P65 Expression Research Articles

Effect of Centella asiatica ethanol extract on zebrafish larvae (Danio rerio) insomnia model through inhibition of Orexin, ERK, Akt and p38

Background: Insomnia is difficulty initiating or maintaining sleep for at least three nights a week or more and lasting for at least 3 months. One of the molecules that play a role in the circadian rhythm of arousal system is hypocretin/orexin. Orexin activates the p38-MAPK signaling pathway and increases phosphorylated ERK1/2 levels. Centella asiatica (CA) has a role in the signal work of the MAPK/ERK, Akt, and p38 path in many various diseases. Methods: The research method used is true laboratory experimental. The research approach used was randomized control group post-test only. Zebrafish embryos aged 0-7 dpf were used in this study. The treatment group consisted of 5 groups: normal, insomnia, insomnia + 2.5 μg/mL CA, insomnia + 5 μg/mL CA, and insomnia + 10 μg/mL CA. The locomotor motion of zebrafish larvae was observed using Basler cameras on days five-, six- and seven-day post fertilization (dpf), then analyzed by using Western Blot method. Results: The results proved that exposure to CA extract was able to reduce the expression of orexin (91963 ± 9129) and p38 (117425 ± 6398) as an arousal trigger in the sleep-wake cycle, with the most optimal concentration of CA 5 μg/mL. Exposure to CA extract was also able to reduce the expression of ERK (94795 ± 30830) and Akt (60113.5 ± 27833.5) with an optimum concentration of CA 2.5 μg/mL. Conclusion: Exposure to CA extract was able to improve the sleep activity of zebrafish larvae insomnia model by extending the total inactivity time (cumulative duration) and shortening the duration of first sleep (latency to first) in light and dark phases through inhibition of orexin, ERK, p38, and Akt.

O-Sialoglycoprotein Endopeptidase Deficiency Impairs Proteostasis and Induces Autophagy in Human Embryonic Stem Cells.

The OSGEP gene encodes O-sialoglycoprotein endopeptidase, a catalytic unit of the highly conserved KEOPS complex (Kinase, Endopeptidase, and Other Proteins of small Size) that regulates the second biosynthetic step in the formation of N-6-threonylcarbamoyladenosine (t6A). Mutations in KEOPS cause Galloway-Mowat syndrome (GAMOS), whose cellular function in mammals and underlying molecular mechanisms are not well understood. In this study, we utilized lentivirus-mediated OSGEP knockdown to generate OSGEP-deficient human embryonic stem cells (hESCs). OSGEP-knockdown hESCs exhibited reduced stemness factor expression and G2/M phase arrest, indicating a potential role of OSGEP in the regulation of hESC fate. Additionally, OSGEP silencing led to enhanced protein synthesis and increased aggregation of proteins, which further induced inappropriate autophagy, as evidenced by the altered expression of P62 and the conversion of LC3-I to LC3-II. The above findings shed light on the potential involvement of OSGEP in regulating pluripotency and differentiation in hESCs while simultaneously highlighting its crucial role in maintaining proteostasis and autophagy, which may have implications for human disease.

Kidney Injury in a Murine Hemorrhagic Shock/Resuscitation Model Is Alleviated by sulforaphane's Anti-Inflammatory and Antioxidant Action.

Hemorrhagic shock/resuscitation (HS/R) can lead to acute kidney injury, mainly manifested as oxidative stress and inflammatory injury in the renal tubular epithelial cells, as well as abnormal autophagy and apoptosis. Sulforaphane (SFN), an agonist of the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) signaling pathway, is involved in multiple biological activities, such as anti-inflammatory, antioxidant, autophagy, and apoptosis regulation. This study investigated the effect of SFN on acute kidney injury after HS/R in mice. Hemorrhagic shock was induced in mice by controlling the arterial blood pressure at a range of 35-45mmHg for 90min within arterial blood withdrawal. Fluid resuscitation was carried out by reintroducing withdrawn blood and 0.9% NaCl. We found that SFN suppressed the elevation of urea nitrogen and serum creatinine levels in the blood induced by HS/R. SFN mitigated pathological alterations including swollen renal tubules and renal casts in kidney tissue of HS/R mice. Inflammation levels and oxidative stress were significantly downregulated in mouse kidney tissue after SFN administration. In addition, the kidney tissue of HS/R mice showed high levels of autophagosomes as observed by electron microscopy. However, SFN can further enhance the formation of autophagosomes in the HS/R + SFN group. SFN also increased autophagy-related proteins Beclin1 expression and suppressed P62 expression, while increasing the ratio of microtubule-associated protein 1 light chain 3 (LC3)-II and LC3-I (LC3-II/LC3-I). SFN also effectively decreased cleaved caspase-3 level and enhanced the ratio of anti-apoptotic protein B cell lymphoma 2 and Bcl2-associated X protein (Bcl2/Bax). Collectively, SFN effectively inhibited inflammation and oxidative stress, enhanced autophagy, thereby reducing HS/R-induced kidney injury and apoptosis levels in mouse kidneys.

