Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis.

BACKGROUND: Influenza A viruses (IAV) are extremely common respiratory viruses for the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), in which IAV infection may further evoke abnormal macrophage polarization, amplify cytokine storms. Melatonin exerts potential effects of anti-inflammation and anti-IAV infection, while its effects on IAV infection-induced AECOPD are poorly understood. METHODS: COPD mice models were established through cigarette smoke exposure for consecutive 24 weeks, evaluated by the detection of lung function. AECOPD mice models were established through the intratracheal atomization of influenza A/H3N2 stocks in COPD mice, and were injected intraperitoneally with melatonin (Mel). Then, The polarization of alveolar macrophages (AMs) was assayed by flow cytometry of bronchoalveolar lavage (BAL) cells. In vitro, the effects of melatonin on macrophage polarization were analyzed in IAV-infected Cigarette smoking extract (CSE)-stimulated Raw264.7 macrophages. Moreover, the roles of the melatonin receptors (MTs) in regulating macrophage polarization and apoptosis were determined using MTs antagonist luzindole. RESULTS: The present results demonstrated that IAV/H3N2 infection deteriorated lung function (reduced FEV20,50/FVC), exacerbated lung damages in COPD mice with higher dual polarization of AMs. Melatonin therapy improved airflow limitation and lung damages of AECOPD mice by decreasing IAV nucleoprotein (IAV-NP) protein levels and the M1 polarization of pulmonary macrophages. Furthermore, in CSE-stimulated Raw264.7 cells, IAV infection further promoted the dual polarization of macrophages accompanied with decreased MT1 expression. Melatonin decreased STAT1 phosphorylation, the levels of M1 markers and IAV-NP via MTs reflected by the addition of luzindole. Recombinant IL-1ß attenuated the inhibitory effects of melatonin on IAV infection and STAT1-driven M1 polarization, while its converting enzyme inhibitor VX765 potentiated the inhibitory effects of melatonin on them. Moreover, melatonin inhibited IAV infection-induced apoptosis by suppressing IL-1ß/STAT1 signaling via MTs. CONCLUSIONS: These findings suggested that melatonin inhibited IAV infection, improved lung function and lung damages of AECOPD via suppressing IL-1ß/STAT1-driven macrophage M1 polarization and apoptosis in a MTs-dependent manner. Melatonin may be considered as a potential therapeutic agent for influenza virus infection-induced AECOPD.

Qingjie decoction attenuated E.coli-induced diarrhea by regulating energy metabolism and alleviating inflammation based on network analysis and metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Diarrhea is a frequently encountered gastrointestinal complication in clinical practice, and E. coli is one of the main causative agents. Although Qingjie decoction (QJD) has been shown to be highly effective in treating diarrhea by eliminating heat-toxin, the underlying molecular mechanisms and pathways of QJD remain unclear. AIM OF REVIEW: The aim of this research was to explore the effects and fundamental mechanism of QJD on diarrhea induced by E.coli in rats. MATERIALS AND METHODS: Initially, we used UHPLC-MS/MS analysis to identify the chemical composition of QJD. Then, we constructed a visualization network using network pharmacology. Next, we utilized metabolomics to identify differentially expressed metabolites of QJD that are effective in treating diarrhea. RESULTS: The chemical composition of QJD was analyzed using UHPLC-MS/MS, which identified a total of 292 components. Using a network pharmacology approach, 127 bioactive compounds of QJD were screened, targeting 171 potential diarrhea treatment targets. TNF-α, IL-6, IL-1ß, and CAT were identified as important targets through visualizing the PPI network. Enrichment analysis demonstrated significant enrichment in the TNF signaling pathway, IL-17 signaling pathway, and PI3K-Akt signaling pathway. QJD showed beneficial effects, such as increased body weight, decreased fecal water content, and reduced inflammatory cell infiltration in the duodenum and colon, as well as maintaining the structure of the duodenum and colon. Metabolomic analysis revealed 32 differentially expressed metabolites in the control, model and QJD-H groups, including glucose, valine, and cysteine. Functional analysis indicated that differential metabolites were related to energy metabolism, including glucose metabolism, TCA cycle, and amino acid metabolism. CONCLUSION: QJD significantly increased body weight, decreased water content in feces, relieved inflammatory cell infiltration, maintained the structure of duodenum and colon. Combining network analysis and metabolomics, QJD exerted therapeutic effects by inhibiting inflammation and oxidative stress, regulating glucose metabolism, tricarboxylic acid metabolism, and amino acid metabolism.

