Kaempferitrin attenuates unilateral ureteral obstruction-induced renal inflammation and fibrosis in mice by inhibiting NOX4-mediated tubular ferroptosis.

Tubular ferroptosis significantly contributes to renal inflammation and fibrosis, critical factors in chronic kidney disease (CKD). This study aims to investigate Kaempferitrin, a potent flavonoid glycoside from Bauhinia forficata leaves, renowned for its anti-inflammatory and antitumor effects, and to elucidate its potential mechanisms in mitigating inflammation and fibrosis induced by tubular ferroptosis. The study investigated Kaempferitrin's impact on tubular ferroptosis using a unilateral ureteral obstruction (UUO) model-induced renal inflammation and fibrosis. In vitro, erastin-induced ferroptosis in primary tubular epithelial cells (TECs) was utilized to further explore Kaempferitrin's effects. Additionally, NADPH oxidase 4 (NOX4) transfection in TECs and cellular thermal shift assay (CETSA) were conducted to identify Kaempferitrin's target protein. Kaempferitrin effectively improved renal function, indicated by reduced serum creatinine and blood urea nitrogen levels. In the UUO model, it significantly reduced tubular necrosis, inflammation, and fibrosis. Its renoprotective effects were linked to ferroptosis inhibition, evidenced by decreased iron, 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA) levels, and increased glutathione (GSH). Kaempferitrin also normalized glutathione peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11(SLC7A11) expression, critical ferroptosis mediators. In vitro, it protected TECs from ferroptosis and consistently suppressed NOX4 expression. NOX4 transfection negated Kaempferitrin's antiferroptosis effects, while CETSA confirmed Kaempferitrin-NOX4 interaction. Kaempferitrin shows promise as a nephroprotective agent by inhibiting NOX4-mediated ferroptosis in tubular cells, offering potential therapeutic value for CKD.

Sirtuin1 Mediates the Protective Effects of Echinacoside against Sepsis-Induced Acute Lung Injury via Regulating the NOX4-Nrf2 Axis.

Currently, the treatment for sepsis-induced acute lung injury mainly involves mechanical ventilation with limited use of drugs, highlighting the urgent need for new therapeutic options. As a pivotal aspect of acute lung injury, the pathologic activation and apoptosis of endothelial cells related to oxidative stress play a crucial role in disease progression, with NOX4 and Nrf2 being important targets in regulating ROS production and clearance. Echinacoside, extracted from the traditional Chinese herbal plant Cistanche deserticola, possesses diverse biological activities. However, its role in sepsis-induced acute lung injury remains unexplored. Moreover, although some studies have demonstrated the regulation of NOX4 expression by SIRT1, the specific mechanisms are yet to be elucidated. Therefore, this study aimed to investigate the effects of echinacoside on sepsis-induced acute lung injury and oxidative stress in mice and to explore the intricate regulatory mechanism of SIRT1 on NOX4. We found that echinacoside inhibited sepsis-induced acute lung injury and oxidative stress while preserving endothelial function. In vitro experiments demonstrated that echinacoside activated SIRT1 and promoted its expression. The activated SIRT1 was competitively bound to p22 phox, inhibiting the activation of NOX4 and facilitating the ubiquitination and degradation of NOX4. Additionally, SIRT1 deacetylated Nrf2, promoting the downstream expression of antioxidant enzymes, thus enhancing the NOX4-Nrf2 axis and mitigating oxidative stress-induced endothelial cell pathologic activation and mitochondrial pathway apoptosis. The SIRT1-mediated anti-inflammatory and antioxidant effects of echinacoside were validated in vivo. Consequently, the SIRT1-regulated NOX4-Nrf2 axis may represent a crucial target for echinacoside in the treatment of sepsis-induced acute lung injury.

Orange peels and Chlorella vulgaris supplementation ameliorate gamma radiation-induced oxidative stress by regulating TGF-ß and NOX2/NOX4 signaling pathways.

