treatment ameliorated ethanol-induced gastritis. ELISA analysis showed that baicalin inhibited the levels of TNF-α, IL-8 and IL-1ß. Besides, Akt, p-Akt, IκBα, p-IκBα, NF-κBp65 and NF-κBp-p65 expression were significantly suppressed by baicalin. Meanwhile, baicalin suppressed the translocation of NF-κBp65 to the cell nucleus through immunofluorescence staining, molecular docking analysis showed that baicalin had affinity with Akt and NF-κBp65. SIGNIFICANCE: All results demonstrated that baicalin effectively alleviated chronic gastritis via suppressing the levels of inflammatory regulators and inhibiting Akt/NF-κB activation.

Knockout of MARCH2 inhibits the growth of HCT116 colon cancer cells by inducing endoplasmic reticulum stress.

Membrane-associated RING-CH protein 2 (MARCH2), a member of the MARCH family, functions in vesicle trafficking and autophagy regulation. In this study, we established MARCH2 knockout HCT116 cell lines using CRISPR/Cas9-mediated genome editing to evaluate the role of MARCH2 in colon cancer in vitro and in vivo. Knockout of MARCH2 suppressed cell proliferation, and promoted autophagy, apoptosis and G2/M phase cell cycle arrest. These effects were associated with activation of endoplasmic reticulum (ER) stress. In addition, loss of MARCH2 sensitized HCT116 cells to the chemotherapy drugs etoposide and cisplatin. Moreover, we analyzed the clinical significance of MARCH2 in human colon carcinoma (n=100). High MARCH2 expression was significantly associated with advanced clinicopathological features and poorer overall survival in colon carcinoma. MARCH2 expression correlated negatively with expression of the unfolded protein response molecule p-PERK in colon cancer. Collectively, these data reveal a relationship between MARCH2, ER stress and colon cancer, and indicates MARCH2 may have an important role in the development and progression of colon cancer.

Time-dependent changes of autophagy and apoptosis in lipopolysaccharide-induced rat acute lung injury.

OBJECTIVES: Abnormal lung cell death including autophagy and apoptosis is the central feature in acute lung injury (ALI). To identify the cellular mechanisms and the chronology by which different types of lung cell death are activated during lipopolysaccharide (LPS)-induced ALI, we decided to evaluate autophagy (by LC3-II and autophagosome) and apoptosis (by caspase-3) at different time points after LPS treatment in a rat model of LPS-induced ALI. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into two groups: control group and LPS group. ALI was induced by LPS intraperitoneal injection (3 mg/kg). The lung tissues were collected to measure lung injury score by histopathological evaluation, the protein expression of LC3-II and caspase-3 by Western blot, and microstructural changes by electron microscopy analysis. RESULTS: During ALI, lung cell death exhibited modifications in the death process at different stages of ALI. At early stages (1 hr and 2 hr) of ALI, the mode of lung cell death started with autophagy in LPS group and reached a peak at 2 hr. As ALI process progressed, apoptosis was gradually increased in the lung tissues and reached its maximal level at later stages (6 hr), while autophagy was time-dependently decreased. CONCLUSION: These findings suggest that activated autophagy and apoptosis might play distinct roles at different stages of LPS-induced ALI. This information may enhance the understanding of lung pathophysiology at the cellular level during ALI and pulmonary infection, and thus help optimize the timing of innovating therapeutic approaches in future experiments with this model.