Exosome-derived circKIF20B suppresses gefitinib resistance and cell proliferation in non-small cell lung cancer.

BACKGROUND: The gefitinib resistance mechanism in non-small cell lung cancer (NSCLC) remains unclear, albeit exosomal circular RNA (circRNA) is known to possibly play a vital role in it. METHODS: We employed high-throughput sequencing techniques to detect the expressions of exosomal circRNA both in gefitinib-resistant and gefitinib-sensitive cells in this study. The circKIF20B expression was determined in serum exosomes and tissues of patients by qRT-PCR. The structure, stability, and intracellular localization of circKIF20B were verified by Sanger sequencing, Ribonuclease R (RNase R)/actinomycin D (ACTD) treatments, and Fluorescence in situ hybridization (FISH). The functions of circKIF20B were investigated by 5-Ethynyl-20-deoxyuridine (EdU), flow cytometry, Cell Counting Kit-8 (CCK-8), oxygen consumption rate (OCR), and xenograft model. Co-culture experiments were performed to explore the potential ability of exosomal circKIF20B in treating gefitinib resistance. The downstream targets of circKIF20B were determined by luciferase assay, RNA pulldown, and RNA immunoprecipitation (RIP). RESULTS: We found that circKIF20B was poorly expressed in the serum exosomes of gefitinib-resistant patients (n = 24) and the tumor tissues of patients with NSCLC (n = 85). CircKIF20B was negatively correlated with tumor size and tumor stage. Decreasing circKIF20B was found to promote gefitinib resistance by accelerating the cell cycle, inhibiting apoptosis, and enhancing mitochondrial oxidative phosphorylation (OXPHOS), whereas increasing circKIF20B was found to restore gefitinib sensitivity. Mechanistically, circKIF20B is bound to miR-615-3p for regulating the MEF2A and then altering the cell cycle, apoptosis, and mitochondrial OXPHOS. Overexpressing circKIF20B parental cells can restore sensitivity to gefitinib in the recipient cells by upregulating the exosomal circKIF20B expression. CONCLUSIONS: This study revealed a novel mechanism of circKIF20B/miR-615-3p/MEF2A signaling axis involving progression of gefitinib resistance in NSCLC. Exosomal circKIF20B is expected to be an easily accessible and alternative liquid biopsy candidate and potential therapeutic target in gefitinib-resistant NSCLC. The schematic diagram of mechanism in this study. Exosomal circKIF20B inhibits gefitinib resistance and cell proliferation by arresting the cell cycle, promoting apoptosis, and reducing OXPHOS via circKIF20B/miR-615-3p/MEF2A axis in NSCLC.

Melatonin Suppresses Macrophage M1 Polarization and ROS-Mediated Pyroptosis via Activating ApoE/LDLR Pathway in Influenza A-Induced Acute Lung Injury.

