Andrographolide Treatment Research Articles

Targeting TLR4 and regulating the Keap1/Nrf2 pathway with andrographolide to suppress inflammation and ferroptosis in LPS-induced acute lung injury

Acute lung injury (ALI) is a severe inflammatory condition with a high mortality rate, often precipitated by sepsis. The pathophysiology of ALI involves complex mechanisms, including inflammation, oxidative stress, and ferroptosis, a novel form of regulated cell death. This study explores the therapeutic potential of andrographolide (AG), a bioactive compound derived from Andrographis, in mitigating Lipopolysaccharide (LPS)-induced inflammation and ferroptosis. Our research employed in vitro experiments with RAW264.7 macrophage cells and in vivo studies using a murine model of LPS-induced ALI. The results indicate that AG significantly suppresses the production of pro-inflammatory cytokines and inhibits ferroptosis in LPS-stimulated RAW264.7 cells. In vivo, AG treatment markedly reduces lung edema, decreases inflammatory cell infiltration, and mitigates ferroptosis in lung tissues of LPS-induced ALI mice. These protective effects are mediated via the modulation of the Toll-like receptor 4 (TLR4)/Kelch-like ECH-associated protein 1(Keap1)/Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Molecular docking simulations identified the binding sites of AG on the TLR4 protein (Kd value: -33.5 kcal·mol-1), and these interactions were further corroborated by Cellular Thermal Shift Assay (CETSA) and SPR assays. Collectively, our findings demonstrate that AG exerts potent anti-inflammatory and anti-ferroptosis effects in LPS-induced ALI by targeting TLR4 and modulating the Keap1/Nrf2 pathway. This study underscores AG's potential as a therapeutic agent for ALI and provides new insights into its underlying mechanisms of action.

Anti-inflammatory and anti-oxidative activities of andrographolide determined using atherosclerosis induced mice in Malaysia

Atherosclerosis and relative cardiovascular complications remain the main reasons for death worldwide. This study stimulated atherosclerosis in C57BL/6J mice using P-407 via intraperitoneal injection, and treatment with Andrographolide (AGP) (15, 30 and 45 mg/kg BW) was carried out for six weeks. The heart and aorta were harvested after six weeks and assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and histological studies. The results demonstrated that the treatment with AGP reversed the effects of P-407 induced atherosclerosis. The doses of AGP correlated with the reduction of atherosclerosis biomarkers, and a high dose (45 mg/kg BW) was the most significant dose. The Low-Density Lipoprotein (LDL), Triglycerides (TG), and Atherogenic Index (AI) were significantly reduced by the AGP treatment. The histological results showed a reduction in inflammation, fibrosis and hypertrophy in the heart tissues of the groups treated with AGP compared to the disease control. In addition, AGP treatment significantly decreased Reactive Oxygen Species (ROS) and the inflammation marker (NF-kB). Furthermore, the AGP-treated groups showed typical morphological characteristics of the aorta, while the disease control cells were highly affected. The results demonstrated that AGP is highly recommended as a natural treatment to reduce the symptoms of atherosclerosis by reducing oxidative stress and inflammation.

Safeguarding Neuronal Integrity: Unveiling Possible Role of NFκB in the Neuroprotective Efficacy of Andrographolide Contrary to Aluminium Chloride-induced Neurotoxicity and Associated Spatial Memory Impairments in Rats.

The current study was structured to evaluate the neuroprotective properties of andrographolide in the context of aluminum chloride (AlCl3)-induced neurotoxicity, along with its concurrent impact on spatial memory impairment in Wistar rats. The present investigation elucidated the biochemical and neurobehavioral outcomes of andrographolide treatment in rats, emphasizing the areas of the brain associated with memory, i.e., the cortex and the hippocampus. Prolonged dosing of AlCl3 (7 mg/kg) intraperitoneally for 10 days exhibited a substantial enhancement in the values of oxidative stress markers associated with a reduction in the concentrations of antioxidant enzymes within the brain. The selection of andrographolide doses (1, 2, and 3 mg/kg) was grounded in precedent safety and toxicity investigations, with subsequent oral administration. The evaluation of behavioral parameters, specifically spatial memory, was conducted through the utilization of the Radial Eight Arm Maze (RAM) test. On the concluding day of the experiment, the assessment encompassed biochemical parameter analysis and histological scrutiny of the brain tissue. The oral dosing of andrographolide at 1, 2, and 3 mg/kg, in conjunction with AlCl3, effectively mitigated the behavioral deficits induced by aluminum exposure. Notably, a significant suppression of NFκB was uncovered in the rats treated with andrographolide. Furthermore, histopathological examinations of the cortex and hippocampus of rat brains provided corroborative evidence, demonstrating that andrographolide substantially alleviated the toxic impact of AlCl3, thereby maintaining the typical histoarchitectural arrangement of these regions. These findings collectively suggest that andrographolide holds the potential to counteract memory impairment instigated by aluminum toxicity, accomplished through the modulation of NFκB activity and the amelioration of the adverse consequences of AlCl3 exposure.