Numerous studies revealed that Chlorella vulgaris and orange peels are potential sources for many valuable compounds such as flavonoids, which are natural polyphenols with antioxidant capacities that lessen oxidative stress via suppressing ROS levels. Thus, this study was designed to investigate their radioprotective efficiency either alone or in combination as natural food supplements. Sixty-four male Albino rats were divided into eight groups (n = 8) as follows: control, orange peel (10% in diet), C. vulgaris (1% in diet), orange peel + C. vulgaris, gamma irradiated (2Gy twice per week up to 8Gy), orange peel + gamma irradiation, C. vulgaris + gamma irradiation, and orange peel + C. vulgaris + gamma irradiation. After the experiment, blood serums were collected for biochemical analysis, whole bloods were collected for blood picture, bone marrows were collected for GSH, MDA, TGF-ß, NOX2 and NOX4, and liver tissues were collected for histopathological evaluation. Current study revealed that exposure to gamma irradiation induced a significant disturbance in liver function markers (ALT and AST), kidney function markers (urea and creatinine), cholesterol and triglycerides levels in serum. In addition, a significant decrease in WBCs, RBCs, PLT, and Hb in blood of irradiated rats. Moreover, a significant elevation in TGF-ß, NOX2, NOX4 activities, and MDA level, while showed a marked decrease in GSH concentration. Furthermore, hepatic inflammation appeared in the histopathological examination. Orange peels or C. vulgaris treatments showed acceptable amelioration in all measured parameters, combination between orange peels and C. vulgaris showed statistically significant additive amelioration in radiation induced disturbance.

Chronic SIRT1 supplementation in diabetic mice improves endothelial function by suppressing oxidative stress.

AIMS: Enhancing SIRT1 activity exerts beneficial cardiovascular effects. In diabetes, plasma SIRT1 levels are reduced. We aimed to investigate the therapeutic potential of chronic recombinant murine SIRT1 (rmSIRT1) supplementation to alleviate endothelial and vascular dysfunction in diabetic mice (db/db). METHODS AND RESULTS: Left internal mammary arteries obtained from patients undergoing coronary artery bypass grafting with or without a diagnosis of diabetes were assayed for SIRT1 protein levels. Twelve-week-old male db/db mice and db/+ controls were treated with vehicle or rmSIRT1 intraperitoneally for 4 weeks, after which carotid artery pulse wave velocity (PWV) and energy expenditure/activity were assessed by ultrasound and metabolic cages, respectively. Aorta, carotid, and mesenteric arteries were isolated to determine endothelial and vascular function using the myograph system.Arteries obtained from diabetic patients had significantly lower levels of SIRT1 relative to non-diabetics. In line, aortic SIRT1 levels were reduced in db/db mice compared to db/+ mice, while rmSIRT1 supplementation restored SIRT1 levels. Mice receiving rmSIRT1 supplementation displayed increased physical activity and improved vascular compliance as reflected by reduced PWV and attenuated collagen deposition. Aorta of rmSIRT1-treated mice exhibited increased endothelial nitric oxide (eNOS) activity, while endothelium-dependent contractions of their carotid arteries were significantly decreased, with mesenteric resistance arteries showing preserved hyperpolarization. Ex vivo incubation with reactive oxygen species (ROS) scavenger Tiron and NADPH oxidase inhibitor apocynin revealed that rmSIRT1 leads to preserved vascular function by suppressing NADPH oxidase (NOX)-related ROS synthesis. Chronic rmSIRT1 treatment resulted in reduced expression of both NOX1 and NOX4, in line with a reduction in aortic protein carbonylation and plasma nitrotyrosine levels. CONCLUSIONS: In diabetic conditions, arterial SIRT1 levels are significantly reduced. Chronic rmSIRT1 supplementation improves endothelial function and vascular compliance by enhancing eNOS activity and suppressing NOX-related oxidative stress. Thus, SIRT1 supplementation may represent novel therapeutic strategy to prevent diabetic vascular disease.

6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells.

In women, ovary cancer is already the fifth leading cause of mortality worldwide. The use of cancer therapies, such as surgery, radiotherapy, and chemotherapy, may be a powerful anti-cancer therapeutic strategy; however, these therapies still have many problems, including resistance, toxicity, and side effects. Therefore, natural herbal medicine has the potential to be used for cancer therapy because of its low toxicity, fewer side effects, and high success. This study aimed to investigate the anti-cancer effect of 6-shogaol in ovarian cancer cells. 6-shogaol induces ER stress and cell death via the reduction in cell viability, the increase in LDH cytotoxicity, caspase-3 activity, and Ca2+ release, and the upregulation of GRP78, p-PERK, p-eIF2α, ATF-4, CHOP, and DR5. Moreover, 6-shogaol treatment medicates endoplasmic reticulum (ER) stress and cell death by upregulating Nox4 and releasing ROS. The knockdown of Nox4 in ovarian cancer cells inhibits ER stress and cell death by blocking the reduction in cell viability and the enhancement of LDH cytotoxicity, caspase-3 activity, Ca2+, and ROS release. In gefitinib-resistant ovarian cancer cells, A2780R and OVCAR-3R, 6-shogaol/gefitinib overcomes gefitinib resistance by inhibiting EMT phenomena such as the reduction in E-cadherin, and the increase in N-cadherin, vimentin, Slug, and Snail. Therefore, our results suggest that 6-shogaol exerts a potential anti-cancer effect in ovarian cancer and combination treatment with 6-shogaol and gefitinib may provide a novel anti-tumor therapeutic strategy in gefitinib-resistant ovarian cancer.