Influenza virus infection is one of the strongest pathogenic factors for the development of acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS). However, the underlying cellular and molecular mechanisms have not been clarified. In this study, we aim to investigate whether melatonin modulates macrophage polarization, oxidative stress, and pyroptosis via activating Apolipoprotein E/low-density lipoprotein receptor (ApoE/LDLR) pathway in influenza A-induced ALI. Here, wild-type (WT) and ApoE-/- mice were instilled intratracheally with influenza A (H3N2) and injected intraperitoneally with melatonin for 7 consecutive days. In vitro, WT and ApoE-/- murine bone marrow-derived macrophages (BMDMs) were pretreated with melatonin before H3N2 stimulation. The results showed that melatonin administration significantly attenuated H3N2-induced pulmonary damage, leukocyte infiltration, and edema; decreased the expression of proinflammatory M1 markers; enhanced anti-inflammatory M2 markers; and switched the polarization of alveolar macrophages (AMs) from M1 to M2 phenotype. Additionally, melatonin inhibited reactive oxygen species- (ROS-) mediated pyroptosis shown by downregulation of malonaldehyde (MDA) and ROS levels as well as inhibition of the NLRP3/GSDMD pathway and lactate dehydrogenase (LDH) release. Strikingly, the ApoE/LDLR pathway was activated when melatonin was applied in H3N2-infected macrophages and mice. ApoE knockout mostly abrogated the protective impacts of melatonin on H3N2-induced ALI and its regulatory ability on macrophage polarization, oxidative stress, and pyroptosis. Furthermore, recombinant ApoE3 (re-ApoE3) inhibited H3N2-induced M1 polarization of BMDMs with upregulation of MT1 and MT2 expression, but re-ApoE2 and re-ApoE4 failed to do this. Melatonin combined with re-ApoE3 played more beneficial protective effects on modulating macrophage polarization, oxidative stress, and pyroptosis in H3N2-infected ApoE-/- BMDMs. Our study indicated that melatonin attenuated influenza A- (H3N2-) induced ALI by inhibiting the M1 polarization of pulmonary macrophages and ROS-mediated pyroptosis via activating the ApoE/LDLR pathway. This study suggested that melatonin-ApoE/LDLR axis may serve as a novel therapeutic strategy for influenza virus-induced ALI.

Mori fructus aqueous extracts attenuate carbon tetrachloride-induced renal injury via the Nrf2 pathway and intestinal flora.

Mori fructus aqueous extracts (MFAEs) have been used as a traditional Chinese medicine for thousands of years with the function of strengthening the liver and tonifying the kidney. However, its inner mechanism to alleviative renal injury is unclear. To investigate the attenuation of MFAEs on nephrotoxicity and uncover its potential molecular mechanism, we established a nephrotoxicity model induced by carbon tetrachloride (CCl4). The mice were randomly divided into control group, CCl4 model group (10% CCl4), CCl4 + low and high MFAEs groups (10% CCl4 + 100 mg/kg and 200 mg/kg MFAEs). We found that MFAEs decreased the kidney index of mice, restored the pathological changes of renal structure induced by CCl4, reduced cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (Kim-1) blood urea nitrogen and creatinine contents in serum, promoted the nuclear transportation of Nrf2 (nuclear factor erythroid derived 2 like 2), elevated the expression of HO-1 (heme oxygenase 1), GPX4 (glutathione peroxidase 4), SLC7A11 (solute carrier family 7 member 11), ZO-1 (zonula occludens-1) and Occludin, suppressed the expression of Keap1 (kelch-like ECH-associated protein 1), HMGB1 (High Mobility Group Protein 1), ACSL4 (acyl-CoA synthetase long chain family member 4) and TXNIP (thioredoxin interacting protein), upregulated the flora of Akkermansia, Anaerotruncus, Clostridium_sensu_stricto, Ihubacter, Alcaligenes, Dysosmobacter, and downregulated the flora of Clostridium_XlVa, Helicobacter, Paramuribaculum. Overlapped with Disbiome database, Clostridium_XlVa, Akkermansia and Anaerotruncus may be the potential genera treated with renal injury. It indicated that MFAEs could ameliorate kidney injury caused by CCl4 via Nrf2 signaling.

Interleukin-6 neutralizing antibody attenuates the hypersecretion of airway mucus via inducing the nuclear translocation of Nrf2 in chronic obstructive pulmonary disease.

