Ginsenoside Rg1 alleviates chronic inflammation-induced neuronal ferroptosis and cognitive impairments via regulation of AIM2 - Nrf2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng is a valuable herb in traditional Chinese medicine. Modern research has shown that it has various benefits, including tonifying vital energy, nourishing and strengthening the body, calming the mind, improving cognitive function, regulating fluids, and returning blood pressure, etc. Rg1 is a primary active component of ginseng. It protects hippocampal neurons, improves synaptic plasticity, enhances cognitive function, and boosts immunity. Furthermore, it exhibits anti-aging and anti-fatigue properties and holds great potential for preventing and managing neurodegenerative diseases (NDDs). AIM OF THE STUDY: The objective of this study was to examine the role of Rg1 in treating chronic inflammatory NDDs and its molecular mechanisms. MATERIALS AND METHODS: In vivo, we investigated the protective effects of Rg1 against chronic neuroinflammation and cognitive deficits in mice induced by 200 µg/kg lipopolysaccharide (LPS) for 21 days using behavioral tests, pathological sections, Western blot, qPCR and immunostaining. In vitro experiments involved the stimulation of HT22 cells with 10 µg/ml of LPS, verification of the therapeutic effect of Rg1, and elucidation of its potential mechanism of action using H2DCFDA staining, BODIPY™ 581/591 C11, JC-1 staining, Western blot, and immunostaining. RESULTS: Firstly, it was found that Rg1 significantly improved chronic LPS-induced behavioral and cognitive dysfunction in mice. Further studies showed that Rg1 significantly attenuated LPS-induced neuronal damage by reducing levels of IL-6, IL-1ß and ROS, and inhibiting AIM2 inflammasome. Furthermore, chronic LPS exposure induced the onset of neuronal ferroptosis by increasing the lipid peroxidation product MDA and regulating the ferroptosis-associated proteins Gpx4, xCT, FSP1, DMT1 and TfR, which were reversed by Rg1 treatment. Additionally, Rg1 was found to activate Nrf2 and its downstream antioxidant enzymes, such as HO1 and NQO1, both in vivo and in vitro. In vitro studies also showed that the Nrf2 inhibitor ML385 could inhibit the anti-inflammatory, antioxidant, and anti-ferroptosis effects of Rg1. CONCLUSIONS: This study demonstrated that Rg1 administration ameliorated chronic LPS-induced cognitive deficits and neuronal ferroptosis in mice by inhibiting neuroinflammation and oxidative stress. The underlying mechanisms may be related to the inhibition of AIM2 inflammasome and activation of Nrf2 signaling. These findings provide valuable insights into the treatment of chronic neuroinflammation and associated NDDs.

