CGRP is essential for protection against alveolar epithelial cell necroptosis by activating the AMPK/L-OPA1 signaling pathway during acute lung injury.

Alveolar epithelial cell (AEC) necroptosis is critical to disrupt the alveolar barrier and provoke acute lung injury (ALI). Here, we define calcitonin gene-related peptide (CGRP), the most abundant endogenous neuropeptide in the lung, as a novel modulator of AEC necroptosis in lipopolysaccharide (LPS)-induced ALI. Upon LPS-induced ALI, overexpression of Cgrp significantly mitigates the inflammatory response, alleviates lung tissue damage, and decreases AEC necroptosis. Similarly, CGRP alleviated AEC necroptosis under the LPS challenge in vitro. Previously, we identified that long optic atrophy 1 (L-OPA1) deficiency mediates mitochondrial fragmentation, leading to AEC necroptosis. In this study, we discovered that CGRP positively regulated mitochondrial fusion through stabilizing L-OPA1. Mechanistically, we elucidate that CGRP activates AMP-activated protein kinase (AMPK). Furthermore, the blockade of AMPK compromised the protective effect of CGRP against AEC necroptosis following the LPS challenge. Our study suggests that CRGP-mediated activation of the AMPK/L-OPA1 axis may have potent therapeutic benefits for patients with ALI or other diseases with necroptosis.

[Study on the mechanism of electroacupuncture repairing intestinal barrier via regulating mast cell in rats with diarrhea-predominant irritable bowel syndrome].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on mast cell activation-related substances and intestinal barrier function in diarrhea-predominant irritable bowel syndrome (IBS-D) model rats, so as to explore its underlying mechanisms. METHODS: Thirty female SD rats were randomly divided into control group, model group and EA group, with 10 rats in each group. IBS-D model was established by chronic unpredictable mild stress combined with senna solution gavage. Rats in the EA group received EA treatment (2 Hz/15 Hz，0.1-1.0 mA) at "Zusanli" (ST36), "Taichong"(LR3) and "Tianshu"(ST25), 20 min per day, for a total of 14 days, with sides alternated daily. Visceral pain threshold was used to evaluate visceral hypersensitivity, diarrhea index was used to evaluate diarrhea degree. After all treatments, the pathological scores of colon were recorded after HE staining, the contents of cholecystokinin (CCK), substance P (SP), tryptase (TPS) and adenosine triphosphate (ATP) in colon were detected by ELISA, and the expressions of colonic tight junction protein ZO-1 and occludin were detected by Western blot. RESULTS: Compared with the control group, the visceral pain threshold, the expression levels of colonic ZO-1 and occludin proteins decreased (P<0.01), while the diarrhea index, the contents of colonic CCK, SP, TPS and ATP were significantly increased (P<0.01) in the model group. After intervention, in comparison with the model group, the visceral pain thre-shold, the protein expression levels of colonic ZO-1 and occludin protein increased (P<0.01), while the diarrhea index, the contents of colonic CCK, SP, TPS and ATP were significantly decreased (P<0.01) in the EA group. CONCLUSION: EA can significantly alleviate the symptoms of visceral hypersensitivity and diarrhea in IBS-D rats. Its mechanism may be related to down-regulating colonic CCK, SP, TPS and ATP, inhibiting mast cell activation and degranulation, and up-regulating colonic barrier tight junction proteins.

Mitochondrial citrate accumulation drives alveolar epithelial cell necroptosis in lipopolysaccharide-induced acute lung injury.

Necroptosis is the major cause of death in alveolar epithelial cells (AECs) during acute lung injury (ALI). Here, we report a previously unrecognized mechanism for necroptosis. We found an accumulation of mitochondrial citrate (citratemt) in lipopolysaccharide (LPS)-treated AECs because of the downregulation of Idh3α and citrate carrier (CIC, also known as Slc25a1). shRNA- or inhibitor-mediated inhibition of Idh3α and Slc25a1 induced citratemt accumulation and necroptosis in vitro. Mice with AEC-specific Idh3α and Slc25a1 deficiency exhibited exacerbated lung injury and AEC necroptosis. Interestingly, the overexpression of Idh3α and Slc25a1 decreased citratemt levels and rescued AECs from necroptosis. Mechanistically, citratemt accumulation induced mitochondrial fission and excessive mitophagy in AECs. Furthermore, citratemt directly interacted with FUN14 domain-containing protein 1 (FUNDC1) and promoted the interaction of FUNDC1 with dynamin-related protein 1 (DRP1), leading to excessive mitophagy-mediated necroptosis and thereby initiating and promoting ALI. Importantly, necroptosis induced by citratemt accumulation was inhibited in FUNDC1-knockout AECs. We show that citratemt accumulation is a novel target for protection against ALI involving necroptosis.

