Schisandrin B Protects against Acute Ethanol-Induced Cardiac Injury by Downregulating Autophagy via the NOX4/ROS Pathway.

INTRODUCTION: Although oxidative stress has been demonstrated to mediate acute ethanol-induced changes in autophagy in the heart, the precise mechanism behind redox regulation in acute ethanol heart disease remains largely unknown. METHODS: Wild-type C57BL/6 mice were intraperitoneally injected with ethanol (3 g/kg/day) for 3 consecutive days. The effects of ethanol on cultured primary cardiomyocytes and H9c2 myoblasts were also studied in vitro. Levels of autophagic flux, cardiac apoptosis and function, reactive oxygen species (ROS) accumulation, NOX4, and NOX2 were examined. The NOX4 gene was knocked down with NOX4 siRNA. RESULTS: In this study, we demonstrated that schisandrin B inhibited acute ethanol-induced autophagy and sequent apoptosis. In addition, schisandrin B treatment improved cardiac function in ethanol-treated mice. Furthermore, NOX4 protein expression was increased during acute ethanol exposure, and the upregulation of NOX4 was significantly inhibited by schisandrin B treatment. The knockdown of NOX4 prevented ROS accumulation, cell autophagy, and apoptosis. CONCLUSION: These results highlight that NOX4 is a critical mediator of ROS and elaborate the role of the NOX4/ROS axis in the effect of schisandrin B on autophagy and autophagy-mediated apoptosis in acute ethanol exposure, which suggests a therapeutic strategy for acute alcoholic cardiomyopathy.

Sodium butyrate enhances titanium nail osseointegration in ovariectomized rats by inhibiting the PKCα/NOX4/ROS/NF-κB pathways.

BACKGROUND: Elevated levels of oxidative stress as a consequence of estrogen deficiency serve as a key driver of the onset of osteoporosis (OP). In addition to increasing the risk of bone fractures, OP can reduce the bone volume proximal to titanium nails implanted to treat these osteoporotic fractures, thereby contributing to titanium nail loosening. Sodium butyrate (NaB) is a short-chain fatty acid produced by members of the gut microbiota that exhibits robust antioxidant and anti-inflammatory properties. METHODS: OP fracture model rats parameters including bone mineral density (BMD), new bone formation, and the number of bonelets around the implanted nail were analyzed via micro-CT scans, H&E staining, and Masson's staining. The protective effects of NaB on such osseointegration and the underlying mechanisms were further studied in vitro using MC3T3-E1 cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce oxidative stress. Techniques including Western immunoblotting, electron microscopy, flow cytometry, alkaline phosphatase (ALP) staining, and osteoblast mineralization assays were employed to probe behaviors such as reactive oxygen species production, mineralization activity, ALP activity, protein expression, and the ability of cells to attach to and survive on titanium plates. RESULTS: NaB treatment was found to enhance ALP activity, mineralization capacity, and Coll-I, BMP2, and OCN expression levels in CCCP-treated MC3T3-E1 cells, while also suppressing PKC and NF-κB expression and enhancing Nrf2 and HO-1 expression in these cells. NaB further suppressed intracellular ROS production and malondialdehyde levels within the cytosol while enhancing superoxide dismutase activity and lowering the apoptotic death rate. In line with these results, in vivo work revealed an increase in BMD in NaB-treated rats that was associated with enhanced bone formation surrounding titanium nails. CONCLUSION: These findings indicate that NaB may represent a valuable compound that can be postoperatively administered to aid in treating OP fractures through the enhancement of titanium nail osseointegration.

Suppressive effect erythropoietin on oxidative stress by targeting AMPK/Nox4/ROS pathway in renal ischemia reperfusion injury.

OBJECTIVE: To explore the effect of erythropoietin (EPO) on the AMP-activated protein kinase (AMPK)/nicotinamide adenine dinucleotide phosphatase oxidase 4 (NOX4) signaling pathway during renal ischemia reperfusion injury (RIRI) in rats. METHODS: A rat model of RIRI was established by clamping the left renal pedicle and removing the right kidney. The rats in the sham group did not have their left renal pedicle clamped. Rats with a model of RIRI were randomly divided into RIRI alone (control), erythropoietin treatment (EPO/RIRI), and Compound C treatment (CPC/RIRI) groups. Hematoxylin-eosin (H&E) staining was used to examine pathological kidney damage. Serum creatinine and urea nitrogen levels were measured to evaluate renal function. Western blotting was performed to detect the expression levels of phosphorylated p-AMPK and total AMPK protein in the kidneys. RT-PCR was used to evaluate the mRNA levels of Nox4 and p22 in the kidneys. Oxidative stress-related indices (ROS, CAT, GSH, SOD, and MDA) were also measured. RESULTS: EPO treatment improved kidney function by preventing kidney damage induced by the RIRI model. Preventing ischemia/reperfusion injury in the RIRI model was correlated with an increased p-AMPK/AMPK ratio and elevated activity of CAT, GSH, and SOD, which ameliorated the expression of NOX4, p22, ROS, and MDA. Moreover, treatment with CPC (an AMPK inhibitor) reduced the effects of EPO in the RIRI model. CONCLUSION: EPO treatment protected rats against RIRI in the RIRI model by alleviating oxidative stress by triggering the AMPK/NOX4/ROS pathway.

Waterfall Forest Environment Regulates Chronic Stress via the NOX4/ROS/NF-κB Signaling Pathway.

