Study on Intestinal Flora and Expression of NOX1 in Patients With Irritable Bowel Syndrome / 胃肠病学

Background: Studies have shown that intestinal flora and nicotinamide-adenosine dinucleotide phosphate oxidase 1 (NOX1) play important roles in the occurrence of chronic visceral pain, however, the roles in irritable bowel syndrome (IBS) are not clear. Aims: To study the correlation of intestinal flora mucosal characteristics with NOX1 expression in patients with IBS. Methods: Colonic mucosa specimens of IBS patients diagnosed by Rome criteria and healthy subjects from July 2017 to December 2017 at Peace Hospital Affiliated to Changzhi Medical College were collected. High-throughput sequencing was used to detect intestinal mucosal flora, and the species diversity and abundance of the intestinal flora were analyzed. The expression of NOX1 in colonic mucosa was determined by immunohistochemistry. Results: The intestinal mucosal flora of IBS group was mainly composed of Bacteroides, Proteobacteria and Firmicutes. There was no significant difference in diversity of flora among IBS-D group, IBS-U group and control group (P>0.05). Compared with the control group, the abundance of Tissierella was significantly increased in the IBS group (H=6.688, P0.05). Conclusions: Compared with healthy subjects, there is no significant change in the diversity of intestinal mucosal flora in IBS patients. The abundance of some flora is changed, and the change of abundance of flora varies in different subtypes. The expression of NOX1 in colonic mucosa of IBS patients is up-regulated, and there was a certain correlation with the abundance of Tissierella.

Effect of NF-κB binding element deletion on transcriptional regulation of NOX1 / 中国病理生理杂志

AIM:To investigate the effect of NF-κB binding element deletion on transcriptional regulation of (NOX1).METHODS:pGL3-Basic vector inserted with the NOX1 proximal promoter, and the same vector inserted with the NOX1 proximal promoter in the absence of the positive NF-κB binding element, were constructed.After cloning, diges-tion and purification, NOX1 proximal promoter (≈1 415 bp) was inserted into the multicloning site of the pGL3-Basic vec-tor and then sequenced ( pGL3-NOX1-1415) .NF-κB binding elements in the NOX1 promoters were predicted by Alibaba 2.1 software.The positive element was deleted by overlapping PCR.The deletion mutant was inserted into the pGL3 vector in the same way (pGL3-NOX1-1327).The plasmids were transfected into A549 cells, and then the cells were stimulated with TNF-α.The luciferase activity was monitored on MD SpectraMax M5 enzyme-labeled instrument.RESULTS:The se-quences of pGL3-NOX1-1415 and pGL3-NOX1-1327 were identified to be correct.Compared with control group, the lucif-erase activity was significantly higher in the cells transfected with pGL3-NOX1-1327 (P<0.05), but it was significantly lower than that in the cells transfected with pGL3-NOX1-1415 (P<0.05).CONCLUSION: NF-κB plays an essential role in the transcriptional regulation of NOX1 in TNF-α-induced A549 cells.Activated NF-κB binds to specific elements in NOX1 promoter regions to control the transcription.

Entamoeba histolytica Induces Cell Death of HT29 Colonic Epithelial Cells via NOX1-Derived ROS

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.

Resveratrol inhibits foam cell formation via NADPH oxidase 1-mediated reactive oxygen species and monocyte chemotactic protein-1

Resveratrol is a polyphenolic compound in red wine that has anti-oxidant and cardioprotective effects in animal models. Reactive oxygen species (ROS) and monocyte chemotactic protein-1 (MCP-1) play key roles in foam cell formation and atherosclerosis. We studied LPS-mediated foam cell formation and the effect of resveratrol. Resveratrol pretreatment strongly suppressed LPS-induced foam cell formation. To determine if resveratrol affected the expression of genes that control ROS generation in macrophages, NADPH oxidase 1 (Nox1) was measured. Resveratrol treatment of macrophages inhibited LPS-induced Nox1 expression as well as ROS generation, and also suppressed LPS-induced MCP-1 mRNA and protein expression. We investigated the upstream targets of Nox1 and MCP-1 expression and found that Akt-forkhead transcription factors of the O class (FoxO3a) is an important signaling pathway that regulates both genes. These inhibitory effects of resveratrol on Nox1 expression and MCP-1 production may target to the Akt and FoxO3a signaling pathways.

Prevention of TNF-induced necrotic cell death by rottlerin through a Nox1 NADPH oxidase

Previous studies have demonstrated that rottlerin, a specific PKCdelta inhibitor, potentiates death receptor- mediated apoptosis through a cytochrome c-dependent or -independent pathway. However, its ability to regulate necrotic cell death, as well as the underlying mechanism, remains unknown. We found that in murine fibrosarcoma L929 cells, treatment with rottlerin protected the cells against TNF-induced necrosis, whereas it sensitized the cells to apoptosis induced by co-treatment with Hsp90 inhibitor geldanamycin and TNF, in a manner independent of its ability to inhibit PKC-delta. TNF treatment induced rapid accumulation of mitochondrial superoxide (O2") through the Nox1 NADPH oxidase when cells undergo necrosis. Moreover, pretreatment with rottlerin failed to induce the GTP-bound form of small GTPase Rac1 by TNF treatment, and subsequently suppressed mitochondrial O2(-) production and poly(ADP-ribose) polymerase activation, thus inhibiting necrotic cell death. Therefore, our study suggests that Nox1 NADPH oxidase is a new molecular target for anti-necrotic activity of rottlerin upon death-receptor ligation.