Astaxanthin attenuates diabetic kidney injury through upregulation of autophagy in podocytes and pathological crosstalk with mesangial cells

Objectives: Astaxanthin (ATX) is a strong antioxidant drug. This study aimed to investigate the effects of ATX on podocytes in diabetic nephropathy and the underlying renal protective mechanism of ATX, which leads to pathological crosstalk with mesangial cells.Methods: In this study, diabetic rats treated with ATX exhibited reduced 24-h urinary protein excretion and decreased blood glucose and lipid levels compared to vehicle-treated rats. Glomerular mesangial matrix expansion and renal tubular epithelial cell injury were also attenuated in ATX-treated diabetic rats compared to control rats.Results: ATX treatment markedly reduced the α-SMA and collagen IV levels in the kidneys of diabetic rats. Additionally, ATX downregulated autophagy levels. In vitro, compared with normal glucose, high glucose inhibited LC3-II expression and increased p62 expression, whereas ATX treatment reversed these changes. ATX treatment also inhibited α-SMA and collagen IV expression in cultured podocytes. Secreted factors (vascular endothelial growth factor B and transforming growth factor-β) generated by high glucose-induced podocytes downregulated autophagy in human mesangial cells (HMCs); however, this downregulation was upregulated when podocytes were treated with ATX.Conclusions: The current study revealed that ATX attenuates diabetes-induced kidney injury likely through the upregulation of autophagic activity in podocytes and its antifibrotic effects. Crosstalk between podocytes and HMCs can cause renal injury in diabetes, but ATX treatment reversed this phenomenon.

Envafolimab Inhibits the Growth of Gastric Cancer Cells with Low PD-L1 Expression through the DDX20/NF-κB/TNF-α Signaling Pathway.

The mechanism of action of envafolimab (also known as KN035), a programmed death ligand 1 (PD-L1) inhibitor, in gastric adenocarcinoma patients with low PD-L1 expression is not well understood. This study aimed to observe the efficacy of envafolimab in gastric adenocarcinoma with low PD-L1 expression and explore the underlying mechanisms. The objective of this study was to explore the underlying mechanism of envafolimab in gastric cancer with low PD-L1 expression. Cytotoxicity and proliferation were evaluated by a CCK8 assay. Transwell assays were used to detect the migration and invasion ability of gastric cancer cells. The effect of envafolimab on the apoptosis of gastric cancer cells was detected by flow cytometry. The effect of envafolimab on gastric cancer cells with low PD-L1 expression was investigated via proteomics and bioinformatics analysis. A total of 19 patients with advanced gastric adenocarcinoma who received envafolimab monotherapy or combination therapy were reviewed. Among them, 4 patients had low PD-L1 expression, the objective response rate (ORR) was 75% (3/4), and the disease control rate (DCR) was 100% (4/4). In vitro experiments showed that envafolimab inhibited the proliferation, invasion, and migration of gastric cancer cells with low expression of PD-L1 and induced cell apoptosis. DDX20 may be the target of envafolimab in gastric cancer cells, and it is related to the NF-κB signaling pathway. Western blot results showed that the protein expressions of DDX20, NF-κB p65, and TNF-α in gastric cancer cells were decreased after adding envafolimab. Furthermore, the DDX20 gene was silenced by small interfering RNA to further study the effect of DDX20 on PDL1 low expression in gastric cancer cells. This study confirmed that envafolimab could inhibit the growth of gastric cancer cells with low PD-L1 expression by down-regulating DDX20 expression and regulating the NFκB/TNF-α signaling pathway.

