Gypenoside XLIX Activates the Sirt1/Nrf2 Signaling Pathway to Inhibit NLRP3 Inflammasome Activation to Alleviate Septic Acute Lung Injury.

Currently, treatment options for acute lung injury (ALI) are limited. Gypenoside XLIX (Gyp-XLIX) is known for its anti-inflammatory properties, but there is a lack of extensive research on its effects against ALI. This study induced ALI in mice through cecal ligation and puncture surgery and investigated the biological activity and potential mechanisms of Gypenoside XLIX (40 mg/kg) by intraperitoneal injection. The in vitro ALI model was established using mouse lung epithelial (MLE-12) cells stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Various methods, including Hematoxylin and Eosin (H&E) staining, biochemical assay kits, Quantitative Polymerase Chain Reaction (qPCR) analysis, Western blotting, Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay, immunofluorescence, and flow cytometry, were employed for this research. The results indicated that pretreatment with Gypenoside XLIX significantly alleviated pathological damage in mouse lung tissues and reduced the expression levels of inflammatory factors. Additionally, Gypenoside XLIX inhibited ROS levels and NLRP3 inflammasome, possibly mediated by the Sirt1/Nrf2 signaling pathway. Moreover, Gypenoside XLIX significantly inhibited sepsis-induced lung cell apoptosis and excessive autophagy of mitochondria. Specifically, it suppressed mitochondrial pathway apoptosis and the Pink1/Parkin pathway of mitochondrial autophagy. These findings reveal the multifaceted effects of Gypenoside XLIX in anti-inflammatory, antioxidative, and inhibition of cell apoptosis and autophagy. This provides strong support for its therapeutic potential in sepsis-related lung injuries.

Gypenoside XLIX alleviates intestinal injury by inhibiting sepsis-induced inflammation, oxidative stress, apoptosis, and autophagy.

Intestinal barrier dysfunction is a significant complication induced by sepsis, yet therapeutic strategies targeting such dysfunction remain inadequate. This study investigates the protective effects of Gypenoside XLIX (Gyp XLIX) against intestinal damage induced by sepsis. Septic intestinal injury in mice was induced by cecum ligation and puncture (CLP) surgery. The biological activity and potential mechanisms of Gyp XLIX were explored through intraperitoneal injection of Gyp XLIX (40 mg/kg). The study demonstrates that Gyp XLIX improves the pathological structural damage of the intestine and increases tight junction protein expression as well as the number of cup cells. Through activation of the nuclear factor erythroid 2-related factor 2 - Kelch-like ECH-associated protein 1 (Nrf2-Keap1) pathway, Gyp XLIX enhances antioxidant enzyme levels while reducing the excessive accumulation of reactive oxygen species (ROS). In addition, Gyp XLIX effectively alleviates sepsis-induced intestinal inflammation by inhibiting the nuclear factor kappa B (NF-κB) pathway and activation of the NLRP3 inflammasome. Moreover, Gyp XLIX inhibits cell death through modifying phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, further enhancing its ability to shield the intestinal barrier. The combined action of these molecular mechanisms promotes the restoration of immune balance and reduces excessive autophagy activity induced under septic conditions. In summary, Gyp XLIX exhibits a significant preventive action against intestinal damage brought on by sepsis, with its mechanisms involving the improvement of intestinal barrier function, antioxidative stress, inhibition of inflammatory response, and cell apoptosis. This research offers a potential strategy for addressing intestinal barrier impairment brought on by sepsis.

Gypenoside XLIX alleviates acute liver injury: Emphasis on NF-κB/PPAR-α/NLRP3 pathways.