Synergistic properties of garlic and Citrullus colocynthis on reproductive injury caused by diabetes in male rats: Structural and molecular evidence.

This study evaluates the synergistic effect of garlic and Citrullus colocynthis on diabetic reproductive damage by suppressing the AGEs/RAGE/Nox-4 signaling pathway. Thirty-five male Wistar rats were divided into five groups (n = 7/group): Control, Diabetic, Diabetic+G (Garlic, 1 mL/100 g b.w), Diabetic+C (C. colocynthis, 10 mg/kg b.w) and Diabetic+GC (Garlic, 1 mL/100 g b.w and C. colocynthis, 10 mg/kg b.w) groups. At the end of the experimental period (30 days), in diabetic rats, glucose increased, and body & testis weight, luteinizing hormone (LH) and testosterone levels, and sperm count decreased significantly and histopathological injuries were observed. In addition, they have increased testicular apoptosis and oxidative stress. Also, the mechanism based on advanced glycation end products (AGEs)/receptors for advanced glycation end products (RAGE)/NADPH oxidase-4 (Nox-4) was activated in diabetic rats. Separate consumption of garlic and C. colocynthis in Diabetic+G and Diabetic+C groups alleviated the negative adverse effect of diabetes to some extent, but when they were used in the combination form (Diabetic+GC) improvement was profound. Testis histopathology, increased body and testis weight, and enhanced capacity in protecting diabetic reproductive injury was seen. Decreases in testosterone and LH concentration and sperm count in diabetic rats were also reversed by combined administration of garlic and C. colocynthis. It regulated oxidative stress markers, meanwhile reducing caspase-3 immunoexpression. In addition, overexpression of RAGE, Nox-4 and nuclear transcription factor-κB (NF-κB) was inhibited by the combination of garlic and C. colocynthis. PRACTICAL APPLICATIONS: Diabetes mellitus is wide spread all around the world with variety of complications in body including reproductive system in which patients suffer from physical and psychological aspects. Despite many efforts in providing agents for controlling diabetes and its complications, economic conditions of some countries make it difficult for people to provide costly medicine and as a result, they have to bear the complications until they pass away. However, traditional medicine is still finding its way, especially in poor countries with emphasis on medicinal plants. There have been many studies on plants to alleviate diabetes or its side effects. But, using one plant for long term, may be not so effective. Here, we attempted to find whether two plants from two different species can show more efficacy than each one alone. We noticed garlic and Citrullus colocynthis despite having beneficial effects when used alone, they could show synergistic effects in combination.

Polydatin Ameliorates High Fructose-Induced Podocyte Oxidative Stress via Suppressing HIF-1α/NOX4 Pathway.

Long-term high fructose intake drives oxidative stress, causing glomerular podocyte injury. Polydatin, isolated from Chinese herbal medicine Polygonum cuspidatum, is used as an antioxidant agent that protects kidney function. However, it remains unclear how polydatin prevents oxidative stress-driven podocyte damage. In this study, polydatin attenuated high fructose-induced high expression of HIF-1α, inhibited NOX4-mediated stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 (SDF-1α/CXCR4) axis activation, reduced reactive oxygen species (ROS) production in rat glomeruli and cultured podocytes. As a result, polydatin up-regulated nephrin and podocin, down-regulated transient receptor potential cation channel 6 (TRPC6) in these animal and cell models. Moreover, the data from HIF-1α siRNA transfection showed that high fructose increased NOX4 expression and aggravated SDF-1α/CXCR4 axis activation in an HIF-1α-dependent manner, whereas polydatin down-regulated HIF-1α to inhibit NOX4 and suppressed SDF-1α/CXCR4 axis activation, ameliorating high fructose-induced podocyte oxidative stress and injury. These findings demonstrated that high fructose-driven HIF-1α/NOX4 pathway controlled podocyte oxidative stress damage. Intervention of this disturbance by polydatin could help the development of the therapeutic strategy to combat podocyte damage associated with high fructose diet.