Fn14 exacerbates acute lung injury by activating the NLRP3 inflammasome in mice.

BACKGROUND: Uncontrolled inflammation is an important factor in the occurrence and development of acute lung injury (ALI). Fibroblast growth factor-inducible 14 (Fn14), a plasma membrane-anchored receptor, takes part in the pathological process of a variety of acute and chronic inflammatory diseases. However, the role of Fn14 in ALI has not yet been elucidated. This study aimed to investigate whether the activation of Fn14 exacerbated lipopolysaccharide (LPS)-induced ALI in mice. METHODS: In vivo, ALI was induced by intratracheal LPS-challenge combined with/without Fn14 receptor blocker aurintricarboxylic acid (ATA) treatment in C57BL/6J mice. Following LPS administration, the survival rate, lung tissue injury, inflammatory cell infiltration, inflammatory factor secretion, oxidative stress, and NLRP3 inflammasome activation were assessed. In vitro, primary murine macrophages were used to evaluate the underlying mechanism by which Fn14 activated the NLRP3 inflammasome. Lentivirus was used to silence Fn14 to observe its effect on the activation of NLRP3 inflammasome in macrophages. RESULTS: In this study, we found that Fn14 expression was significantly increased in the lungs of LPS-induced ALI mice. The inhibition of Fn14 with ATA downregulated the protein expression of Fn14 in the lungs and improved the survival rate of mice receiving a lethal dose of LPS. ATA also attenuated lung tissue damage by decreasing the infiltration of macrophages and neutrophils, reducing inflammation, and suppressing oxidative stress. Importantly, we found that ATA strongly inhibited the activation of NLRP3 inflammasome in the lungs of ALI mice. Furthermore, in vitro, TWEAK, a natural ligand of Fn14, amplified the activation of NLRP3 inflammasome in the primary murine macrophage. By contrast, inhibition of Fn14 with shRNA decreased the expression of Fn14, NLRP3, Caspase-1 p10, and Caspase-1 p20, and the production of IL-1ß and IL-18. Furthermore, the activation of Fn14 promoted the production of reactive oxygen species and inhibited the activation of Nrf2-HO-1 in activated macrophages. CONCLUSIONS: Our study first reports that the activation of Fn14 aggravates ALI by amplifying the activation of NLRP3 inflammasome. Therefore, blocking Fn14 may be a potential way to treat ALI.

COX-2/sEH Dual Inhibitor PTUPB Attenuates Epithelial-Mesenchymal Transformation of Alveolar Epithelial Cells via Nrf2-Mediated Inhibition of TGF-ß1/Smad Signaling.

Background: Arachidonic acid (ARA) metabolites are involved in the pathogenesis of epithelial-mesenchymal transformation (EMT). However, the role of ARA metabolism in the progression of EMT during pulmonary fibrosis (PF) has not been fully elucidated. The purpose of this study was to investigate the role of cytochrome P450 oxidase (CYP)/soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) metabolic disorders of ARA in EMT during PF. Methods: A signal intratracheal injection of bleomycin (BLM) was given to induce PF in C57BL/6 J mice. A COX-2/sEH dual inhibitor PTUPB was used to establish the function of CYPs/COX-2 dysregulation to EMT in PF mice. In vitro experiments, murine alveolar epithelial cells (MLE12) and human alveolar epithelial cells (A549) were used to explore the roles and mechanisms of PTUPB on transforming growth factor (TGF)-ß1-induced EMT. Results: PTUPB treatment reversed the increase of mesenchymal marker molecule α-smooth muscle actin (α-SMA) and the loss of epithelial marker molecule E-cadherin in lung tissue of PF mice. In vitro, COX-2 and sEH protein levels were increased in TGF-ß1-treated alveolar epithelial cells (AECs). PTUPB decreased the expression of α-SMA and restored the expression of E-cadherin in TGF-ß1-treated AECs, accompanied by reduced migration and collagen synthesis. Moreover, PTUPB attenuated TGF-ß1-Smad2/3 pathway activation in AECs via Nrf2 antioxidant cascade. Conclusion: PTUPB inhibits EMT in AECs via Nrf2-mediated inhibition of the TGF-ß1-Smad2/3 pathway, which holds great promise for the clinical treatment of PF.