Liver is one of the vital organs in the human body and liver injury will have a very serious impact on human damage. Gypenoside XLIX is a PPAR-α activator that inhibits the activation of the NF-κB signaling pathway. The components of XLIX have pharmacological effects such as cardiovascular protection, antihypoxia, anti-tumor and anti-aging. In this study, we used cecum ligation and puncture (CLP) was used to induce in vivo mice hepatic injury, and lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells, evaluated whether Gypenoside XLIX could have a palliative effect on sepsis-induced acute liver injury via NF-κB/PPAR-α/NLRP3. In order to gain insight into these mechanisms, six groups were created in vivo: the Contol group, the Sham group, the CLP group, the CLP + XLIX group (40 mg/kg) and the Sham + XLIX (40 mg/kg) group, and the CLP + DEX (2 mg/kg) group. Three groups were created in vitro: Control, LPS, LPS + XLIX (40 µM). The analytical methods used included H&E staining, qPCR, reactive oxygen species (ROS), oil red O staining, and Western Blot. The results showed that XLIX attenuated hepatic inflammatory injury in mice with toxic liver disease through inhibition of the TLR4-mediated NF-κB pathway, attenuated lipid accumulation through activation of PPAR-α, and attenuated hepatic pyroptosis by inhibiting NLRP3 production. Regarding the imbalance between oxidative and antioxidant defenses due to septic liver injury, XLIX reduced liver oxidative stress-related biomarkers (ALT, AST), reduced ROS accumulation, decreased the amount of malondialdehyde (MDA) produced by lipid peroxidation, and increased the levels of antioxidant enzymes such as glutathione (GSH) and catalase (CAT). Our results demonstrate that XLIX can indeed attenuate septic liver injury. This is extremely important for future studies on XLIX and sepsis, and provides a potential pathway for the treatment of acute liver injury.

Gypenoside XLIX attenuates sepsis-induced splenic injury through inhibiting inflammation and oxidative stress.

BACKGROUND: To investigate the effect of Gypenoside XLIX (Gyp-XLIX) on acute splenic injury (ASI) induced by cecal ligation and puncture (CLP) in septic mice, a study was conducted. METHODS: Sixty healthy mice were randomly divided into six groups: the NC group, the Sham group, the Sham + Gyp-XLIX group, the CLP group, the CLP + Gyp-XLIX group, and the CLP + Dexamethasone (DEX) group. The NC group did not undergo any operation, while the rest of the groups underwent CLP to establish the sepsis model. The Sham group only underwent open-abdominal suture surgery without cecum puncture. After the operation, the groups were immediately administered the drug for a total of 5 days. Various methods such as hematoxylin and eosin (HE) staining, biochemical kits, qRT-PCR, and reactive oxygen species (ROS) were used for analysis. RESULTS: The results demonstrated that Gyp-XLIX effectively mitigated the splenic histopathological damage, while reducing the malondialdehyde (MDA) lipid peroxidation index and enhancing the antioxidant activities of catalase (CAT), glutathione (GSH) and total antioxidant capacity (T-AOC). The utilization of Dihydroethidium (DHE) fluorescent probe revealed that Gyp-XLIX inhibited the acute splenic accumulation of ROS induced by CLP in septic mice. Further investigations revealed that Gyp-XLIX exhibited a down-regulatory effect on the protein levels of inflammatory mediators iNOS and COX-2, consequently leading to the suppression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1ß. Additionally, it up-regulated the expression of anti-inflammatory factor IL-10. CONCLUSION: In conclusion, Gyp-XLIX was significantly effective in attenuating CLP-induced acute splenic inflammation and oxidative stress in septic mice.

An effective and high-throughput sample preparation method involving demalonylation followed by an ultrahigh-performance liquid chromatography-charged aerosol detector for analyzing gypenoside XLIX and gypenoside A in Gynostemma longipes.

Gypenosides (Gps) are the major bioactive components in Gynostemma species. They include neutral Gps and acidic malonylgypenosides (MGps). MGps are abundant in Gynostemma species and can be transformed into corresponding Gps via extraction, concentration, and drying. If only the Gps were quantified and MGps were ignored, the quality of Gynostemma species would be underestimated. This study aimed to develop a sample preparation method involving demalonylation and ultrahigh-performance liquid chromatography-charged aerosol detector (UHPLC-CAD) analysis to determine the contents of gypenoside XLIX (Gp XLIX) and gypenoside A (Gp A). First, the optimized ultrasonic extraction method was established to extract G. longipes powder ultrasonically. Then, the extracted solution was put into a closed container (centrifuge tube) and heated in a water bath at 95 °C. Then, MGps were converted into corresponding Gps. The proposed preparation method was compared with the other three methods, including water bath reflux heating, alkali hydrolysis, and extraction of heated powder, and was shown to exhibit higher conversion and better convenience. Subsequently, an UHPLC-CAD method was established and validated. Gp XLIX and Gp A showed excellent linear correlations between 15.55 and 248.8 µg/mL and 24.10-385.5 µg/mL, respectively (R2 > 0.999). The limit of detection was 1.40 ng (Gp XLIX) and 2.41 ng (Gp A), and the limit of quantification was 7.77 ng and 14.46 ng, respectively. The relative standard deviation for precision, stability, and repeatability was 0.63-3.15%. The average recovery of Gp XLIX and Gp A was 98.97% and 98.23%, respectively. The established method was applied for determining Gp XLIX and Gp A contents in wild or cultivated G. longipes samples collected from the Qinba Mountains area. The contents of Gp XLIX and Gp A were 5.16-23.02 mg/g and 15.78-54.55 mg/g, respectively. Conclusively, the proposed sample preparation and analysis method could be used for the quality control and evaluation of G. longipes.