The role of quercetin in ameliorating bleomycin-induced pulmonary fibrosis: insights into autophagy and the SIRT1/AMPK signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by a constant incidence rate. Unfortunately, effective pharmacological treatments for this condition are lacking and the identification of novel therapeutic approaches and underlying pathological mechanisms are required. This study investigated the potential of quercetin in alleviating pulmonary fibrosis by promoting autophagy and activation of the SIRT1/AMPK pathway. Mouse models of IPF were divided into four treatment groups: control, bleomycin (BLM), quercetin (Q), and quercetin + EX-527 (Q + E) treatment. Pulmonary fibrosis was induced in the mouse models through intratracheal instillation of BLM. Various indexes were identified through histological staining, Western blotting analysis, enzyme-linked immunosorbent assay, immunohistochemistry, and transmission electron microscopy. Quercetin treatment ameliorated the pathology of BLM-induced pulmonary fibrosis of mice by reducing α-smooth muscle actin (α-SMA), collagen I (Col I), and collagen III (Col III) levels, and also improved the level of E-cadherin in lung tissue. Furthermore, Quercetin significantly enhanced LC3II/LC3I levels, decreased P62 expression, and increased the number of autophagosomes in lung tissue. These effects were accompanied by the activation of the SIRT1/AMPK pathway. Treatment with EX-527, an inhibitor for SIRT1, reversed all effects induced by quercetin. This study showed that quercetin could alleviate pulmonary fibrosis and improve epithelial-mesenchymal transition by acting on the SIRT1/AMPK signaling pathway, which may be achieved by regulating the level of autophagy.

DLK1 regulates periodontal inflammation by inhibiting NF-κB p65 and JNK signaling pathways.

The roles and molecular mechanisms of Delta-like 1 (DLK1) in periodontitis remain largely unknown. Here, we investigated the expression of DLK1 and NF-κB p65 in Porphyromonas gingivalis (Pg.)-induced periodontitis in vivo. Periodontal inflammation and alveolar bone resorption were analyzed using western blotting, micro-computed tomography, TRAP staining, immunohistochemistry, and immunofluorescence. Raw246.7 cells were stimulated with 1μg/ml Porphyromonas gingivalis lipopolysaccharide (Pg.LPS) to assess DLK1 expression in vitro. DLK1 overexpression was achieved, and transfection efficiency was confirmed using western blotting and immunofluorescence. The NF-κB and MAPK pathways were activated by treating cells with 1μg/ml Pg.LPS to explore related mechanisms. Compared with normal tissues, both DLK1 and NF-κB p65 expression increased in periodontitis gingival tissues. DLK1-positive expression was observed in inflammatory infiltrating cells and osteoclasts in the marginal lacunae of the alveolar bone. DLK1 expression in CD68-positive macrophages was detected by immunofluorescence. However, DLK1 expression in Raw246.7 cells decreased after Pg.LPS stimulation and during osteoclast differentiation. DLK1 levels negatively correlated with TNF-α, IL-1β, and NFATC1. Increased DLK1 in Raw246.7 cells further inhibited COX2 and iNOS expressions. Mechanistically, DLK1 overexpression down-regulated NF-κB p65 and JNK levels. In summary, these findings suggest that DLK1 overexpression inhibits periodontal inflammation through the NF-κB p65 and JNK pathways. Interventions targeting increased DLK1 levels may have therapeutic implications for periodontitis.

Inhibition of autophagy induced by tetrandrine promotes the accumulation of reactive oxygen species and sensitizes efficacy of tetrandrine in pancreatic cancer

Pancreatic cancer, characterized by its poor prognosis, exhibits a marked resistance to conventional chemotherapy and immunotherapy, underscoring the urgent need for more effective treatment modalities. In light of this, the present study is designed to assess the potential antineoplastic efficacy of a combined regimen involving tetrandrine, a plant-derived alkaloid, and autophagy inhibitors in the context of pancreatic cancer. Electron microscopy and immunoblots showed that tetrandrine promoted the formation of autophagosomes and the upregulation of LC3II and the downregulation of p62 expression, indicating that tetrandrine induced autophagy in pancreatic cancer cells. Western blot revealed that tetrandrine inhibited the phosphorylation of AKT and mTOR, as well as the expression of Bcl-2, while upregulating Beclin-1 expression. Moreover, tetrandrine promoted the transcription and protein expression of ATG7. Following the combination of autophagy inhibitors and tetrandrine, the apoptotic rate and cell death significantly increased in pancreatic cancer cells. Consistent results were obtained when ATG7 was silenced. Additionally, tetrandrine induced the generation of ROS, which was involved in the activation of autophagy and apoptosis. Further investigation revealed that upon autophagy inhibition, ROS accumulated in pancreatic cancer cells, resulting in decreased mitochondrial membrane potential and further induction of apoptosis. The results of treating subcutaneous xenograft tumors with a combination of tetrandrine and chloroquine validated that autophagy inhibition enhances the toxicity of tetrandrine against pancreatic cancer in vivo. Altogether, our study demonstrates that tetrandrine induces cytoprotective autophagy in pancreatic cancer cells. Inhibiting tetrandrine-induced autophagy promotes the accumulation of ROS and enhances its toxicity against pancreatic cancer.