Elucidation of the key therapeutic targets and potential mechanisms of Andrographolide multi-targets against osteoarthritis via network pharmacological analysis and experimental validation

ObjectiveOur purpose is to unveil Andrographolide's potential multi-target and multi-mechanism therapeutic effects in treating OA via systematic network pharmacological analysis and cell experimental validation. Materials and MethodsInitially, we gathered data from Andrographolide and OA-related databases to obtain information on Andrographolide's biological properties and the targets linked with OA. We developed a bioinformatic network about Andrographolide and OA, whereby we analyzed the network to identify potential therapeutic targets and mechanisms of action of Andrographolide. Subsequently, we used molecular docking to analyze the binding sites of Andrographolide to the target proteins. At the same time, SDF-1 was used to construct an OA cell model to verify the therapeutic effect of Andrographolide on OA and its effect on target proteins. ResultsOur experimental results show that Andrographolide has excellent pharmaceutical properties, by Lipinski's rules for drugs, suggesting that this compound can be considered to have a high therapeutic potential in drug development. 233 targets were preliminarily investigated, the mechanisms through which Andrographolide targets OA primarily involve the TNF signaling pathway, PI3K-AKT signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. These mechanisms target OA by influencing immune and inflammatory responses in the joints, regulating apoptosis to prevent chondrocyte death. Finally, TNF-α, STAT3, TP53, IL-6, JUN, IL-1β, HIF-1α, TGF-β1, and AKT1 were identified as 9 key targets of Andrographolide anti-OA. In addition, our molecular docking analyzes with cell experimental validation further confirm the network pharmacology results. According to our molecular docking results, Andrographolide can bind to all the hub target proteins and has a good binding ability (binding energy < -5 kcal/mol), with the strongest binding affinity to AKT1 of −9.2 kcal/ mol. The results of cell experiments showed that Andrographolide treatment significantly increased the cell viability and the expression of COL2A1 and ACAN proteins. Moreover, 30 μM Andrographolide significantly reversed SDF-1-induced increases in the protein expression of TNF-α, STAT3, TP53, IL-6, JUN, IL-1β, HIF-1α, and TGF-β1, and decreases in the protein expression of AKT1. ConclusionThis study provides a comprehensive understanding of the potential therapeutic targets and mechanisms of action of Andrographolide in OA treatment. Our findings suggest that Andrographolide is a promising candidate for drug development in the management of OA.

Synergistic inhibition effects of andrographolide and baicalin on coronavirus mechanisms by downregulation of ACE2 protein level

The SARS-CoV-2 virus, belonging to the Coronavirus genus, which poses a threat to human health worldwide. Current therapies focus on inhibiting viral replication or using anti-inflammatory/immunomodulatory compounds to enhance host immunity. This makes the active ingredients of traditional Chinese medicine compounds ideal therapies due to their proven safety and minimal toxicity. Previous research suggests that andrographolide and baicalin inhibit coronaviruses; however, their synergistic effects remain unclear. Here, we studied the antiviral mechanisms of their synergistic use in vitro and in vivo. We selected the SARS-CoV-2 pseudovirus for viral studies and found that synergistic andrographolide and baicalein significantly reduced angiotensin-converting enzyme 2 protein level and viral entry of SARS-CoV-2 into cells compared to singal compound individually and inhibited the major protease activity of SARS-CoV-2. This mechanism is essential to reduce the pathogenesis of SARS-CoV-2. In addition, their synergistic use in vivo also inhibited the elevation of pro-inflammatory cytokines, including IL-6 and TNF-α—the primary cytokines in the development of acute respiratory distress syndrome (the main cause of COVID-19 deaths). In conclusion, this study shows that synergistic andrographolide and baicalein treatment acts as potent inhibitors of coronavirus mechanisms in vitro and in vivo—and is more effective together than in isolation.

Andrographolide regulates H3 histone lactylation by interfering with p300 to alleviate aortic valve calcification.