Complanatoside A targeting NOX4 blocks renal fibrosis in diabetic mice by suppressing NLRP3 inflammasome activation and autophagy.

BACKGROUND: Diabetic nephropathy (DN) is an important cause of end-stage renal disease. Complanatoside A (CA), an active component from Semen Astragali Complanati, has been reported to be a potential candidate for the treatment of kidney diseases. However, the underlying mechanisms and protective effects of CA in DN remain unclear. PURPOSE: In this paper, the effects and the mechanism of CA against ameliorating DN were investigated in vivo and in vitro. STUDY DESIGN: Here, a high-fat diet/streptozotocin-induced diabetic model and TGF-ß1-induced HK-2 cells were used to explore the protective effects and mechanisms of CA on DN in vivo and in vitro. METHODS: Major biochemical indexes, Histopathological morphology, and Immunohistochemistry have explored the therapeutic effect of CA on DN. Subsequently, TGF-ß1-induced HK-2 cells were utilized to investigate the anti-renal fibrosis effect of CA. Finally, the mechanism of CA against renal fibrosis was studied via western blotting, immunofluorescence, transfection, and molecular docking. RESULTS: The results showed that CA attenuated glomerular hypertrophy, collagen matrix deposition, and tubular interstitial fibrosis in diabetic mice. Moreover, the activation of TGF-ß1-inducible epithelial-mesenchymal transition (EMT) was hindered by CA treatment in HK-2 cells. Mechanistically, the data suggested that upregulated NOX4 during diabetes and TGF-ß1 in HK-2 cells was prominently diminished after CA treatment. Furthermore, CA exposure inhibited NLRP3 inflammasome activation and downstream inflammation gene expression such as IL-18 and IL-1ß in vivo and vitro. These findings indicated that CA obstructed the EMT to protect renal tubular epithelial cells against fibrosis via blocking NLRP3 activation, which was associated with inhibiting NOX4. Besides, the markedly raised autophagy levels in the diabetic model characterized by increasing LC3II/LC3I and Beclin1 were reversed after CA treatment, which is also a pivotal mechanism against renal fibrosis. More importantly, specific NOX4 overexpressed in HK-2 cells abolished that CA exposure blocked TGF-ß1-induced-EMT, ROS generation, NLRP3, and autophagy activation. Meanwhile, the inhibition of cell migration, ROS generation, autophagy, and renal inflammation after CA treatment was more pronounced in NOX4-deficient HK-2 cells. CONCLUSION: Our findings provided evidence that CA might be a potential therapeutic agent for DN by ameliorating NLRP3 inflammasome and autophagy activation via targeting NOX4 inhibition.

The effect of Kappaphycus alvarezii active fraction on oxidative stress and inflammation in streptozotocin and nicotinamide-induced diabetic rats.

BACKGROUND: High glucose concentration increases the glycation process which leads to oxidative stress and inflammation, that can cause complications in diabetes. Several medicinal plants have been used in the treatment of diabetes and its complications. One of them is Kappaphycus alvarezii, an algae that has known antidiabetic abilities. This study aimed to examine the effect of K. alvarezii active fraction on plasma hydrogen peroxide (H2O2) and Tumor Necrosis Factor α (TNFα) levels, renal NADPH oxidase 4 (NOX4) and Nuclear Factor κ B (NFκB) gene expressions. METHODS: Active fraction was obtained from bioassay-guided fractionation with antiglycation ability. In vivo study was performed on twenty Wistar male rats. The level of H2O2 was measured using H2O2 Assay Kit, the Optical Density value measured using spectrophotometer at a wavelength of 405 nm. Plasma TNFα level was measured using ELISA. Renal NOX4 and NFκB gene expression was analyzed using qPCR. RESULTS: Active fraction significantly reduced plasma H2O2 but not TNFα levels. Furthermore, renal NOX4 gene expression was lower in the diabetic rat group treated with active fraction compared to the untreated group but not NFκB gene expression. CONCLUSIONS: K. alvarezii active fraction has an activity to reduce plasma H2O2 as well as renal NOX4 gene expression. Therefore, this fraction could be developed as a potential candidate for diabetes treatment through oxidative stress mechanisms.