Effects of Gypenoside XLIX on fatty liver cell gene expression in vitro: a genome-wide analysis.

OBJECTIVE: To perform Genome-wide analysis of Gypenoside XLIX (Gyp-XLIX) in the treatment of fatty liver cells. METHODS: The gene profiles of 3 normal liver cells, 3 fatty liver cells, and 3 fatty liver cells treated with Gyp-XLIX were detected by high-throughput sequencing to identify the differentially expressed genes (DEGs) in fatty liver treated by Gyp-XLIX. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to explore the biological functions of DEGs. By constructing lncRNA-mRNA co-expression network of DEGs, network node genes were mined. Possible target genes of differentially expressed lncRNA were predicted by cis regulation. RESULTS: 782 DEGs were screened out; that is, 172 genes were highly expressed in fatty liver cells, and the expression decreased to the level of normal liver cells after Gyp-XLIX treatment; 610 genes were under expressed in fatty liver cells, and the expression increased to the level of normal liver cells after Gyp-XLIX treatment. Functional analysis of KEGG and GO showed that DEGs process DNA-binding transcription factor activity and ion transmembrane transporter activity in the plasma membrane region. This mediates glycerophospholipid metabolism, bile secretion, fatty acid degradation and other signaling pathways. lncRNA analysis showed that the expression of 16 lncRNAs was low in fatty liver cells, and the expression was increased to the level of normal liver cells after Gyp-XLIX treatment. Target gene prediction showed that 16 differentially expressed lncRNAs had cis potential to regulate target genes, among which lncRNA RPARP-AS1 had a high degree of relationship with other genes. lncRNA-mRNA co-expression network results showed that lncRNA RPARP-AS1 may acted on NFKB2. CONCLUSION: LncRNA was differentially expressed in fatty liver cells and Gyp-XLIX treated fatty liver cells, and lncRNA RPARP-AS1 may be a regulatory gene in Gyp-XLIX treated fatty liver.

Gypenoside XLIX Ameliorate High-Fat Diet-Induced Atherosclerosis via Regulating Intestinal Microbiota, Alleviating Inflammatory Response and Restraining Oxidative Stress in ApoE-/- Mice.

A high-fat choline diet (HFCD)-induced atherosclerosis model in ApoE-/- mice was established to explore the anti-atherosclerotic effects of gypenoside XLIX (GPE). It was found that HFCD-induced atherosclerotic index such as dyslipidemia, atherosclerotic plaque, inflammation, and gut microbiota dysfunction could be reduced by GPE treatment. GPE treatment could decrease Verrucomicrobia, Proteobacteria, and Actinobacteria abundance, and increase Firmicutes and Bacteroidetes population. Moreover, the Firmicutes/Bacteroidetes ratio increased significantly after treatment with GPE. After treatment with GPE, the relative abundance of trimethylamine-producing intestinal bacteria Clostridioides and Desulfovibrionaceae decreased while butyrate-producing bacteria such as Eubacterium, Roseburia, Bifidobacterium, Lactobacillus, and Prevotella increased significantly. The GPE group demonstrated higher SCFAs concentrations in the fecal sample, such as Acetic Acid, Propionic Acid, and Butyric Acid. Further pathway analysis showed that 29 metabolic pathways were appreciably disturbed during GPE treatment, including citrate cycle (TCA cycle); galactose and glycero-lipid-metabolism biosynthesis of unsaturated fatty acids, fatty acid biosynthesis. This study suggests that the anti-atherosclerotic effect of GPE is related to the substantial changes in intestinal microbiota and anti-inflammatory activity.