Early growth response factor 3 may regulate coronary atherosclerosis through the NF-κB signaling pathway and VEGF expression

AimThe present study was conducted to measure the expression of early growth response factor 3 (Egr3), inflammatory cytokines (IL-1β, IL-6), vascular endothelial growth factor (VEGF) and NF-κB in patients with coronary artery disease (CAD) to investigate the relationships of these molecules and Egr3 gene expression. MethodsWe recruited 132 CAD patients and 63 healthy individuals. The expression levels of Egr3, VEGF, p50 and p65 were measured by reverse transcription quantitative polymerase chain reaction and the levels of Egr3, IL-1β and IL-6 in patients serum and in human coronary artery endothelial cells (HCAECs) were measured by enzyme-linked immunosorbent assay (ELISAs) in CAD patients. HCAECs were treated with ox-LDL to establish an in vitro atherosclerosis model. An oil red O staining assay was used to assess the lipid droplet formation. A colloidal external lumen formed by Matrigel was used to test the migration of HCAECs. The expression of Egr3, VEGF and NF-κB was determined by Western blotting. ResultsThe levels of serum Egr3 and IL-6 in the severe stenosis group were greater than those in the mild stenosis group and controls (p < 0.05). The level of serum IL-1β in the severe stenosis group was greater than that in the control group (p < 0.05). Moreover, Egr3 expression was positively associated with IL-6 levels (r = 0.55, p < 0.001), IL-1β levels (r = 0.21, p = 0.004) and the Gensini score (r = 0.20, p = 0.02). We also found that Egr3 expression was significantly greater in CAD patients than that in controls. And its expression was highest in the mild patients. The expression of VEGF, P50 and P65 was also greater in CAD patients. In the in vitro experiment, we found that the inhibition of Egr3 expression significantly reduced the expression levels of p50, p65, IL-6 and CRP. Moreover, the inhibition of Egr3 expression significantly reduced the lipid droplet formation and decreased capability of lumen formation. ConclusionsIn the pathogenesis of atherosclerosis, Egr3 gene expression may induce the expression of inflammatory factors and lipid droplet formation and lumen formation, which could promote the atherosclerosis development.

Erythrocyte membrane biomimetic EGCG nanoparticles attenuate renal injury induced by diquat through the NF-κB/NLRP3 inflammasome pathway.

Diquat (DQ) poisoning can cause multiple organ damage, and the kidney is considered to be the main target organ. Increasing evidence shows that alleviating oxidative stress and inflammatory response has promising application prospects. Epigallocatechin gallate (EGCG) has potent antioxidant and anti-inflammatory effects. In this study, red blood cell membrane (RBCm)-camouflaged polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) were synthesized to deliver EGCG (EGCG-RBCm/NPs) for renal injury induced by DQ. Human renal tubular epithelial cells (HK-2 cells) were stimulated with 600μM DQ for 12h and mice were intraperitoneally injected with 50mg/kg b.w. DQ, followed by 20mg/kg b.w./day EGCG or EGCG-RBCM/NPs for 3days. The assessment of cellular vitality was carried out using the CCK-8 assay, while the quantification of reactive oxygen species (ROS) was performed through ROS specific probes. Apoptosis analysis was conducted by both flow cytometry and TUNEL staining methods. Pathological changes in renal tissue were observed. The expressions of NLRP3, IL-1β, IL-18, NFκB and Caspase1 were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, immunofluorescence, and Western blot. The results showed that the DQ group had increased ROS expression, increased the level of oxidative stress, and increased apoptosis rate compared with the control group. Histopathological analysis of mice in the DQ group showed renal tubular injury and elevated levels of blood urea nitrogen (BUN), serum creatinine (SCr), kidney injury molecule-1 (KIM-1), and cystatin C (Cys C). Furthermore, the DQ group exhibited heightened expression of NLRP3, p-NFκB p65, Caspase1 p20, IL-1β, and IL-18. However, EGCG-RBCm/NPs treatment mitigated DQ-induced increases in ROS, apoptosis, and oxidative stress, as well as renal toxicity and decreases in renal biomarker levels. Meanwhile, the expression of the above proteins were significantly decreased, and the survival rate of mice was ultimately improved, with an effect better than that of the EGCG treatment group. In conclusion, EGCG-RBCm/NPs can improve oxidative stress, inflammation, and apoptosis induced by DQ. This effect is related to the NF-κB/NLRP3 inflammasome pathway. Overall, this study provides a new approach for treating renal injury induced by DQ.