Ginsenoside Rb1 alleviates diabetic kidney podocyte injury by inhibiting aldose reductase activity.

Panax notoginseng, a traditional Chinese medicine, exerts beneficial effect on diabetic kidney disease (DKD), but its mechanism is not well clarified. In this study we investigated the effects of ginsenoside Rb1 (Rb1), the main active ingredients of Panax notoginseng, in alleviating podocyte injury in diabetic nephropathy and the underlying mechanisms. In cultured mouse podocyte cells, Rb1 (10 µM) significantly inhibited high glucose-induced cell apoptosis and mitochondrial injury. Furthermore, Rb1 treatment reversed high glucose-induced increases in Cyto c, Caspase 9 and mitochondrial regulatory protein NOX4, but did not affect the upregulated expression of aldose reductase (AR). Molecular docking analysis revealed that Rb1 could combine with AR and inhibited its activity. We compared the effects of Rb1 with eparestat, a known aldose reductase inhibitor, in high glucose-treated podocytes, and found that both alleviated high glucose-induced cell apoptosis and mitochondrial damage, and Rb1 was more effective in inhibiting apoptosis. In AR-overexpressing podocytes, Rb1 (10 µM) inhibited AR-mediated ROS overproduction and protected against high glucose-induced mitochondrial injury. In streptozotocin-induced DKD mice, administration of Rb1 (40 mg·kg-1·d-1, ig, for 7 weeks) significantly mitigated diabetic-induced glomerular injuries, such as glomerular hypertrophy and mesangial matrix expansion, and reduced the expression of apoptotic proteins. Collectively, Rb1 combines with AR to alleviate high glucose-induced podocyte apoptosis and mitochondrial damage, and effectively mitigates the progression of diabetic kidney disease.

The Herbal Formula JI017 Induces ER Stress via Nox4 in Breast Cancer Cells.

Chemotherapy is a powerful anti-tumor therapeutic strategy; however, resistance to treatment remains a serious concern. To overcome chemoresistance, combination therapy with anticancer drugs is a potential strategy. We developed a novel herbal extract, JI017, with lower toxicity and lesser side effects. JI017 induced programmed cell death and excessive unfolded protein response through the release of intracellular reactive oxygen species (ROS) and calcium in breast cancer cells. JI017 treatment increased the expression of endoplasmic reticulum (ER) stress markers, including p-PERK, p-eIF2α, ATF4, and CHOP, via the activation of both exosomal GRP78 and cell lysate GRP78. The ROS inhibitors diphenyleneiodonium and N-acetyl cysteine suppressed apoptosis and excessive ER stress by inhibiting Nox4 in JI017-treated breast cancer cells. Furthermore, in paclitaxel-resistant breast cancer cell lines, MCF-7R and MDA-MB-231R, a combination of JI017 and paclitaxel overcame paclitaxel resistance by blocking epithelial-mesenchymal transition (EMT) processes, such as the downregulation of E-cadherin expression and the upregulation of HIF-1α, vimentin, Snail, and Slug expression. These findings suggested that JI017 exerts a powerful anti-cancer effect in breast cancer and a combination therapy of JI017 and paclitaxel may be a potential cancer therapy for paclitaxel resistant breast cancer.

JI017, a Complex Herbal Medication, Induces Apoptosis via the Nox4-PERK-CHOP Axis in Ovarian Cancer Cells.

Many anti-cancer drugs, including paclitaxel and etoposide, have originated and been developed from natural products, and traditional herbal medicines have fewer adverse effects and lesser toxicity than anti-tumor reagents. Therefore, we developed a novel complex herbal medicine, JI017, which mediates endoplasmic reticulum (ER) stress and apoptosis through the Nox4-PERK-CHOP signaling pathway in ovarian cancer cells. JI017 treatment increases the expression of GRP78, ATF4, and CHOP and the phosphorylation of PERK and eIF2α via the upregulation of Nox4. Furthermore, it increases the release of intracellular reactive oxygen species (ROS), the production of intracellular Ca2+, and the activation of exosomal GRP78 and cell lysate GRP78. Combination treatment using the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG) and JI017 reportedly induces increased ER stress and cell death in comparison to the control; however, knockdown experiments of PERK and CHOP indicated suppressed apoptosis and ER stress in JI017-treated ovarian cancer cells. Furthermore, targeting Nox4 using specific siRNA and pharmacological ROS inhibitors, including N-acetylcystein and diphenylene iodonium, blocked apoptosis and ER stress in JI017-treated ovarian cancer cells. In the radioresistant ovarian cancer model, when compared to JI017 alone, JI017 co-treatment with radiation induced greater cell death and resulted in overcoming radioresistance by inhibiting epithelial-mesenchymal-transition-related phenomena such as the reduction of E-cadherin and the increase of N-cadherin, vimentin, Slug, and Snail. These findings suggest that JI017 is a powerful anti-cancer drug for ovarian cancer treatment and that its combination treatment with radiation may be a novel therapeutic strategy for radioresistant ovarian cancer.