Airway mucus hypersecretion is a vital pathophysiologic feature in chronic obstructive pulmonary disease (COPD) patients in which airflow limitation result, and it is key to strategizing in the management of COPD. To investigate the mechanisms underlying the action of interleukin-6 neutralizing antibody (IL-6 Ab) in attenuating airway mucus hypersecretion in COPD, human and mouse primary bronchial epithelial cells from COPD patients and mice were isolated, human organoid model of trachea was established and all treated with IL-6 and/or IL-6 Ab. The differential expression of Muc5ac and Nrf2 were determined in pDHBE compared to pNHBE cells via high-throughput sequencing of transcriptome. The serum concentration of Muc5ac was significantly elevated and positively correlated with IL-6 in COPD patients using ELISA, and the excessive mucus secretion was observed in the trachea of COPD patients using HE, AB-PAS and IHC staining. The levels of Muc5ac were significantly elevated in the IL-6-treated group, and diminished with IL-6 Ab treatment, both in vitro and in the organoid model using qRT-PCR, WB and IF. The expression levels of protein Muc5ac were significantly reduced in cells transfected with the IL-6 small interfering RNA (siRNA-IL-6), which was in contrast to the levels of protein Nrf2, and the protective effects of IL-6 Ab were inhibited in cells transfected with Nrf2 short hairpin RNA (shRNA-Nrf2). IL-6 Ab significantly attenuated hypersecretion of airway mucus by inducing nuclear translocation of Nrf2 in COPD. These findings indicated that IL-6 Ab may constitute a novel therapeutic agent for IL-6-induced airway mucus hypersecretion by improving airflow limitation in COPD patients.

Alternatones A and B, two polyketides possessing novel skeletons from entophyte Alternaria alternate L-10.

On our ongoing searching for bioactive natural products derived from entophytes, two polyketides possessing novel skeletons, alternatones A-B (1-2), were identified from the culture of Alternaria alternate L-10. Their structures were established by a combination of spectroscopic and single-crystal X-ray diffraction with Cu Ka radiation. Alternatone A (1) exhibited cytotoxic activity against human hepatoma carcinoma HepG-2 cell line. The putative biosynthetic pathways for compounds 1-2 were also proposed.

Shaoyao decoction attenuates DSS-induced ulcerative colitis, macrophage and NLRP3 inflammasome activation through the MKP1/NF-κB pathway.

BACKGROUND: Shaoyao decoction (SYD), a traditional Chinese medicine prescription that originated in the Jin-Yuan Dynasty, has shown effects in treating ulcerative colitis. However, the underlying mechanism is unclear. We combined network pharmacology with molecular biology technology to detect the mechanism underlying the effect of SYD on ulcerative colitis. We combined network pharmacology with molecular biology technology to detected the further mechanism in SYD effect on ulcerative colitis. PURPOSE: In this study, we investigated the mechanism by which SYD exerts a protective effect against ulcerative colitis in vivo and in vitro. STUDY DESIGN AND METHODS: We focused on two aspects of the mechanism by which SYD relieves ulcerative colitis, regulation of the MAPK cascade and the NF-κB signaling pathway, through analysis of the "active ingredient-target-disease" network followed by GO enrichment and KEGG pathway analysis according to network pharmacology. Mice with ulcerative colitis underwent 5% dextran sulfate sodium (DSS), and the RAW 264.7 cell model was used to identify important targets. RESULTS: We found that after 5% DSS treatment, the inflammation indexes and the expression of NLRP3-related proteins were increased concomitant with the loss of mucins and occludin. Treatment with SYD (2.25 g/kg, BW) significantly improved the expression of mucins and occludin after DSS at the protein and transcriptional levels. Furthermore, SYD treatment significantly reduced NF-κB P65 and P38 expression, thus exerting a great antinecrotic effect, as revealed by TUNEL staining and Western blotting. The beneficial effects of SYD were almost canceled by NSC 95397 (an inhibitor of mitogen-activated protein kinase phosphatase-1 (MKP1)) after DSS treatment in vivo or LPS treatment in vitro. In addition, treatment with SYD reduced caspase-1 activity and rescued the release of ASC and GSDMD, thus inhibiting the assembly of NLRP3 and maintaining the integrity of the intestinal barrier. We also conducted in vitro experiments in the LPS-induced RAW 264.7 cell model and found that cells incubated with 1 mg/ml SYD for 24 h possessed the highest cell viability. Next, we incubated 1 mg/ml SYD for 24 h after treatment with 1 µg/ml LPS for 6 h. We showed that 1 mg/ml SYD displayed anti-inflammatory and anti-necrotic effects through the NLRP3, NF-κB P65 and P38 pathways, and the effects of SYD were also inhibited by 10 nM NSC 95397. CONCLUSION: These results demonstrate that SYD has protective effects against ulcerative colitis and alleviates pyroptosis by inhibiting the MKP1/NF-κB/NLRP3 pathway.