Our previous studies have found that andrographolide (AGP) alleviates calcific aortic valve disease (CAVD), but the underlying mechanism is unclear. This study explores the molecular target and signal mechanisms of AGP in inhibiting CAVD. The anti-calcification effects of the aortic valve with AGP treatment were evaluated by alizarin red staining in vitro and ultrasound and histopathological assessment of a high-fat (HF)-fed ApoE-/- mouse valve calcification model. A correlation between the H3 histone lactylation (H3Kla) and calcification was detected. Molecular docking and surface plasmon resonance (SPR) experiments were further used to confirm p300 as a target for AGP. Overexpression (oe) and silencing (si) of p300 were used to verify the inhibitory effect of AGP targeting p300 on the H3Kla in vitro and ex vivo. AGP significantly inhibited calcium deposition in valve interstitial cells (VICs) and ameliorated aortic valve calcification. The multi-omics analysis revealed the glycolysis pathway involved in CAVD, indicating that AGP interfered with lactate production by regulating lactate dehydrogenase A (LDHA). In addition, lactylation, a new post-translational modification, was shown to have a role in promoting aortic valve calcification. Furthermore, H3Kla and H3K9la site were shown to correlate with Runx2 expression inhibition by AGP treatment. Importantly, we found that p300 transferase was the molecular target of AGP in inhibiting H3Kla. Our findings, for the first time, demonstrated that AGP alleviates calcification by interfering with H3Kla via p300, which might be a powerful drug to prevent CAVD.

Abstract C072: Andrographolide exhibits anti-cancer activity via restoring DNMT3B-suppressed ZNF382 expression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy. Due to the lack of early diagnosis and the limited response to current clinical treatments, PDAC has a high recurrence rate and a poor 5-year survival rate. Andrographolide, a labdane diterpenoid compound and the main active ingredient found in the medicinal plant Andrographis paniculata, is known for its anti-cancer effects against a variety of cancer types. Nevertheless, there are few studies on the anti-cancer activity and underlying mechanism of andrographolide against PDAC. We recently found that andrographolide suppressed cell viability, proliferation and migration abilities of PDAC cells, as well as impaired tumor growth in vivo. Notably, andrographolide induced the mRNA and protein expression of Zinc finger protein 382 (ZNF382) in PDAC cells. ZNF382 functions as a transcription repressor but its biological function in pancreatic cancer remains unclear. Through analysis of pancreatic adenocarcinoma (PAAD) patient data from TCGA, the expression of ZNF382 is lower in tumor samples than in normal tissues, and patients with higher ZNF382 expression showed better overall survival rates. Recently, we confirmed that ZNF382 expression was significantly decreased in clinical tumor tissues compared with matched normal tissues in 9 out of 10 pancreatic cancer patients. In addition, we demonstrated that overexpression of ZNF382 inhibited malignant phenotypes of PDAC cells and suppressed tumor growth in mice. Based on TCGA database analysis, the ZNF382 promoter is hypermethylated in primary pancreatic tumors, and this hypermethylation pattern correlates with its low expression. We further showed that andrographolide treatment inhibited the expression of DNA methyltransferase 3 beta (DNMT3B) and promoted the demethylation of ZNF382 promoter in PDAC cells. Notably, overexpression of DNMT3B attenuated both the andrographolide-induced expression of ZNF382 and its associated anti-cancer effects. In summary, our findings indicate that ZNF382 serves as a tumor suppressor in pancreatic cancer and suggest that andrographolide exerts its anticancer effects in pancreatic cancer through the suppression of DNMT3B-dependent DNA methylation, leading to the reactivation of ZNF382 expression. Citation Format: Kai-Ru Zhuang, Hsin-Yu Chan, Chian-Feng Chen, Shu-Ling Fu. Andrographolide exhibits anti-cancer activity via restoring DNMT3B-suppressed ZNF382 expression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C072.

Combination of Pirfenidone and Andrographolide Ameliorates Hepatic Stellate Cell Activation and Liver Fibrosis by Mediating TGF-β/Smad Signaling Pathway.

Biliary atresia (BA) is a devastating congenital disease characterized by inflammation and progressive liver fibrosis. Activation of hepatic stellate cells (HSCs) plays a central role in the pathogenesis of hepatic fibrosis. Our study aimed to investigate the pharmacological effect and potential mechanism of pirfenidone (PFD) and andrographolide (AGP) separately and together on liver fibrosis of BA. The bile ducts of male C57BL/6J mice were ligated or had the sham operation. The in vivo effects of PFD and/or AGP on liver fibrosis of BA were evaluated. Human hepatic stellate cells (LX-2) were also treated with PFD and/or AGP in vitro. PFD and/or AGP ameliorates liver fibrosis and inflammation in the mice model of BA, as evidenced by significant downregulated in the accumulation of collagen fibers, hepatic fibrosis markers (α-SMA, collagen I, and collagen IV), and inflammatory markers (IL-1β, IL-6, and TNF-α). Moreover, compared with monotherapy, these changes are more obvious in the combined treatment of PFD and AGP. Consistent with animal experiments, hepatic fibrosis markers (α-SMA, collagen I, and CTGF) and inflammatory markers (IL-1β, IL-6, and TNF-α) were significantly decreased in activated LX-2 cells after PFD and/or AGP treatment. In addition, PFD and/or AGP inhibited the activation of HSCs by blocking the TGF-β/Smad signaling pathway, and the combined treatment of PFD and AGP synergistically inhibited the phosphorylation of Smad2 and Smad3. The combined application of PFD and AGP exerted superior inhibitive effects on HSC activation and liver fibrosis by mediating the TGF-β/Smad signaling pathway as compared to monotherapy. Therefore, the combination of PFD and AGP may be a promising treatment strategy for liver fibrosis in BA.