The processed Euphorbia lathyris L. alleviates the inflammatory injury via regulating LXRα/ABCA1 expression and TLR4 positioning to lipid rafts

Euphorbia lathyris L. (EL) is a traditional poisonous herbal medicine used to treat dropsy, ascites, amenorrhea, anuria and constipation. Processing to reduce toxicity of EL is essential for its safe and effective application. However, there is little known regarding the molecular mechanism of reducing toxicity after EL processing. This research aimed to screen the differential markers for EL and PEL, explore the differential mechanisms of inflammatory injury induced by EL and processed EL (PEL) to expound the mechanism of alleviating toxicity after EL processing. The results showed that 15 potential biomarkers, mainly belonging to diterpenoids, were screened to distinguish EL from PEL. EL promoted the expressions of TLR4, NLRP3, NF-κB p65, IL-1β and TNF-α, increased lipid rafts abundance and promoted TLR4 positioning to lipid rafts. Meanwhile, EL decreased LXRα and ABCA1 expression, and reduced cholesterol efflux. In contrast to EL, the effects of PEL on these indicators were markedly weakened. In addition, Euphorbia factors L1, L2, and L3 affected LXRα, ABCA1, TLR4, NLRP3, NF-κB p65, TNF-α and IL-1β expression, influenced cholesterol efflux and lipid rafts abundance, and interfered with the colocalization of TLR4 and lipid rafts. The inflammatory injury caused by processed EL was significantly weaker than that caused by crude EL, and reduction of Euphorbia factors L1, L2, and L3 as well as attenuation of inflammatory injury participated in processing-based detoxification of EL. Our results provide valuable insights into the attenuated mechanism of EL processing and will guide future research on the processing mechanism of toxic traditional Chinese medicine.

Spexin inhibits excessive autophagy-induced ferroptosis to alleviate doxorubicin-induced cardiotoxicity by upregulating Beclin 1.

Doxorubicin is widely used in the treatment of malignant tumours, but doxorubicin-induced cardiotoxicity severely limits its clinical application. Spexin is a neuropeptide that acts as a novel biomarker in cardiovascular disease. However, the effects of spexin on doxorubicin-induced cardiotoxicity is unclear. We established a model of doxorubicin-induced cardiotoxicity both in vivo and in vitro. Levels of cardiac damage in mice was assessed through cardiac function assessment, determination of serum cardiac troponin T and CKMB levels and histological examination. CCK8 and PI staining were used to assess the doxorubicin-induced toxicity in cultures of cardiomyocytes in vitro. Ferroptosis was assessed using FerroOrange staining, determination of MDA and 4-HNE content and ferroptosis-associated proteins SLC7A11 and GPX4. Mitochondrial membrane potential and lipid peroxidation levels were measured using TMRE and C11-BODIPY 581/591 probes, respectively. Myocardial autophagy was assessed by expression of P62 and Beclin1. Spexin treatment improved heart function of mice with doxorubicin-induced cardiotoxicity, and attenuated doxorubicin-induced cardiotoxicity by decreasing iron accumulation, abnormal lipid metabolism and inhibiting ferroptosis. Interestingly, doxorubicin caused excessive autophagy in cardiomyocyte in culture, which could be alleviated by treatment with spexin. Knockdown of Beclin 1 eliminated the protective effects of spexin in mice with DIC. Spexin ameliorated doxorubicin-induced cardiotoxicity by inhibiting excessive autophagy-induced ferroptosis, suggesting that spexin could be a drug candidate against doxorubicin-induced cardiotoxicity. Beclin 1 might be critical in mediating the protective effect of spexin against doxorubicin-induced cardiotoxicity.

Molecular Targets of Plant-based Alkaloids and Polyphenolics in Liver and Breast Cancer- An Insight into Anticancer Drug Development.