The effects of nuclear factor-kappa B in pancreatic stellate cells on inflammation and fibrosis of chronic pancreatitis.

The activation of pancreatic stellate cells (PSCs) plays a critical role in the progression of pancreatic fibrosis. Nuclear factor-kappa B (NF-κB) is associated with chronic pancreatitis (CP). Previous evidence indicated that NF-κB in acinar cells played a double-edged role upon pancreatic injury, whereas NF-κB in inflammatory cells promoted the progression of CP. However, the effects of NF-κB in PSCs have not been studied. In the present study, using two CP models and RNAi strategy of p65 in cultured PSCs, we found that the macrophage infiltration and MCP-1 expression were increased, and the NF-κBp65 protein level was elevated. NF-κBp65 was co-expressed with PSCs. In vitro, TGF-ß1 induced overexpression of the TGF-ß receptor 1, phosphorylated TGF-ß1-activated kinase 1 (p-TAK1) and NF-κB in the PSCs. Moreover, the concentration of MCP-1 in the supernatant of activated PSCs was elevated. The migration of BMDMs was promoted by the supernatant of activated PSCs. Further knockdown of NF-κBp65 in PSCs resulted in a decline of BMDM migration, accompanied by a lower production of MCP-1. These findings indicate that TGF-ß1 can induce the activation of NF-κB pathway in PSCs by regulating p-TAK1, and the NF-κB pathway in PSCs may be a target of chronic inflammation and fibrosis.

Niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells.

Our previous study found that the anthelmintic drug niclosamide relaxed the constricted arteries and inhibited proliferation and migration of vascular smooth muscle cells. Here, we investigated the effect of niclosamide ethanolamine (NEN) on trachea function and the proliferation and migration of trachea smooth muscle cells. Isometric tension of trachea was recorded by multi-channel myograph system. The cell proliferation was detected by using BrdU cell proliferation assay. The cell migration ability was evaluated by using scratch assay. The protein level was measured by using western blot technique. Acute treatment with NEN dose-dependently relaxed acetylcholine chloride (Ach)- and High K+ physiological salt solution (KPSS)-induced constriction of mice trachea. Pre-treatment with NEN inhibited Ach- and KPSS-induced constriction of mice trachea. NEN treatment inhibited proliferation of human bronchial smooth muscle cells (HBSMCs), inhibited migration of HBSMCs and rat primary trachea smooth muscle cells. NEN treatment activated adenosine monophosphate activated protein kinase (AMPK) activity and inhibited signal transducer and activator of transcription 3 (STAT3) activity in HBSMCs. In conclusion, niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells, indicating that niclosamide might be a potential drug for chronic asthma treatment.

PFP alleviates nonalcoholic steatohepatitis fatty liver in both Apo E-/- mice and Changliver cell[S].