Protective effect of andrographolide against ulcerative colitis by activating Nrf2/HO-1 mediated antioxidant response.

Ulcerative colitis (UC) is a recurring inflammatory bowel disease, in which oxidative stress plays a role in its progression, and regulation of the oxidative/antioxidative balance has been suggested as a potential target for the treatment of UC. The aim of this study was to evaluate the protective effect of andrographolide against UC and its potential antioxidant properties by modulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Dextran sulfate sodium (DSS) -induced UC mice and the LPS-induced HT29 inflammatory cell model were established to uncover the potential mechanisms of andrographolide. ML385, a Nrf2 inhibitor, was used in both models to assess whether andrographolide exerts a protective effect against UC through the Nrf2/HO-1 pathway. The in vivo experiment showed that andrographolide ameliorated the symptoms and histopathology of DSS-induced mice and restored the expressions of ZO-1, Occludin-1 and Claudin-1. Meanwhile, DSS-induced oxidative stress and inflammation were suppressed by andrographolide treatment, along with the upregulation of key proteins in the Nrf2/HO-1 pathway. In vitro experiments showed that andrographolide attenuated LPS-induced excessive generation of ROS in HT29 cells, reduced inflammatory factors, and upregulated the expression of proteins related to tight junctions and Nrf2/HO-1 pathway. In addition, ML385 abolished the beneficial effect of andrographolide. In conclusion, the protective effect of andrographolide against UC may involve the suppression of oxidative stress and inflammation via the Nrf2/HO-1 pathway.

Andrographolide protect against lipopolysacharides induced vascular endothelium dysfunction by abrogation of oxidative stress and chronic inflammation in Sprague-Dawley rats.

The development of heart disease involves interconnected factors such as oxidative stress, inflammation, and vascular dysfunction. Andrographolide (AG), known for its potent antioxidant and anti-inflammatory properties, has the potential to counteract lipopolysaccharides (LPS)-induced endothelial dysfunction by reducing oxidative stress and inflammation. Our research aimed to investigate the effects of AG on alleviating vascular endothelium dysfunction, oxidative stress, and inflammation in an experimental model induced by LPS. To create chronic vascular endothelium dysfunction, inflammation, and oxidative stress, rats received weekly injections of LPS via their tail vein over a 6-week period. The study evaluated the therapeutic effects of orally administered AG (50 mg/kg/day) on diseased conditions. We conducted aortic histology and measured nitric oxide (NO) thresholds, superoxide dismutase (SOD) activity, constitutive nitric oxide (cNOS) activity, and inducible nitric oxide (iNOS) levels, alongside several inflammatory biomarkers. To evaluate endothelial dysfunction, we assessed endothelium-dependent and endothelium-independent vasorelaxation in aortas through histopathological and various immunoassays examinations. Vascular Endothelial inflammatory activity was consequently enhanced in LPS groups animals when compared to normal control, also endothelial performance were dependently improved by AG therapy. IL-1β and tumors necrosis factor levels in the aorta decreased in a dose-dependent manner after exogenous AG delivery to LPS-treated rats. However, in current research work aortic SOD activity, NO levels, and cNOS activity increased, whereas aortic malondialdehyde levels and iNOS activity decreased after the AG treatment. These findings suggest that long-term AG therapy could be considered as a potential therapy to avoid vascular endothelial dysfunction and major nonobstructive coronary artery disease.