Liver and Breast cancer are ranked as the most prevailing cancers that cause high cancer-related mortality. As cancer is a life-threatening disease that affects the human population globally, there is a need to develop novel therapies. Among the available treatment options include radiotherapy, chemotherapy, surgery, and immunotherapy. The most superlative modern method is the use of plant-derived anticancer drugs that target the cancerous cells and inhibit their proliferation. Plant-derived compounds are generally considered safer than synthetic drugs/traditional therapies and could serve as potential novel targets to treat liver and breast cancer to revolutionize cancer treatment. Alkaloids and Polyphenols have been shown to act as anticancer agents through molecular approaches. They disrupt various cellular mechanisms, inhibit the production of cyclins and CDKs to arrest the cell cycle, and activate the DNA repairing mechanism by upregulating p53, p21, and p38 expression. In severe cases, when no repair is possible, they induce apoptosis in liver and breast cancer cells by activating caspase-3, 8, and 9 and increasing the Bax/Bcl-2 ratio. They also deactivate several signaling pathways, such as PI3K/AKT/mTOR, STAT3, NF-kB, Shh, MAPK/ERK, and Wnt/β-catenin pathways, to control cancer cell progression and metastasis. The highlights of this review are the regulation of specific protein expressions that are crucial in cancer, such as in HER2 over-expressing breast cancer cells; alkaloids and polyphenols have been reported to reduce HER2 as well as MMP expression. This study reviewed more than 40 of the plant-based alkaloids and polyphenols with specific molecular targets against liver and breast cancer. Among them, Oxymatrine, Hirsutine, Piperine, Solamargine, and Brucine are currently under clinical trials by qualifying as potent anticancer agents due to lesser side effects. As a lot of research is there on anticancer compounds, there is a desideratum to compile data to move towards clinical trials phase 4 and control the prevalence of liver and breast cancer.

Cai's herbal tea enhances mitochondrial autophagy of type 1 diabetic mellitus β cells through the AMPK/mTOR pathway and alleviates inflammatory response.

This study investigates the therapeutic mechanisms of Cai's Herbal Tea in Type 1 Diabetes Mellitus (T1DM) mice, focusing on its effects on mitochondrial change and autophagy via the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway. The composition of Cai's Herbal Tea was analyzed by Ultra-High Performance Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry (UHPLC-Q/TOF-MS). C57BL/6 mice and Min6 pancreatic beta cells were divided into control, diabetic mellitus (DM)/high glucose (HG), and treatment groups (low, medium, and high doses of Cai's Tea, and Metformin). Key physiological parameters, pancreatic islet health, Min6 cell morphology, viability, and insulin (INS) secretion were assessed. Small Interfering RNA-AMPK (si-AMPK) was utilized to confirm the pathway involvement. Cai's Herbal Tea improved body weight, pancreatic islet pathological injury, and INS secretion whereas reduced total triglycerides, fasting blood sugar, and Interferon gamma (INF-γ) in T1DM mice, particularly at higher doses. In Min6 cells, Cai's Tea mitigated HG-induced damage and proinflammatory response, enhancing cell viability and INS secretion. Notably, it reduced swelling and improved cristae structure in treated groups of mitochondria and promoted autophagy via the AMPK-mTOR pathway, evidenced by increased LC3II/LC3I and P-AMPK/AMPK ratios, and decreased P-mTOR/mTOR and P62 expressions in pancreatic islet β-cells. Furthermore, these effects were converted by si-AMPK interference. Cai's Herbal Tea exhibits significant therapeutic efficacy in T1DM mice by improving mitochondrial health and inducing autophagy through the AMPK-mTOR pathway in pancreatic islet β-cells. These findings highlight its potential as a therapeutic approach for T1DM management.

Investigation into the anti-inflammatory mechanism of Pothos chinensis (Raf.) Merr. By regulating TLR4/MyD88/NF-κB pathway: Integrated network pharmacology, serum pharmacochemistry, and metabolomics