High-calorie food leads to nonalcoholic fatty liver disease (NAFLD) through the dysregulation of genes involved in lipid metabolism, but the precise mechanism is still unknown. Pomegranate flowers are used to treat diabetes mellitus in traditional Uighur medicine. Here we sought to investigate the effect and mechanism of pomegranate flower polyphenols (PFP) on NAFLD Apo E-/- mice induced by a high-fat diet (HFD) and whether PFP improves NAFLD through decreasing oxidative stress. PFP supplementation in mice significantly reduced the HFD-induced gains in body weight compared with the mice fed only with HFD. It also significantly reduced HFD-induced increases in serum lipids, including cholesterol and triglyceride. Consistent with the reduced liver weight, hepatic lipid accumulation, and the size of lipid droplets in the epididymal fat pads were also reduced by PFP supplementation. To further investigate how PFP may reduce obesity, we analyzed lipid metabolism-related genes in the liver. PFP supplementation altered expression profiles of several lipid metabolism-related genes, including ACC, AMPK, CPT-1α, FAS, LDLR, Leptin, LXR, PON1, PPAR, SirT3, and SREBP, relative to those in HFD control mice. The expression patterns of these genes observed by quantitative reverse transcriptase-polymerase chain reaction and AMPK, SirT3, ACC2, and CPT-1A expression were confirmed by immunohistochemical assays. Collectively, our results indicate that PFP prevents HFD-induced obesity in Apo E-/- mice, and its anti-obesity effects may be related to the regulation of lipogenesis at the level of transcription.

[Effects of pomegranate flowers polyphenols on liver PON expression of diabetes combining non-alcoholic fat liver diseases rats].

To investigate the protective effects and possible mechanism of pomegranate flowers polyphenols (PFP) on liver function of rats with diabetes combining non-alcoholic fat liver diseases, diabetes combining nonalcoholic fat liver disease model rats were established with high calorie feeding and small dose intraperitoneal injection of streptozotocin (STZ). Model rats were randomly divided into: model group, metformin group, pomegranate flowers polyphenols low, medium and high dose group (75, 150 and 300 mg x kg(-1)). After four weeks treatment, the levels of FPG, blood fat profiles and serum insulin, ALT, AST levels, SOD and MDA in the liver and serum separately were analyzed with biochemical methods. Paraoxonase (PON1 and PON3) mRNA and protein expression in liver were checked by RT-PCR and immunohistochemical method. Pathological changes of the liver were observed. FPG, IRI, non-HDL-C and transaminase significantly reduced and HDL-C raised in the each PFP dose group; Furthermore, compared with model group, fat drops in liver cells significantly reduced, antioxidant ability enhanced, PON1 mRNA and protein expression level in liver increased significantly. The protective effects of PFP against diabetes combining non-alcoholic fat liver diseases rats might through the increase liver PON1 mRNA and protein expression further enhanced the body antioxidant capacity and reduced IRI so as to ameliorate the rat hepatic steatosis.

Serologic analysis of anti-porcine endogenous retroviruses immune responses in humans after ex vivo transgenic pig liver perfusion.

Improvements in xenotransplantation may significantly increase the availability of organs for human transplantation. The use of porcine organs, however, has raised concern about possible transmission of porcine endogenous retroviruses (PERV) to the recipients. The authors developed monoclonal antibodies specific to the PERV Gag viral product and show that these antibodies can detect PERV antigen under a variety of assay conditions, including enzyme linked immunosorbent assay (ELISA), Western blot, and immunofluorescence staining methods. Two patients in fulminant hepatic failure were treated by extracorporeal perfusion using transgenic porcine livers before receiving orthotopic liver transplants. Despite the use of immune suppression that allowed survival of the allograft, these patients both showed a strong immune response to the xenograft suggesting a largely intact capability to mount a humoral immune response. However, analysis of patient serum samples over a 3 to 4 year period has showed no evidence of an immune response to PERV antigens, suggesting a lack of PERV infection.