L-OPA1 deficiency aggravates necroptosis of alveolar epithelial cells through impairing mitochondrial function during acute lung injury in mice.

Necroptosis, a recently described form of programmed cell death, is the main way of alveolar epithelial cells (AECs) death in acute lung injury (ALI). While the mechanism of how to trigger necroptosis in AECs during ALI has been rarely evaluated. Long optic atrophy protein 1 (L-OPA1) is a crucial mitochondrial inner membrane fusion protein, and its deficiency impairs mitochondrial function. This study aimed to investigate the role of L-OPA1 deficiency-mediated mitochondrial dysfunction in AECs necroptosis. We comprehensively investigated the detailed contribution and molecular mechanism of L-OPA1 deficiency in AECs necroptosis by inhibiting or activating L-OPA1. First, our data showed that L-OPA1 expression was downregulated in the lungs and AECs under the lipopolysaccharide (LPS) challenge. Furthermore, inhibition of L-OPA1 aggravated the pathological injury, inflammatory response, and necroptosis in the lungs of LPS-induced ALI mice. In vitro, inhibition of L-OPA1 induced necroptosis of AECs, while activation of L-OPA1 alleviated necroptosis of AECs under the LPS challenge. Mechanistically, inhibition of L-OPA1 aggravated necroptosis of AECs by inducing mitochondrial fragmentation and reducing mitochondrial membrane potential. While activation of L-OPA1 had the opposite effects. In summary, these findings indicate for the first time that L-OPA1 deficiency mediates mitochondrial fragmentation, induces necroptosis of AECs, and exacerbates ALI in mice.

Electroacupuncture Alleviates Visceral Hypersensitivity in IBS-D Rats by Inhibiting EGCs Activity through Regulating BDNF/TrkB Signaling Pathway.

OBJECTIVE: To determine whether electroacupuncture (EA) could alleviate visceral hypersensitivity in diarrhea-predominant irritable bowel syndrome (IBS-D) rats by inhibiting EGCs activity via the BDNF/TrkB signaling pathway. METHODS: Sprague Dawley rats were randomly divided to a control group (n = 8) and a model preparation group (n = 32), which received Senna solution by gavage and CUMS (chronic unpredictable mild stress) for 14 consecutive days and was further divided to a Model group, an EA group (only electroacupuncture), an EA + TrkB agonist group (electroacupuncture and TrkB), and an EA + DMSO group (electroacupuncture and DMSO, n = 8 for each). Rats in the three EA groups were acupunctured at ST25, ST36, and LR3 for 20 min every day for 14 days. Abdominal withdrawal reflex (AWR) was used to quantify visceral sensitivity; reverse transcription polymerase chain reaction (RT-PCR) and double immunofluorescent staining were used to detect the colocalized expression of GFAP/BDNF and GFAP/TrkB. Western Blot (WB) was used to detect the expression of PLC and SP in the colon. Flow cytometry was used to detect the expression of Ca2+. RESULTS: EA effectively alleviated visceral hypersensitivity in IBS-D rats (P < 0.05). Compared to the control group, the expression of BDNF, TrkB, PLC, SP, and Ca2+ and the colocalized expression of GFAP/BDNF and GFAP/TrkB increased in the Model group (P < 0.05), while all these parameters decreased in the EA group following EA intervention (P < 0.05). In addition, no significant difference was found between the EA + TrkB agonist group and the control group (P > 0.05). CONCLUSIONS: EA alleviates visceral hypersensitivity of IBS-D rats possibly by inhibiting the activity of EGCs through the BDNF/TrkB-PLC-Ca2+ signaling pathway in the colon.

Extracellular citrate serves as a DAMP to activate macrophages and promote LPS-induced lung injury in mice.

Citrate has a prominent role as a substrate in cellular energy metabolism. Recently, citrate has been shown to drive inflammation. However, the role of citrate in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains unclear. Here, we aimed to clarify whether extracellular citrate aggravated the LPS-induced ALI and the potential mechanism. Our findings demonstrated that extracellular citrate aggravated the pathological lung injury induced by LPS in mice, characterized by up-regulation of pro-inflammatory factors and over-activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in the lungs. In vitro, we found that citrate treatment significantly augmented the expression of NLRP3 and pro-IL-1ß and enhanced the translocation of NF-κB/p65 into the nucleus. Furthermore, extracellular citrate plus adenosine-triphosphate (ATP) significantly increased the production of reactive oxygen species (ROS) in primary murine macrophages. Inhibiting the production of ROS with a ROS scavenger N-acetyl-L-cysteine (NAC) attenuated the activation of NLRP3 inflammasome. Altogether, we conclude that extracellular citrate may serve as a damage-associated molecular pattern (DAMP) and aggravates LPS-induced ALI by activating the NLRP3 inflammasome.