Ethnopharmacological relevanceInflammation is directly related to disease progression and contributes significantly to the global burden of disease. Pothos chinensis (Raf.) Merr. (PCM) is commonly used in Yao medicine in China to treat tumors, and orthopedic illnesses such as knee osteoarthritis, and rheumatic bone discomfort. PCM was found to have significant anti-inflammatory properties in previous studies. Aim of the studyTo explore the active compounds of PCM and their anti-inflammatory pharmacological mechanisms through an integrated strategy of serum pharmacochemistry, network pharmacology, and serum metabolomics. Materials and methodsThe qualitative and quantitative analyses of the chemical components of PCM were performed using UPLC-QTOF-MS/MS and UPLC, respectively, and the prototype components of PCM absorbed into the blood were analyzed. Based on the characterized absorbed into blood components, potential targets and signaling pathways of PCM anti-inflammatory were found using network pharmacology. Furthermore, metabolomics studies using UPLC-QTOF-MS/MS identified biomarkers and metabolic pathways related to the anti-inflammatory effects of PCM. Finally, the hypothesized mechanisms were verified by in vivo and in vitro experiments. ResultsForty chemical components from PCM were identified for the first time, and seven of them were quantitatively analyzed, while five serum migratory prototype components were found. Network pharmacology KEGG enrichment analysis revealed that arachidonic acid metabolism, Tyrosine metabolism, TNF signaling pathway, NF-κB signaling pathway, and phenylalanine metabolism were the main signaling pathways of PCM anti-inflammatory. Pharmacodynamic results showed that PCM ameliorated liver injury and inflammatory cell infiltration and downregulated protein expression of IL-1β, NF-κB p65, and MyD88 in the liver. Metabolomics studies identified 53 different serum metabolites, mainly related to purine and pyrimidine metabolism, phenylalanine metabolism, primary bile acid biosynthesis, and glycerophospholipid metabolism. The comprehensive results demonstrated that the anti-inflammatory modulatory network of PCM was related to 5 metabolites, 3 metabolic pathways, 7 targets, and 4 active components of PCM. In addition, molecular docking identified the binding ability between the active ingredients and the core targets, and the anti-inflammatory efficacy of the active ingredients was verified by in vitro experiments. ConclusionOur study demonstrated the anti-inflammatory effect of PCM, and these findings provide new insights into the active ingredients and metabolic mechanisms of PCM in anti-inflammation.

Myeloid-specific Hdac10 deletion protects against LPS-induced acute lung injury via P62 acetylation at lysine 165

BackgroundAberrant activation of macrophages is associated with pathogenesis of acute lung injury (ALI). However, the potential pathogenesis has not been explored.ObjectivesWe aimed to identify whether histone deacetylase (HDAC) 10 is involved in lipopolysaccharide (LPS)-exposed ALI and reveal the underlying pathogenesis by which it promotes lung inflammation in LPS-exposed ALI via modifying P62 with deacetylation.MethodsWe constructed an ALI mice model stimulated with LPS to determine the positive effect of Hdac10 deficiency. Moreover, we cultured murine alveolar macrophage cell line (MH-S cells) and primary bone marrow-derived macrophages (BMDMs) to explore the pro-inflammatory activity and mechanism of HDAC10 after LPS challenge.ResultsHDAC10 expression was increased both in mice lung tissues and macrophage cell lines and promoted inflammatory cytokines production exposed to LPS. Hdac10 deficiency inhibited autophagy and inflammatory response after LPS stimulation. In vivo, Hdac10fl/fl-LysMCre mice considerably attenuated lung inflammation and inflammatory cytokines release exposed to LPS. Mechanistically, HDAC10 interacts with P62 and mediates P62 deacetylation at lysine 165 (K165), by which it promotes P62 expression and increases inflammatory cytokines production. Importantly, we identified that Salvianolic acid B (SAB), an HDAC10 inhibitor, reduces lung inflammatory response in LPS-stimulated ALI.ConclusionThese results uncover a previously unknown role for HDAC10 in regulating P62 deacetylation and aggravating lung inflammation in LPS-induced ALI, implicating that targeting HDAC10 is an effective therapy for LPS-exposed ALI.

Effect of Peitu Yifei Granules on autophagy mechanism in rats with idiopathic pulmonary fibrosis based on PI3K/Akt/mTOR signaling pathway

To investigate the mechanism by which Peitu Yifei Granules inhibit idiopathic pulmonary fibrosis(IPF) in rats, fifty specific-pathogen-free(SPF) grade male Wistar rats were randomly divided into blank group and modeling group. IPF was induced in the modeling group rats by tracheal infusion of 5 mg·kg~(-1) bleomycin(BLM) and then randomly divided into model group, pirfenidone group, and high-dose, medium-dose, and low-dose groups treated with Peitu Yifei Granules. After 24 hours of modeling, the treatment groups received intragastric administration of either Peitu Yifei Granules or pirfenidone as a positive control drug; meanwhile, the model group received an equal volume of normal saline. After 21 days of treatment administration, lung tissue samples were collected for analysis. Pathological changes in lung tissues were assessed using hematoxylin-eosin(HE) staining and Masson's trichrome staining. The expression levels of protein kinase B(Akt), mammalian target of rapamycin(mTOR), their phosphorylated forms, and sequestosome 1(p62) were determined through Western blot(WB). Fluorescent quantitative real-time polymerase chain reaction(RT-qPCR) was used to measure messenger ribonucleic acid(mRNA) expression levels of Beclin-1, microtubule-associated proteins 1A/1B light chain 3B(LC3B), and p62. Immunohistochemistry was performed to assess protein expression levels of Beclin-1 and LC3B in lung tissue samples. RESULTS:: demonstrated that lung tissue structure appeared normal without significant collagen deposition in the blank group rats. In contrast, rats from the model group exhibited thickened alveolar septa along with evident inflammatory changes and collagen deposition. Compared to the model group rats, those treated with Peitu Yifei Granules or pirfenidone showed significantly improved lung tissue structure with reduced inflammation and collagen deposition observed histologically. Furthermore, compared with those of the blank group, the expressions of p62 and its mRNA, p-Akt and p-mTOR protein in lung tissues of the model group were significantly increased, while Beclin-1, LC3B and their mRNA levels were significantly decreased. Compared with those of the model group, the expressions of p62 and its mRNA, p-Akt and p-mTOR in lung tissues of the pirfenidone group and Peitu Yifei Granules high-dose and medium-dose groups were significantly decreased, while Beclin-1, LC3B and their mRNA expressions were significantly increased. The above results indicate that Peitu Yifei Granules can improve autophagy levels in lung tissues by inhibiting the phosphoinositide 3-kinase(PI3K)/Akt/mTOR signaling pathway and delay the development of IPF disease.