Ethanol extract of Andrographis paniculata alleviates aluminum-induced neurotoxicity and cognitive impairment through regulating the p62-keap1-Nrf2 pathway

BackgroundAlzheimer’s disease (AD) is the most prevalent neurodegenerative and remains incurable. Aluminum is a potent neurotoxin associated with AD. The main pathological features of AD are extracellular amyloid-β protein deposition and intracellular hyperphosphorylated Tau protein. A body of evidence suggest that oxidative stress and autophagy are involved in the pathogenesis of AD. Andrographis paniculata (AP) is a native plant with anti-inflammatory, anti-oxidative stress, and regulation of autophagy properties. AP significantly alleviated cognitive impairments, reduced Aβ deposition and has neuroprotective effect. However, its effects on aluminum-induced AD model have not been studied much. In this study, we investigated whether AP protect against aluminum-induced neurotoxicity through regulation of p62-Kelch-like ECH-associated protein 1(Keap1)-Nuclear factor E2 related factor 2 (Nrf2) pathway and activation autophagy in vivo and in vitro.MethodsUPLC-ESI-qTOF-MS/MS was used to identify the chemical constituents of AP ethanol extract. The mice with cognitive deficit were established by injecting aluminum chloride and D-galactose, and treated with either AP extract (200, 400, or 600 mg/kg/d) or andrographolide (2 mg/kg/2d).The spatial memory ability was detected by Morris water maze, HE staining were used to detect in brain tissue,Oxidative stress indexs and SOD activity in both serum and brain tissue were detected by kit.The expression of p62-Nrf2 pathway proteins were measured via western blotting. Furthermore, the neurotoxicity model was induced by aluminum maltolate (700 µM) in PC12 cells. Following AP and andrographolide treatment, the cell viability was detected. The relevant mRNA and protein expressions were detected in cells transfected with the p62 siRNA.ResultsThe main active components of AP included andrographolide, neoandrographolide and deoxyandrographolide as identified. AP and andrographolide significantly improved the spatial memory ability of mice, attenuated pathological changes of hippocampal cells, reduced the level of malondialdehyde, and increased superoxide dismutase activity in serum or brain tissue as compared to model control. In addition, the Nrf2, p62 and LC3B-II proteins expression were increased, and p-Tau and Keap1 proteins were decreased in the hippocampus after AP and andrographolide treatment.Furthermore, AP increased aluminum maltolate-induced cell viability in PC12 cells. Silencing p62 could reverse the upregulation expression of Nrf2 and downregulation of Keap1 and Tau proteins induced by AP in aluminum maltolate-treated cells.ConclusionsAP had neuroprotective effects against aluminum -induced cognitive dysfunction or cytotoxicity, which was involved in the activation of the p62-keap1-Nrf2 pathway and may develop as therapeutic drugs for the treatment of AD. However, this study has certain limitations, further optimize the protocol or model and study the molecular mechanism of AP improving AD.

The Dual Functions of Andrographolide in the Epstein–Barr Virus-Positive Head-and-Neck Cancer Cells: The Inhibition of Lytic Reactivation of the Epstein–Barr Virus and the Induction of Cell Death

Andrographolide, a medicinal compound, exhibits several pharmacological activities, including antiviral and anticancer properties. Previously, we reported that andrographolide inhibits Epstein–Barr virus (EBV) lytic reactivation, which is associated with viral transmission and oncogenesis in epithelial cancers, including head-and-neck cancer (HNC) cells. However, the underlying mechanism through which andrographolide inhibits EBV lytic reactivation and affects HNC cells is poorly understood. Therefore, we investigated these mechanisms using EBV-positive HNC cells and the molecular modeling and docking simulation of protein. Based on the results, the expression of EBV lytic genes and viral production were significantly inhibited in andrographolide-treated EBV-positive HNC cells. Concurrently, there was a reduction in transcription factors (TFs), myocyte enhancer factor-2D (MEF2D), specificity protein (SP) 1, and SP3, which was significantly associated with a combination of andrographolide and sodium butyrate (NaB) treatment. Surprisingly, andrographolide treatment also significantly induced the expression of DNA Methyltransferase (DNMT) 1, DNMT3B, and histone deacetylase (HDAC) 5 in EBV-positive cells. Molecular modeling and docking simulation suggested that HDAC5 could directly interact with MEF2D, SP1, and SP3. In our in vitro study, andrographolide exhibited a stronger cytotoxic effect on EBV-positive cells than EBV-negative cells by inducing cell death. Interestingly, the proteome analysis revealed that the expression of RIPK1, RIPK3, and MLKL, the key molecules for necroptosis, was significantly greater in andrographolide-treated cells. Taken together, it seems that andrographolide exhibits concurrent activities in HNC cells; it inhibits EBV lytic reactivation by interrupting the expression of TFs and induces cell death, probably via necroptosis.

Andrographolide Attenuates Inflammation Due to Intra-Abdominal Sepsis by Enhancing Bacterial Clearance in Mice.