Bioconversion of steviol glycosides into steviol by Microbacterium barkeri.

The strain which degraded steviol glycosides to steviol (STE) was screened and isolated from soil samples. A phylogenetic tree was constructed and used to determine the taxonomic status of the strain. 16S rDNA sequence was ultimately used to identify the strain as Microbacterium barkeri XJ. The transformation product was detected and identified as STE by HPLC/LC-MS/IR analysis. The bioconversion rate of 1% (v/v) steviol glycosides (stevioside, rebaudioside A, rebaudioside C) into STE in basic medium were 100% within 24 h, 84 h and 144 h, respectively. The results indicated XJ was more effective than mixed flora in the bioconversion of steviol glycosides to STE.

A COX-2/sEH dual inhibitor PTUPB ameliorates cecal ligation and puncture-induced sepsis in mice via anti-inflammation and anti-oxidative stress.

Arachidonic acid can be metabolized to prostaglandins and epoxyeicosatrienoic acids (EETs) by cyclooxygenase-2 (COX-2) and cytochrome P450 (CYP), respectively. While protective EETs are degraded by soluble epoxide hydrolase (sEH) very fast. We have reported that dual inhibition of COX-2 and sEH with specific inhibitor PTUPB shows anti-pulmonary fibrosis and renal protection. However, the effect of PTUPB on cecal ligation and puncture (CLP)-induced sepsis remains unclear. The current study aimed to investigate the protective effects of PTUPB against CLP-induced sepsis in mice and the underlying mechanisms. We found that COX-2 expressions were increased, while CYPs expressions were decreased in the liver, lung, and kidney of mice undergone CLP. PTUPB treatment significantly improved the survival rate, reduced the clinical scores and systemic inflammatory response, alleviated liver and kidney dysfunction, and ameliorated the multiple-organ injury of the mice with sepsis. Besides, PTUPB treatment reduced the expression of hypoxia-inducible factor-1α in the liver, lung, and kidney of septic mice. Importantly, we found that PTUPB treatment suppressed the activation of NLRP3 inflammasome in the liver and lung of septic mice. Meanwhile, we found that PTUPB attenuated the oxidative stress, which contributed to the activation of NLRP3 inflammasome. Altogether, our data, for the first time, demonstrate that dual inhibition of COX-2 and sEH with PTUPB ameliorates the multiple organ dysfunction in septic mice.

PTUPB ameliorates high-fat diet-induced non-alcoholic fatty liver disease via inhibiting NLRP3 inflammasome activation in mice.

Non-alcoholic fatty liver disease (NAFLD) affects 25% of the global adult population, and no effective pharmacological treatment has been found. Products of arachidonic acid metabolism have been developed into a novel therapy for metabolic syndrome and diabetes. It has been demonstrated that protective actions of a novel dual cyclooxygenase-2 (COX-2) and soluble epoxide hydrolase (sEH) inhibitor, PTUPB, on the metabolic abnormalities. Here, we investigated the effects of PTUPB on hepatic steatosis in high-fat diet (HFD)-induced obese mice, as well as in hepatocytes in vitro. We found that PTUPB treatment reduced body weight, liver weight, liver triglyceride and cholesterol content, and the expression of lipolytic/lipogenic and lipid uptake related genes (Acc, Cd36, and Cidec) in HFD mice. In addition, PTUPB treatment arrested fibrotic progression with a decrease of collagen deposition and expression of Col1a1, Col1a3, and α-SMA. In vitro, PTUPB decreased palmitic acid-induced lipid deposition and downregulation of lipolytic/lipogenic genes (Acc and Cd36) in hepatocytes. Additionally, we found that PTUPB reduced the production of pro-inflammatory cytokines and suppressed the NLRP3 inflammasome activation in HFD mice and hepatocytes. In conclusion, dual inhibition of COX-2/sEH attenuates hepatic steatosis by inhibiting the NLRP3 inflammasome activation. PTUPB might be a promising potential therapy for liver steatosis associated with obesity.

COX-2/sEH dual inhibitor PTUPB alleviates bleomycin-induced pulmonary fibrosis in mice via inhibiting senescence.