Ginsenoside Rg1 attenuates lipopolysaccharide-induced chronic liver damage by activating Nrf2 signaling and inhibiting inflammasomes in hepatic cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng (Panax ginseng C. A. Meyer) is a precious traditional Chinese medicine with multiple pharmacological effects. Ginsenoside Rg1 is a main active ingredient extracted from ginseng, which is known for its age-delaying and antioxidant effects. Increasing evidence indicates that Rg1 exhibits anti-inflammatory properties in numerous diseases and may ameliorate oxidative damage and inflammation in many chronic liver diseases. AIM OF THE STUDY: Chronic inflammatory injury in liver cells is an important pathological basis of many liver diseases. However, its mechanism remains unclear and therapeutic strategies to prevent its development need to be further explored. Thus, our study is to delve the protective effect and mechanism of Rg1 against chronic hepatic inflammatory injuries induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: The chronic liver damage model in mice was build up by injecting intraperitoneally with LPS (200 µg/kg) for 21 days. Serum liver function indicators and levels of IL-1ß, IL-6 and TNF-α were examined by using corresponding Kits. Hematoxylin and Eosin (H&E), Periodic acid-Schiff (PAS), and Masson stains were utilized to visualize hepatic histopathological damage, glycogen deposition, and liver fibrosis. The nuclear import of p-Nrf2 and the generation of Col4 in the liver were detected by IF, while IHC was employed to detect the expressions of NLRP3 and AIM2 in the hepatic. The Western blot and q-PCR were used to survey the expressions of proteins and mRNAs of fibrosis and apoptosis, and the expressions of Keap1, p-Nrf2 and NLRP3, NLRP1, AIM2 inflammasome-related proteins in mouse liver. The cell viability of human hepatocellular carcinoma cells (HepG2) was detected by Cell Counting Kit-8 to select the action concentration of LPS, and intracellular ROS generation was detected using a kit. The expressions of Nuclear Nrf2, HO-1, NQO1 and NLRP3, NLRP1, and AIM2 inflammasome-related proteins in HepG2 cells were detected by Western blot. Finally, the feasibility of the molecular interlinking between Rg1 and Nrf2 was demonstrated by molecular docking. RESULTS: Rg1 treatment for 21 days decreased the levels of ALT, AST, and inflammatory factors of serum IL-1ß, IL-6 and TNF-α in mice induced by LPS. Pathological results indicated that Rg1 treatment obviously alleviated hepatocellular injury and apoptosis, inflammatory cell infiltration and liver fibrosis in LPS stimulated mice. Rg1 promoted Keap1 degradation and enhanced the expressions of p-Nrf2, HO-1 and decreased the levels of NLRP1, NLRP3, AIM2, cleaved caspase-1, IL-1ß and IL-6 in livers caused by LPS. Furthermore, Rg1 effectively suppressed the rise of ROS in HepG2 cells induced by LPS, whereas inhibition of Nrf2 reversed the role of Rg1 in reducing the production of ROS and NLRP3, NLRP1, and AIM2 expressions in LPS-stimulated HepG2 cells. Finally, the molecular docking illustrated that Rg1 exhibits a strong affinity towards Nrf2. CONCLUSION: The findings indicate that Rg1 significantly ameliorates chronic liver damage and fibrosis induced by LPS. The mechanism may be mediated through promoting the dissociation of Nrf2 from Keap1 and then activating Nrf2 signaling and further inhibiting NLRP3, NLRP1, and AIM2 inflammasomes in liver cells.

Ginsenoside Rg1 treatment protects against cognitive dysfunction via inhibiting PLC-CN-NFAT1 signaling in T2DM mice.

Background: As a complication of Type II Diabetes Mellitus (T2DM), the etiology, pathogenesis, and treatment of cognitive dysfunction are still undefined. Recent studies demonstrated that Ginsenoside Rg1 (Rg1) has promising neuroprotective properties, but the effect and mechanism in diabetes-associated cognitive dysfunction (DACD) deserve further investigation. Methods: After establishing the T2DM model with a high-fat diet and STZ intraperitoneal injection, Rg1 was given for 8 weeks. The behavior alterations and neuronal lesions were judged using the open field test (OFT) and Morris water maze (MWM), as well as HE and Nissl staining. The protein or mRNA changes of NOX2, p-PLC, TRPC6, CN, NFAT1, APP, BACE1, NCSTN, and Aß1-42 were investigated by immunoblot, immunofluorescence or qPCR. Commercial kits were used to evaluate the levels of IP3, DAG, and calcium ion (Ca2+) in brain tissues. Results: Rg1 therapy improved memory impairment and neuronal injury, decreased ROS, IP3, and DAG levels to revert Ca2+ overload, downregulated the expressions of p-PLC, TRPC6, CN, and NFAT1 nuclear translocation, and alleviated Aß deposition in T2DM mice. In addition, Rg1 therapy elevated the expression of PSD95 and SYN in T2DM mice, which in turn improved synaptic dysfunction. Conclusions: Rg1 therapy may improve neuronal injury and DACD via mediating PLC-CN-NFAT1 signal pathway to reduce Aß generation in T2DM mice.

Interruption of TRPC6-NFATC1 signaling inhibits NADPH oxidase 4 and VSMCs phenotypic switch in intracranial aneurysm.