Intra-abdominal infection is a complex pathophysiological process involving multiple systems and organs of the body. Abdominal infections complicated by severe sepsis or septic shock have a high mortality rate of 30-50%. Therefore, novel strategies to treat sepsis are urgently needed. Andrographolide (AD), the main active ingredient of Andrographis paniculata, reportedly exerts beneficial effects on mice with sepsis. However, its exact mechanism of action in attenuating inflammation due to intra-abdominal sepsis remains unclear to date. Hence, this study aimed to examine the therapeutic effects of AD on cecal ligation and puncture (CLP)-induced sepsis and elucidate the underlying mechanisms. Results showed that AD therapy could significantly improve the 7-day survival rate and alleviate pathological organ injury in mice with CLP. In addition, AD treatment decreased the levels of proinflammatory factors, such as tumor necrosis factor-α and interleukin (IL)-6 in the peritoneal cavity fluid and blood and increased the level of anti-inflammatory factor IL-10 in the peritoneal cavity fluid of mice with CLP. Moreover, bacterial counts in the blood and peritoneal lavage fluid were lower in the mice treated with AD than in those untreated. Mechanistically, AD treatment increased the percentage and phagocytic activity of macrophages in the peritoneal cavity. These data showed that AD can improve the survival of mice with intra-abdominal sepsis by enhancing bacterial clearance, as evidenced by the increased percentages and phagocytic activity of macrophages in the peritoneal cavity. This study is the first to demonstrate the protective effects of AD against intra-abdominal sepsis.

Andrographolide inhibits the proliferation and migration of vascular smooth muscle cells via PI3K/AKT signaling pathway and amino acid metabolism to prevent intimal hyperplasia

Andrographolide (AGP) exerts pharmacological effects when used for the treatment of cardiovascular disease, but the molecular mechanisms underlying its inhibitory effects on the proliferation and migration of vascular smooth muscle cells (VSMCs) and intimal hyperplasia (IH) are unknown. The proliferation and migration of VSMCs treated with AGP were examined using the CCK-8, flow cytometry, and wound healing assays. Expression levels of proteins related to cell proliferation and apoptosis were quantified. Multi-omics analysis with RNA-seq and metabolome was used to explore the potential molecular mechanism of AGP treatment. Additionally, an in vivo model was established through ligation of the left common carotid artery to identify the therapeutic potential of AGP in IH. Molecular docking and western blotting were performed to verify the mechanism discovered with multi-omics analysis. The results showed that AGP inhibited the proliferation and migration of cultured VSMCs in a dose-dependent manner and alleviated IH-related vascular stenosis. AGP significantly downregulated the protein levels of CDK1, CCND1, and BCL2 and upregulated the protein level of BAX. Gene expression profiles showed a total of 3,298 differentially expressed genes (DEGs) after AGP treatment, of which 1,709 DEGs had upregulated expression and 1,589 DEGs had downregulated expression. KEGG enrichment analysis highlighted the PI3K/AKT signaling pathway, verified with the detection of the activation of PI3K and AKT phosphorylation. Further GO enrichment combined with metabolomics analysis showed that AGP inhibition in cultured VSMCs involved the amino acid metabolic process, and the expression levels of the two key factors PRDM16 and EZH2, identified with PPI and docking analysis, were significantly inhibited by AGP treatment. In conclusion, our study showed that AGP inhibited VSMCs proliferation and migration by suppressing the PI3K/AKT signaling pathway and amino acid metabolism, which, in turn, improved IH.

Identification of Andrographolide as a novel FABP4 inhibitor for osteoarthritis treatment

Background and PurposeFatty acid binding protein 4 (FABP4) has been identified as a contributor to cartilage degradation in osteoarthritis (OA) patients, and inhibiting FABP4 using small molecules has emerged as a promising approach for developing OA drugs. Our previous research showed that Andrographis paniculata, a medicinal plant, strongly inhibits FABP4 activity. This led us to hypothesize that Andrographis paniculata ingredients might have protective effects on OA cartilage through FABP4 inhibition. MethodsWe analyzed scRNA-seq data from joint tissue of OA patients (GSE152805; GSE145286) using Scanpy 1.9.1 and Single Cell Portal. We conducted docking analysis of FABP4 inhibitors using Autodock Vina v.1.0.2. We evaluated the anti-FABP4 activity using a fluorescence displacement assay and measured the fatty acid oxidation (FAO) activity using the FAOBlue assay. We used H2DCF-DA to measure reactive oxygen species (ROS) levels. We studied signaling pathways using bulk RNA sequencing and western blot analysis in human C28/I2 chondrocytes. We evaluated anti-OA activity in monosodium iodoacetate (MIA)-induced rats. ResultsWe identified Andrographolide (AP) as a novel FABP4 inhibitor. Bulk RNA-sequencing analysis revealed that FABP4 upregulated FAO and ROS in chondrocytes, which was inhibited by AP. ROS generation activated the NF-κB pathway, leading to overexpression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), which is a responsible factor for cartilage degradation in OA patients. AP inhibited FABP4, thereby reducing the overexpression of ADAMTS4 by inhibiting the NF-κB pathway. In MIA rats, AP treatment reduced the overexpression of ADAMTS4, repaired cartilage and subchondral bone, and promoted cartilage regeneration. ConclusionOur results indicate that the inhibition of FABP4 activity by AP explains the anti-OA properties of Andrographis paniculata by protecting against cartilage degradation in OA patients. Additionally, our findings suggest that AP may be a promising therapeutic agent for OA treatment due to its ability to alleviate cartilage damage and bone erosion.