Enzymatic Biotransformation of Gypenoside XLIX into Gylongiposide I and Their Antiviral Roles against Enterovirus 71 In Vitro.

Biotransformation of specific saponins in the valuable medical plants to increase their bioavailability and pharmaceutical activities has attracted more and more attention. A gene encoding a thermophilic glycoside hydrolase from Fervidobaterium pennivorans DSM9078 was cloned and expressed in Escherichia coli. The purified recombinant enzyme, exhibiting endoglucanase cellulase activity, was used to transform gypenoside XLIX into gylongiposide I via highly selective and efficient hydrolysis of the glucose moiety linked to the C21 position in gypenoside XLIX. Under the optimal reaction conditions for large scale production of gylongiposide I, 35 g gypenoside XLIX was transformed by using 20 g crude enzyme at pH 6.0 and 80 °C for 4 h with a molar yield of 100%. Finally, 11.51 g of gylongiposide I was purified using a silica gel column with 91.84% chromatographic purity. Furthermore, inhibitory activities of gypenoside XLIX and gylongiposide I against Enterovirus 71 (EV71) were investigated. Importantly, the EC50 of gypenoside XLIX and gylongiposide I calculated from viral titers in supernatants was 3.53 µM and 1.53 µM, respectively. Moreover, the transformed product gylongiposide I has better anti-EV71 activity than the glycosylated precursor. In conclusion, this enzymatic method would be useful in the large-scale production of gylongiposide I, which would be a novel potent anti-EV71 candidate.

Gypenoside XLIX loaded nanoparticles targeting therapy for renal fibrosis and its mechanism.

Renal fibrosis is the main pathological feature of the occurrence and development of chronic nephropathy. At present, there is no effective treatment, except for renal transplantation and dialysis. Previous studies have shown that nano-preparations can be used as a therapeutic tool to target organs. In this study, we studied the therapeutic effect and mechanism of Chinese medicine monomer Gypenoside (Gyp) XLIX on renal fibrosis and explored the targeting and therapeutic effects of polylactic acid-co-glycoside (PLGA)-Gyp XLIX nanoparticles in unilateral ureteral occlusion (UUO) kidney. Gyp XLIX and PLGA-Gyp XLIX nanoparticles were used to treat UUO mice and Human renal tubular epithelial (HK2) cells stimulated by transforming growth factor-ß (TGF-ß). Histopathological and molecular biological techniques were used to detect the expression of type I collagen and alpha-smooth muscle actin (α-SMA). To investigate the in vivo targeting of PLGA nanoparticles, they were loaded with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide and injected into UUO mice. We evaluated the effect of Gyp XLIX nanoparticles on TGF-ß/Smad3 pathway, a central driver for renal fibrosis in Smad-deficient HK2 cells. Fluorescence imaging showed that the PLGA nanoparticles around 120 nm could be targeted to the UUO kidney. Compared with Gyp XLIX, PLGA-Gyp XLIX nanoparticles could effectively inhibit renal fibrosis and reduce collagen deposition and reduce renal tubular necrosis. Gyp XLIX decreased the phosphorylation of Smad3, but could not further reduce the levels of type I collagen and α-SMA in Smad-deficient cells. This study opens a promising way for targeted drug treatment of renal fibrosis.

Gypenoside XLIX protects against acute kidney injury by suppressing IGFBP7/IGF1R-mediated programmed cell death and inflammation.