Intracranial aneurysm (IA) is a frequent cerebrovascular disorder with unclear pathogenesis. The vascular smooth muscle cells (VSMCs) phenotypic switch is essential for IA formation. It has been reported that Ca2+ overload and excessive reactive oxygen species (ROS) are involved in VSMCs phenotypic switch. The transient receptor potential canonical 6 (TRPC6) and NADPH oxidase 4 (NOX4) are the main pathway to participate in Ca2+ overload and ROS production in VSMCs. Ca2+ overload can activate calcineurin (CN), leading to nuclear factor of activated T cell (NFAT) dephosphorylation to regulate the target gene's transcription. We hypothesized that activation of TRPC6-NFATC1 signaling may upregulate NOX4 and involve in VSMCs phenotypic switch contributing to the progression of IA. Our results showed that the expressions of NOX4, p22phox, p47phox, TRPC6, CN and NFATC1 were significantly increased, and VSMCs underwent a significant phenotypic switch in IA tissue and cellular specimens. The VIVIT (NFATC1 inhibitor) and BI-749327 (TRPC6 inhibitor) treatment reduced the expressions of NOX4, p22phox and p47phox and the production of ROS, and significantly improved VSMCs phenotypic switch in IA rats and cells. Consistent results were obtained from IA Trpc6 knockout (Trpc6-/-) mice. Furthermore, the results also revealed that NFATC1 could regulate NOX4 transcription by binding to its promoter. Our findings reveal that interrupting the TRPC6-NFATC1 signaling inhibits NOX4 and improves VSMCs phenotypic switch in IA, and regulating Ca2+ homeostasis may be an important therapeutic strategy for IA.

Ginsenoside Rg1 treatment alleviates renal fibrosis by inhibiting the NOX4-MAPK pathway in T2DM mice.

Diabetic kidney disease (DKD) is a severe complication of type 2 diabetes mellitus (T2DM). However, the pathogenesis of DKD remains unclear, and effective treatment strategies are still lacking. Ginsenoside Rg1 (Rg1) has been reported to improve DKD, but the mechanism is unclear. NADPH oxidase 4 (NOX4) is an essential reactive oxygen species (ROS) source in the kidney. The mitogen-activated protein kinase (MAPK) signaling may exacerbate renal fibrosis. Therefore, we hypothesized that Rg1 might alleviate renal injury and fibrosis by inhibiting NOX4 and MAPK signaling in T2DM-induced DKD. We found that Rg1 significantly improves lipid deposition, fibrosis, and ROS production and reduces NOX4, p22phox, p47phox, p-ERK, p-JNK, and p-P38 MAPK expressions in the T2DM mice kidneys. We also found that the high-fat diet treatment in mice and the palmitate (PA) and PA + HG (high glucose) exposure in human mesangial cells could significantly induce lipid deposition, ROS production, fibrosis, and the activation of NOX4-MAPK signaling. The results suggest that high lipid and glucose may play a significant role in DKD progression, while Rg1 may attenuate renal fibrosis by inhibiting NOX4-MAPK signaling.

Ginsenoside Rg1 alleviates learning and memory impairments and Aß disposition through inhibiting NLRP1 inflammasome and autophagy dysfunction in APP/PS1 mice.

Alzheimer's disease (AD) is a common neurodegenerative disorder. Amyloid ß (Aß) deposition is considered an important pathological feature of AD. Growing evidence has linked neuroinflammation and autophagy to Aß deposition in the progression of AD. However, there are few drug options for inhibiting neuroinflammation and autophagy to prevent AD. Ginsenoside Rg1 (Rg1), a steroidal saponin extracted from ginseng, has been reported to possess multiple neuroprotective effects. The present study aimed to evaluate whether Rg1 treatment could attenuate cognitive disorders and neuronal injuries by inhibiting NLRP1 inflammasome and autophagy dysfunction in an AD model of APP/PS1 mice. The results of behavioral tests indicated that Rg1 treatment for 12 weeks could significantly improve olfactory dysfunction as well as learning and memory impairments. The results of histopathological tests indicated that Rg1 treatment could reduce Aß deposition and neuronal damages in APP/PS1­9M mice. Additionally, the results of immunoblot, reverse transcription­quantitative PCR or immunohistochemistry demonstrated that Rg1 treatment significantly downregulated the expression levels of inflammation­related proteins of NLRP1, caspase1, IL­1ß and TNF­α, as well as autophagy­related proteins of p­AMPK/AMPK, Beclin1 and LC3 II/LC3 I, and increased the expression levels of p­mTOR/mTOR and P62 in APP/PS1­9M mice. In addition, the molecular docking analysis showed that there was favorable binding result between Rg1 and NLRP1. The present study suggested that Rg1 may alleviate learning and memory impairments and Aß disposition by inhibiting NLRP1 inflammasome and improving autophagy dysfunction, suggesting that Rg1 may be a potential therapeutic agent for delaying AD.