Dual actions of gallic acid and andrographolide trigger AdipoR1 to stimulate insulin secretion in a streptozotocin-induced diabetes rat model.

Common treatments for the management of diabetes have limitations due to side effects, hence the need for continuous research to discover new remedies with better therapeutic efficacy. Previously, we have reported that the combination treatment of gallic acid (20mg/kg) and andrographolide (10mg/kg) for 15 days demonstrated synergistic hypoglycemic activity in the streptozotocin (STZ)-induced insulin-deficient diabetes rat model. Here, we attempt to further elucidate the effect of this combination therapy at the biochemical, histological and molecular levels. Our biochemical analyses showed that the combination treatment significantly increased the serum insulin level and decreased the total cholesterol and triglyceride level of the diabetic animals. Histological examinations of H&E stained pancreas, liver, kidney and adipose tissues of combination-treated diabetic animals showed restoration to the normalcy of the tissues. Besides, the combination treatment significantly enhanced the level of glucose transporter-4 (GLUT4) protein expression in the skeletal muscle of treated diabetic animals compared to single compound treated and untreated diabetic animals. The molecular docking analysis on the interaction of gallic acid and/or andrographolide with the adiponectin receptor 1 (AdipoR1), a key component in the regulation of pancreatic insulin secretion, revealed a greater binding affinity of AdipoR1 to both compounds compared to individual compounds. Taken together, these findings suggest the combination of gallic acid and andrographolide as a potent therapy for the management of diabetes mellitus.

Clinical Efficacy and Immune Function of Andrographolide Sulfonate in Children with Mycoplasma Pneumoniae Pneumonia.

To figure out the effect of andrographolide sulfonate on the clinical efficacy and immune function of children with mycoplasma pneumoniae pneumonia (MPP). From January 2019 to April 2021, a total of 102 children with MPP were selected as the research group. They were assigned into the control and the observation groups by random number table method, with 51 cases/group. The control group was given routine treatment, and the observation was given andrographolide sulfonate treatment. The therapeutic efficacy and improvement of clinical symptoms were compared between the two. The changes of immune function, pulmonary function, myocardial enzymology indexes, MCP-4, IL-1β, IFN-γ, and TNF-α were noticed in the groups before and after treatment. The presence of drug-related adverse reactions of patients was recorded during treatment. The total effective rate of treatment in the observation group was higher, and the time of fever reduction, pulmonary rales disappearance and cough disappearance time were all shorter vs. the control group (p < 0.05). Immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) in the observation group after treatment were higher, and monocyte chemoattractant protein 4 (MCP-4), interleukin-1β (IL-1β), interferon-γ (IFN- γ), and tumor necrosis factor-α (TNF-α) were reduced vs. the control group. The forced expiratory volume in the 1st second (FEV1) and the percentage of forced vital capacity occupied by the forced expiratory volume in the first second (FEV1/FVC) and peak expiratory flow (PEF) values were higher (p < 0.05), but aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) and isoenzyme (CK-MB) were reduced in the observation group after treatment vs. the control group (p < 0.05); No serious adverse reactions took place in the two during treatment. The treatment of andrographolide sulfonate in children with MPP can enhance the therapeutic effect, ameliorate the immune function and lung function of children, and reduce inflammation and myocardial enzymes.