Background: Forest therapy has been proven to have beneficial effects on people with depression and anxiety. However, it remains unknown whether the waterfall forest environment (WF) affects the physical and psychological health of patients with chronic fatigue and how the WF regulates chronic stress. Methods: Twenty-four patients with chronic fatigue were randomly divided into two groups: the WF group and the urban (U) group. Scores on the Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD), and Fatigue Scale-14 (FS-14) were evaluated before and after environmental intervention. Detection of physiological indexes and inflammatory factor levels and immunological analysis were also performed. In addition, the chronic stress rat model was constructed, and the effects of the WF on hopelessness and liver damage of rats were investigated. Results: Patients with chronic fatigue in the WF group showed a significant decrease in FS-14, HAMA, and HAMD scores compared with the U group. The expression levels of glutathione peroxidase and superoxide dismutase were remarkably higher in the WF group than in the U group. However, the expression levels of malondialdehyde and inflammatory factors (IL-1ß, TNF-α, IL-6, and IL-10) were remarkably decreased after the intervention of the WF. In addition, animal experiments confirmed that the WF improved hopelessness, liver damage, and excitability of neurons of chronic stress rats. Mechanistically, the WF reduced the liver damage caused by chronic stress in rats by inhibiting the NOX4/ROS/NF-κB signaling pathway. Conclusions: Collectively, the WF had a positive effect on immune enhancement and physical and psychological health in patients with chronic fatigue and might inhibit chronic stress by regulating the NOX4/ROS/NF-κB signaling pathway.

Methyl ferulic acid attenuates liver fibrosis and hepatic stellate cell activation through the TGF-ß1/Smad and NOX4/ROS pathways.

Liver fibrosis is a pathological wound-healing response caused by chronic liver damage due to a virus, autoimmune disorder, or drugs. Hepatic stellate cells (HSCs) play an essential role in the pathogenesis of liver fibrosis. Methyl ferulic acid (MFA), a biologically active monomer, has a protective effect on liver injury. However, the effects and roles of MFA in liver fibrosis remain unknown. The purpose of the current study was to investigate the effect of MFA on hepatic fibrosis and the underlying mechanisms. Human hepatic stellate LX-2 cells were exposed to 5 µg/L TGF-ß1 for 48 h to stimulate liver fibrosis in vitro. Using MTT, RT-PCR and Western blot analysis, we revealed that MFA significantly inhibited the proliferation of LX-2 cells as well as decreased the expressions of α-SMA and type I collagen in LX-2 cells. SD rats were fed with ethanol, and this combined with the intraperitoneal injection of CCl4 induced liver fibrosis in vivo. We found that the administration of MFA markedly decreased the levels of hyaluronic acid (HA), procollagen type III (PC-III), type IV collagen (CIV) and laminin (LN) in the serum, inhibited the expression of α-smooth muscle actin (α-SMA) as well as type I and type III collagen, and up-regulated the ratio of MMP-2/TIMP-1 in rats. The antifibrotic effects of MFA were also evaluated by H&E staining and Masson's trichrome staining. In addition, further studies suggested that this protection by MFA from liver fibrosis was possibly related to the inhibition of TGF-ß1/Smad and NOX4/ROS signalling. In conclusion, our results demonstrate that MFA attenuated liver fibrosis and hepatic stellate cell activation by inhibiting the TGF-ß1/Smad and NOX4/ROS signalling pathways.

SAA1 increases NOX4/ROS production to promote LPS-induced inflammation in vascular smooth muscle cells through activating p38MAPK/NF-κB pathway.

BACKGROUND: To investigate the effects of serum amyloid A1 (SAA1) on lipopolysaccharide (LPS) -induced inflammation in vascular smooth muscle cells (VSMCs). SAA1 expression was detected in LPS induced VSMCs at different concentrations for different time by using Western blotting. After pre-incubation with recombinant SAA1 protein, VSMCs were treated with 1 µg/ml LPS for 24 h. The VSMCs were then divided into Control, SAA1 siRNA, Nox4 siRNA, LPS, LPS + SAA1 siRNA, LPS + Nox4 siRNA and LPS + SAA1 siRNA + Nox4 groups. MTT was performed to observe the toxicity of VSMCs. Lucigenin-enhanced chemiluminescence method was used to detect superoxide anion (O2-) production and NADPH oxidase activity. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine expressions of inflammatory factors. Western blotting was used to determine expressions of NOX-4 and p38MAPK/NF-κB pathway related proteins. RESULTS: LPS promoted SAA1 protein expression in a concentration-/time-dependent manner. Recombinant SAA1 protein could increase NOX4/ROS production and promote the release of inflammatory factors (IL-1ß, IL-6, IL-8, IL-17, TNF-α and MCP-1) in LPS (1 µg/ml) - induced VSMCs. Besides, both SAA1 siRNA and NOX-4 siRNA could not only enhance the O2- production and NADPH oxidase activity, but also up-regulate the protein expression of NOX4, the release of inflammatory factors, and the levels of p-p38 and p-NF-κB p65 in LPS-induced VSMCs. However, no significant differences in each index were observed between LPS group and LPS + SAA1 siRNA + Nox4 group. CONCLUSION: SAA1-mediated NOX4/ROS pathway could activate p38MAPK/NF-κB pathway, thereby contributing to the release of inflammatory factors in LPS-induced VSMCs.