Pulmonary fibrosis (PF) is a senescence-associated disease with poor prognosis. Currently, there is no effective therapeutic strategy for preventing and treating the disease process. Mounting evidence suggests that arachidonic acid (ARA) metabolites are involved in the pathogenesis of various fibrosis. However, the relationship between the metabolism of ARA and PF is still elusive. In this study, we observed a disorder in the cyclooxygenase-2/cytochrome P450 (COX-2/CYP) metabolism of ARA in the lungs of PF mice induced by bleomycin (BLM). Therefore, we aimed to explore the role of COX-2/CYP-derived ARA metabolic disorders in PF. PTUPB, a dual COX-2 and soluble epoxide hydrolase (sEH) inhibitor, was used to restore the balance of COX-2/CYP metabolism. sEH is an enzyme hydrolyzing epoxyeicosatrienoic acids derived from ARA by CYP. We found that PTUPB alleviated the pathological changes in lung tissue and collagen deposition, as well as reduced senescence marker molecules (p16Ink4a and p53-p21Waf1/Cip1 ) in the lungs of mice treated by BLM. In vitro, we found that PTUPB pretreatment remarkably reduced the expression of senescence-related molecules in the alveolar epithelial cells (AECs) induced by BLM. In conclusion, our study supports the notion that the COX-2/CYP-derived ARA metabolic disorders may be a potential therapeutic target for PF via inhibiting the cellular senescence in AECs.

Exogenous angiotensin (1-7) directly inhibits epithelial-mesenchymal transformation induced by transforming growth factor-ß1 in alveolar epithelial cells.

Accumulating evidence indicates that angiotensin (1-7) [Ang-(1-7)] protects against idiopathic pulmonary fibrosis (IPF) in animal experiments. However, whether Ang-(1-7) effectively inhibits epithelial-mesenchymal transition (EMT) induced by transforming growth factor-ß1 (TGF-ß1) remains unclear. The aim of this study is to examine the eff ;ects of Ang-(1-7) on TGF-ß1-induced EMT in human alveolar epithelial cells. We found that angiotensin-converting enzyme 2 (ACE2) /Ang-(1-7)/MasR were decreased in the lungs of mice with IPF induced by bleomycin, and were negatively correlated with Tgfb1 mRNA expression. In vitro, our data showed that exogenous Ang-(1-7) restored the expression of E-cadherin and decreased the expressions of α-SMA and Vimentin induced by TGF-ß1 in A549 cells. Ang-(1-7) also reduced TGF-ß1-induced migration and synthesis of the extracellular matrix, such as collagen Ⅰ and collagen Ⅲ. Mechanistically, we observed that Ang-(1-7) directly inhibited TGF-ß1-induced phosphorylation of Smad2 and Smad3, and suppressed the expression of the downstream target gene of TGF-ß1-Smad signaling, including ZEB1, ZEB2, TWIST, and SNAIL1. Additionally, phosphorylation of mTOR induced by TGF-ß1 also been suppressed by Ang-(1-7) treatment in A549 cells. Interestingly, we found that TGF-ß1 strongly suppressed the expression of ACE2 in A549 cells through inhibiting SIRT1. In conclusion, our findings indicate that Ang-(1-7) directly inhibits TGF-ß1-induced EMT in alveolar epithelial cells via disruption of TGF-ß1-Smad signaling pathway, contributing to the protective effect against IPF.

Re-examination of the genotoxic activity of water taken from the Songhua River in P. R. China.

The Songhua River, in northeast China, has heavy organic contamination due to domestic sewage and industrial wastewater. Thus, it is important to further determine its genotoxic activity, which is a potential hazard for human health. Short-term genotoxic bioassays using Salmonella, the cytokinesis-block micronucleus cytome assay, and mouse liver cell comet assay were employed to further examine the genotoxic activity of diethyl ether extracts of water samples taken from the Songhua River. Ames test results showed that there were still frame-shift mutagens, both direct and indirect, in water samples at doses of 5.0 or 7.0 L water equivalent/plate. The mutagenicity seems to be less when compared with the results from 2002 to 2003. A dose-response relationship was also obtained between DNA damage in mouse liver cells by comet assay and micronuclei formation by CBMN assay. These results indicate that the water samples showed genotoxic activity with a mutagenic potency. 88 and 104 compounds, respectively, were identified in summer and winter water sample extracts by gas chromatography-mass spectrometry analysis. Four priority pollutants listed by the United States Environmental Protection Agency and six priority pollutants listed by the Chinese Environment Protection Agency were found in summer or winter water samples, respectively. The results indicate that the diethyl ether extracts of surface water samples taken from the Songhua River still show genotoxic activity (≥3.0 L water). The risks of potential carcinogenicity for human health in the Songhua River should be studied further.