Andrographolide promoted ferroptosis to repress the development of non-small cell lung cancer through activation of the mitochondrial dysfunction

BackgroundFerroptosis, a form of regulated cell death by lipid peroxidation, was currently considered as a key factor affecting the occurrence and progression in various cancers. Andrographolide (ADE), a major effective ingredient of Andrographis paniculate, has proven to have a substantial anti-tumor effect on multiple cancer types. However, the function and underlying mechanism of ADE in Non-Small Cell Lung Cancer remain unclear. MethodsCCK8 assay, colony-formation assay, flow cytometry, scratch test, transwell assay, western blotting, ferroptosis analysis and mitochondria analysis were performed to reveal the role and underlying mechanisms of ADE in NSCLC cell lines (H460 and H1650). In vivo, xenograft model and lung metastatic model were performed to verify the effect of ADE on the growth and metastasis of NSCLC. ResultsIn this present study, we demonstrated that treatment with ADE could inhibit cell growth and metastases through eliciting ferroptosis in vitro an in vivo. The IC50 of ADE in H460 and H1650 cells were 33.16 μM and 32.45 μM respectively. In Lewis xenografted animals, i.p. ADE repressed relative tumor growth (p < 0.01) and inhibited metastases (p < 0.01). Notably, the ferroptosis inhibitor Fer-1 abrogated the anti-tumor capacity of ADE. Induction of ferroptosis by ADE was confirmed by elevated levels of reactive oxygen sepsis (ROS), glutathione (GSH), malondialdehyde (MDA), intracellular iron content and lipid ROS reduced glutathione (GSH) accumulation (p < 0.01). Furthermore, ADE inhibited the expression of ferroptosis-related protein GPX4 and SLC7A11. Simultaneously, it also disclosed that ADE enhanced mitochondrial dysfunction, as evidenced by increased mitochondrial ROS release, mitochondrial membrane potential (MMP) depolarization, and decreased mitochondrial ATP. Most interestingly, Mito-TEMPO, a mitochondria-targeted antioxidant, rescued ADE-induced ferroptosis. ConclusionOur data validated that ADE treatment could restrain proliferation and metastases of NSCLC cells through induction of ferroptosis via potentiating mitochondrial dysfunction.

Host resistance to Mycoplasma gallisepticum infection is enhanced by inhibiting PI3K/Akt pathway in Andrographolide-treating chickens.

Mycoplasma gallisepticum (MG) is a pathogenic microorganism that causes chronic respiratory disease (CRD). MG infection has a serious negative impact on the poultry industry. Andrographolide (AG) is known to regulate immune responses, antimicrobial infections, and anti-inflammatory responses. However, the underlying molecular mechanisms of AG action in MG-infected chickens remain unclear. Hence, we constructed models of MG infection by using chickens and chicken macrophage-like (HD11) cells in vivo and in vitro, respectively. The results showed that AG significantly inhibited the mRNA and protein expression of the toxic adhesion protein pMGA1.2 in vivo and in vitro. Meanwhile, AG treatment significantly decreased the mRNA expression of pro-inflammatory such as interleukin-6 (IL-6) and interleukin- 1β (IL-1β), and increased the mRNA expression of an anti-inflammatory such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) in vivo and in vitro. Furthermore, AG treatment down-regulated inflammasome NLRP3 and apoptosis genes caspase3 and caspase9, and up-regulated autophagy protein light chain 3 (LC3) by regulating the PI3K/Akt signaling pathway in vitro. Our results suggest that AG can reduce the expression of NLRP3 and alleviate the inflammatory response from MG infection by inducing autophagy, probably by modulating PI3K/Akt signaling pathway. This study demonstrates that AG can be used as a specific target to prevent and treat MG infection effectively.

Effect of Andrographolide and Resveratrol on OX1R and Prepro-orexin mRNA expression in CIPN-induced hypothalamus of mice with oxaliplatin

Chemotherapy-induced peripheral neuropathy (CIPN) is a one of side effect in cancer patients that receive antineoplastic agent, like oxaliplatin. Orexinergic system in the hypothalamus is the one of system that modulate nociceptive and neuropathy. Because there is flavonoids such as resveratrol and andrographolide that may prevent chemotherapy induced peripheral neuropathy, this study analyzed effects of andrographolide and resveratrol treatment on PPOrx and OX1R mRNA expression in hypothalamic oxaliplatin-induced mice. Materials and Methods This study was conducted for 22 days in mice. Mice injected with oxaliplatin followed by andrographolide or resveratrol. Chemotherapy induced peripheral neuropathic pain was assessed based on withdrawal threshold, mRNA PPOrx expression, and mRNA OX1R expression. Results The results showed that intraperitoneal injection of 100mg/kg resveratrol and 20mg/kg andrographolide increased the withdrawal threshold after oxaliplatin induction. Resveratrol administration also increased the relative expression of PPOrx mRNA significantly, but not the OX1R mRNA relative expression. On the other hand, administration of andrographolide did not cause a change in the expression of PPOrx and OX1R in the hypothalamus. Conclusions Intraperitoneal injection of andrographolide and resveratrol reduces the mechanical allodynia response in oxaliplatin-induced mice significantly. The mechanism of andrographolide increases the withdrawal threshold does not via the orexinergic system, but the mechanism of resveratrol via the orexinergic system.