Smad3-Targeted Therapy Protects against Cisplatin-Induced AKI by Attenuating Programmed Cell Death and Inflammation via a NOX4-Dependent Mechanism.

BACKGROUND: Transforming growth factor-ß (TGF-ß)/Smad signaling is the central mediator in renal fibrosis, yet its functional role in acute kidney injury (AKI) is not fully understood. Recent evidence showed that TGF-ß/Smad3 may be involved in the pathogenesis of AKI, but its functional role and mechanism of action in cisplatin-induced AKI are unclear. OBJECTIVES: Demonstrating that Smad3 may play certain roles in cisplatin nephropathy due to its potential effect on programmed cell death and inflammation. METHODS: Here, we established a cisplatin-induced AKI mouse model with Smad3 knockout mice and created stable in vitro models with Smad3 knockdown tubular epithelial cells. In addition, we tested the potential of Smad3-targeted therapy using 2 in vivo protocols - lentivirus-mediated Smad3 silencing in vivo and use of naringenin, a monomer used in traditional Chinese medicine and a natural inhibitor of Smad3. RESULTS: Disruption of Smad3 attenuated cisplatin-induced kidney injury, inflammation, and NADPH oxidase 4-dependent oxidative stress. We found that Smad3-targeted therapy protected against loss of renal function and alleviated apoptosis, RIPK-mediated necroptosis, renal inflammation, and oxidative stress in cisplatin nephropathy. CONCLUSIONS: These findings show that Smad3 promotes cisplatin-induced AKI and Smad3-targeted therapy protects against this pathological process. These findings have substantial clinical relevance, as they suggest a therapeutic target for AKI.

Clinical efficacies, underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management.

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN.

Clitoria ternatea L. extract prevents kidney damage by suppressing the Ang II/Nox4/oxidative stress cascade in l-NAME-induced hypertension model of rats.

Clitoria ternatia L. (CT) has been reported to have anti-inflammatory and antioxidant effects. This study investigated the effect of CT aqueous flower extract on blood pressure and renal alterations in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats. Male Sprague Dawley rats received l-NAME in drinking water and were treated with CT flower extract or lisinopril. CT aqueous flower extract and lisinopril alleviated l-NAME-induced hypertension (p < 0.05). Glomerular extracellular matrix accumulation, renal fibrosis, and increased serum creatinine levels were observed in l-NAME-induced hypertensive rats and attenuated by CT flower extract or lisinopril co-treatment (p < 0.05). High levels of plasma angiotensin II (Ang II) and upregulated nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) protein expression in the kidneys induced by l-NAME were alleviated by CT flower extract or lisinopril co-treatment (p < 0.05). Furthermore, CT flower extract and lisinopril treatment reduced lipid peroxidation and elevated plasma and kidney malondialdehyde levels in l-NAME-induced hypertensive rats (p < 0.05). In conclusion, CT flower extract prevented l-NAME-induced renal injury and dysfunction in rats. The possible mechanism may be related to the suppression of Ang II-mediated Nox4 expression and the oxidative stress cascade in rats.

Therapeutic potential of diosmin, a citrus flavonoid against arsenic-induced neurotoxicity via suppression of NOX 4 and its subunits.