BACKGROUND: Acute kidney injury (AKI), characterised by excessive inflammatory cell recruitment and programmed cell death, has a high morbidity and mortality; however, effective and specific therapies for AKI are still lacking. OBJECTIVE: This study aimed to evaluate the renoprotective effects of gypenoside XLIX (Gyp XLIX) in AKI. METHODS: The protective effects of Gyp XLIX were tested in two AKI mouse models established using male C57BL/6 mice (aged 6-8 weeks) by a single intraperitoneal injection of cisplatin (20 mg/kg) or renal ischemia-reperfusion for 40 min. Gyp XLIX was administered intraperitoneally before cisplatin administration or renal ischemia-reperfusion. Renal function, tubular injury, renal inflammation and programmed cell death were evaluated. In addition, the renoprotective effects of Gyp XLIX were also evaluated in cisplatin- or hypoxia-treated tubular epithelial cells. The mechanisms underlying these effects were then explored using RNA sequencing. RESULTS: In vivo, Gyp XLIX substantially suppressed the increase in serum creatinine and blood urea nitrogen levels. Moreover, tubular damage was alleviated by Gyp XLIX as shown by periodic acid-Schiff staining, electron microscopy and molecular analysis of KIM-1. Consistently, we found that Gyp XLIX suppressed renal necroptosis though the RIPK1/RIPK3/MLKL pathway. The anti-inflammatory and antinecroptotic effects were further confirmed in vitro. Mechanistically, RNA sequencing showed that Gyp XLIX markedly suppressed the levels of IGF binding protein 7 (IGFBP7). Co-immunoprecipitation and western blot analysis further showed that Gyp XLIX reduced the binding of IGFBP7 to IGF1 receptor (IGF1R). Additionally, picropodophyllin, an inhibitor of IGF1R, abrogated the therapeutic effects of Gyp XLIX on cisplatin-induced renal cell injury; this finding indicated that Gyp XLIX may function by activating IGF1R-mediated downstream signalling Additionally, we also detected the metabolic distribution of Gyp XLIX after injection; Gyp XLIX had a high concentration in the kidney and exhibited a long retention time. These findings may shed light on the application of Gyp XLIX for AKI treatment clinically. CONCLUSION: Gyp XLIX may serve as a potential therapeutic agent for AKI treatment via IGFBP7/ IGF1R-dependent mechanisms.

Conversion of gypenosides by enzymatic hydrolysis with β-glucanase / 国际药学研究杂志

Objective: To investigate the enzymatic hydrolysis of gypenosides by β-glucanase 26130 CN to prepare some hydrolyzed secondary saponins, so as to provide material basis for further biological studies. Methods: Using β-glucanase 26130 CN, the total saponins from Herba Gynostemmatis were hydrolyzed with the enzyme catalysis, and the hydrolytic products were analyzed by ultra high- performance liquid chromatography coupled with quadrupole time- of- flight mass spectrometry(UHPLC- Q- TOF/MSE)to identify the converted products. Then, the main components of Herba Gynostemmatis, gypenosides XLIX and A, were used as substrates of the β-glucanase 26130 CN for convertsion to secondary saponin products. The products were separated by preparative highperformance liquid chromatography(HPLC)and identified by NMR and MS. Results: Twenty eight triterpenoid saponins were identified in the total saponin hydrolysate on the basis of their high-resolution MS data, by comparison with the data in the literature, and seven of them were validated to be the converted products. It was found that the β-glucanase 26130 CN could hydrolyze the glycosidic bond of terminal glucose or xylose in the molecule of gypenosides. By the enzymatic hydrolysis of gypenoside XLIX and gypenoside A, gypenoside I(the one glucosyl-lost gypenoside XLIX)and gypenoside UL1(the one xylosyl-lost gypenoside A)were obtained via the preparative HPLC separation of the gypenoside XLIX and gypenoside A hydrolysates, respectively. Conclusion: β-glucanase 26130 CN could effectively catalyze the hydrolysis of terminal glucosyl and xylosyl groups in gypenosides, with a relatively high hydrolytic conversion rate, which could be used to prepare some secondary saponins or aglycones.

Development of a targeted method for quantification of gypenoside XLIX in rat plasma, using SPE and LC-MS/MS.

A sensitive, selective and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of gypenoside XLIX, a naturally occurring gypenoside of Gynostemma pentaphyllum in rat plasma and then validated according to the US Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation. Plasma samples were prepared by a simple solid-phase extraction. Separation was performed on a Waters XBridgeTM BEH C18 chromatography column (4.6 × 50 mm, 2.5 µm) using a mobile phase of acetonitrile and water (62.5:37.5, v/v). Gypenoside XLIX and the internal standard gypenoside A were detected in the negative ion mode using selection reaction monitoring of the transitions at m/z 1045.6 → 913.5 and 897.5 → 765.4, respectively. The calibration curve was linear (R2 > 0.990) over a concentration range of 10-7500 ng/mL with the lower quantification limit of 10 ng/mL. Intra- and inter-day precision was within 8.6% and accuracy was ≤10.2%. Stability results proved that gypenoside XLIX and the IS remained stable throughout the analytical procedure. The validated LC-MS/MS method was then applied to analyze the pharmacokinetics of gypenoside XLIX after intravenous administration to rats (1.0, 2.0 and 4.0 mg/kg).