Stress level of glucocorticoid exacerbates neuronal damage and Aß production through activating NLRP1 inflammasome in primary cultured hippocampal neurons of APP-PS1 mice.

Glucocorticoid (GC), secreted by adrenal cortex, plays important roles in regulating many physiological functions, while chronic stress level of GC exposure has many adverse effects on the structure and function of hippocampal neurons, and is closely implicated to the deterioration of Alzheimer's disease (AD). Oxidative stress and neuroinflammation play an important role in the occurrence and development of AD. However, it is still unclear whether chronic GC exposure promotes beta-amyloid (Aß) accumulation and neuronal injury by increasing oxidative stress and neuroinflammation. In this study, we investigated the effects of chronic GC exposure on NOX2-NLRP1 inflammasome activation and the protective effects of NLRP1-siRNA against GC-induced neuronal injury in primary hippocampal neurons of APP/PS1 mice. The results showed that chronic dexamethasone (DEX, 1 µM) exposure 72 h had no significant effect on the primary hippocampal neurons of WT mice, but significantly increased Aß1-42 accumulation (2.17 ± 0.19 fold in APP group and 3.06 ± 0.49 fold in APP + DEX group over WT group) and neuronal injury in primary hippocampal neurons of APP/PS1 mice. Meanwhile, chronic DEX exposure significantly increased the levels of reactive oxygen species (ROS) production and IL-1ß, and significantly up-regulated the expressions of NOX2- and NLRP1-related proteins and mRNAs in primary hippocampal neurons of APP/PS1 mice but not in WT mice. Moreover, inhibition of NLRP1 by NLRP1-siRNA treatment also significantly alleviated neuronal injury and Aß1-42 accumulation (1.96 ± 0.11 fold in APP + DEX group and 0.25 ± 0.01 fold in APP + NLRP1-siRNA + DEX group over APP group), and down-regulated the expressions of APP, BACE1, NCSTN and p-TAU/TAU in chronic DEX-induced hippocampal neurons of APP/PS1 mice. The results suggest that chronic GC exposure can accelerate neuronal damage and Aß production by activating oxidative stress and NLRP1 inflammasome in primary hippocampal neurons of APP/PS1 mice, resulting in deterioration of AD.And inhibition of NLRP1 inflammasome may be an important strategy in improving chronic GC-induced neuronal injury.

Ginsenoside Rg1 attenuates LPS-induced chronic renal injury by inhibiting NOX4-NLRP3 signaling in mice.

Chronic renal injury (CRI) is a common pathological damage in chronic renal disease, and the therapeutic options for preventing its progression are limited at present. Ginsenoside Rg1 (Rg1) is reported to have a protective effect on renal injury by improving oxidative stress and inflammation. Lipopolysaccharide (LPS) plays important roles in inducing inflammatory and high-dose LPS is often used to perform acute renal injury. However, little is known about the effect of low-dose LPS on CRI, and the protective effect of Rg1 against chronic LPS-induced CRI. Here, we reported the protective effect and mechanism of Rg1 against LPS-induced CRI in mice. In this study, the results demonstrated that low-dose LPS (0.25 mg/kg) exposure for 14 days significantly induced renal function impairment and renal injury and fibrosis. Meanwhile, LPS exposure significantly increased reactive oxygen species (ROS) generation, NADPH oxidase 4 (NOX4) and NLRP3 inflammasome expression in renal cortex. However, treatment with Rg1, tempol (a superoxide dismutase mimetic), and apocynin (a NOX inhibitor) significantly improved renal function impairment and renal fibrosis, and significantly decreased the levels of TGF-ß, IL-1ß, KIM-1, ß-Gal, and collagen IV in the kidneys. And Rg1 treatment also significantly reduced ROS generation and inhibited the activation of NOX4 and NLRP3 inflammasome. Overall, these results suggest that Rg1 treatment can ameliorate LPS-induced chronic kidney injury and renal fibrosis, the mechanisms may be involved in reducing NOX2-mediated oxidative stress and inhibiting NLRP1 inflammasome.