OBJECTIVES: Water contaminated with arsenic affected millions of people worldwide and arsenic exposure is related to various neurological disorders. Hence, the current study was planned to investigate the neuroprotective activity of diosmin (DSN) against arsenic induced neurotoxicity as an attempt to identify therapeutic intervention to combat arsenicism. MATERIALS AND METHODS: Sodium arsenite an inducer of neurotoxicity was administered orally (13 mg/kg) and DSN treatment at two selected doses (50 and 100 mg/kg) was done for 21 days. Behavioral and biochemical variations were examined by various parameters. Furthermore, histopathological and immunohistochemistry studies were done with the brain sections. RESULTS: The behavioral studies evidenced that arsenic has suppressed the exploratory behavior and motor coordination in rats and DSN treatment has recovered the behavioral changes to normal. Arsenic administration has also found to induce oxidative stress and DSN co-treatment has ameliorated the oxidative stress markers. Interestingly, depleted levels of neurotransmitters were observed with the arsenic and it was restored back by the DSN treatment. Histopathological alterations like pyknosis of the neuronal cells were identified with arsenic exposure and subsided upon DSN co administration. Immunohistochemical studies have revealed the expression of NOX4 and its gp91phox and P47phox subunits and its suppression by DSN treatment may be the key therapeutic factor of it. CONCLUSIONS: Treatment with DSN showed a beneficial effect in protecting against arsenic-induced neurotoxicity by suppressing the toxicity changes and the antioxidant effect of DSN might be attributed to its ability of suppressing NOX4 and its subunits.

[Effect of methyl eugenol on hypoxia/reoxygenation injury of human renal tubular epithelial cells and its mechanism].

This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 µmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.

Effect of methyl eugenol on hypoxia/reoxygenation injury of human renal tubular epithelial cells and its mechanism / 中国中药杂志

This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 μmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.

Functional Food XingJiuTang Attenuates Alcohol-Induced Liver Injury by Regulating SIRT1/Nrf-2 Signaling Pathway.

Lipid accumulation, inflammatory responses and oxidative stress have been implicated in the pathology of alcohol-induced liver injury (ALI). In this work, we evaluated the effects of the functional food XingJiuTang (XJT) on ALI and explored the underlying mechanism. We used alcohol-stimulated human normal hepatocytes L02 for in vitro experiments, while for in vivo experiments, 55 % alcohol was intragastrically administrated to C57BL/6 mice at 16 mL/kg with pre-administration of bifendate and XJT. Liver histology and function, along with the inflammatory cytokines, oxidative mediators and SIRT1/Nrf-2 pathway were evaluated. The results showed that XJT treatment significantly alleviated ALI, ameliorated lipid peroxidation, improved the liver function impaired by alcohol and inhibited the hepatocytes apoptosis in vitro and in vivo. In addition, XJT treatment modulated the activation of the SIRT1/Nrf-2 signaling pathway and suppressed the overexpression of NOX4. Overall, the functional food XJT effectively protects against experimental ALI via activating the SIRT1/Nrf-2 pathway.

Morroniside Inhibits H2O2-Induced Podocyte Apoptosis by Down-Regulating NOX4 Expression Controlled by Autophagy In Vitro.

Podocyte apoptosis is the common pathological basis for the progression of various kidney diseases. The overexpression of NOX4, a key enzyme involved in oxidative stress, has been proved to participate in the occurrence of podocyte apoptosis. Autophagy is a kind of adaptive response of cells under stress. However, as a "double-edged sword", the effect of autophagy on apoptosis in different cells and conditions is complex and variable, which has not been fully explained yet. Morroniside, extracted from the traditional medicinal plant Cornus officinalis, has remarkable antioxidant and anti-apoptosis effects, and has been proven to inhibit the overexpression of NOX4 in kidney tissue. Therefore, H2O2 was used in this study to explore the effects of autophagy on podocyte NOX4 overexpression and apoptosis induced by oxidative stress, as well as the protection mechanism of morroniside in podocytes. The results showed that the autophagy activator rapamycin, as well as the autophagy inhibitor chloroquine, could induce podocyte apoptosis cultured in normal condition, and chloroquine could also significantly increase the NOX4 expression. The NOX4 expression and apoptosis rate of podocytes increased after H2O2 treatment, the expression of LC3-II decreased, and the expressions of p62, mTOR, and p-mTOR increased. The intervention of morroniside and rapamycin improved autophagy activity and inhibited NOX4 overexpression and apoptosis induced by H2O2. And chloroquine reversed the inhibitory effect of morroniside on NOX4 overexpression and podocyte apoptosis. Taken together, our results suggest that the expression level of NOX4 in podocytes is regulated by autophagy activity. Morroniside can reduce oxidative stress induced podocyte apoptosis by restoring the damaged autophagy flux and inhibit the overexpression of NOX4.