Effects and mechanisms of Gegen Qinlian Decoction in inhibiting M1 polarization of macrophages under inflammatory hypoxia microenvironment

To explore the effect and mechanism of Gegen Qinlian Decoction(GQD) in inhibiting M1 polarization of macrophages under inflammatory hypoxia by simulating intestinal hypoxia microenvironment in vitro. A tri-gas incubator was used to simulate normal physiological hypoxia of the colon and inflammatory hypoxia microenvironments of ulcerative colitis(UC). RAW264.7 macrophages were divided into 18.5% O_(2 )(normoxia group), 4% O_2(physiological hypoxia group), and 1% O_2(inflammatory hypoxia group), and they were induced by lipopolysaccharide(LPS) for 24 h. M1 polarization was detected by flow cytometry. Under the condition of 1% inflammatory hypoxia, they were divided into blank group, model group, and GQD-containing serum low, medium, and high dose groups. Flow cytometry was used to detect M1 polarization marker CD86, and ELISA was used to detect the expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in cell supernatant. The mRNA expression of hypoxia-inducible factor-1α(HIF-1α), TNF-α, and IL-1β was detected by qRT-PCR. Western blot was used to detect the expression of HIF-1α/nuclear transcription factor-κB(NF-κB) signaling pathway-related proteins. The nuclear translocation of NF-κB p65 was detected by immunofluorescence. The results showed that the positive rate of CD86 in the 1% O_2 group was the highest. Under the condition of 1% inflammatory hypoxia, compared with the blank group, the expression of CD86, TNF-α, IL-1β, and HIF-1α in the model group increased. Compared with the model group, each group of GQD could reduce the expression of CD86, TNF-α, IL-1β, and HIF-1α. Compared with the blank group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/β, and p-IκBα in the model group increased. Compared with the model group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/β, and p-IκBα in GQD groups was significantly decreased. Compared with the blank group, NF-κB p65 in the model group entered the nucleus significantly. Compared with the model group, the nuclear expression of NF-κB p65 was decreased in each GQD group. Studies have shown that GQD may protect the intestine by down-regulating the HIF-1α/NF-κB signaling pathway to inhibit M1 polarization of macrophages and secretion of related inflammatory factors under 1% inflammatory hypoxia.

Intervention mechanism of ginsenoside Rb_1 on liver steatosis in db/db obese mice based on TLR4/MyD88/NF-κB signaling pathway

Based on the Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor kappaB(NF-κB) signaling pathway, this study observed the regulatory effect of ginsenoside Rb_1(Rb_1) on liver lipid metabolism in db/db obese mice and explored its potential mechanism. Thirty 6-week-old male db/db mice were randomly divided into a model group, a metformin group, and Rb_1 groups with low, medium, and high doses, with six mice in each group. Additionally, six age-matched male db/m mice were assigned to the normal group. The intervention lasted for five weeks. Body weight, fasting blood glucose, and food intake were mea-sured weekly. At the end of the experiment, serum lipid levels and liver function were detected. Hematoxylin-eosin(HE) staining and oil red O staining were performed to observe pathological changes in liver tissue. Real-time quantitative PCR and immunohistochemistry on paraffin sections were used to detect the mRNA and protein expression of TLR4, MyD88, and NF-κB p65. RESULTS:: showed that compared with the normal group, the model group exhibited significant increases in body weight, liver weight, liver index, epididymal fat mass, epididymal fat index, total cholesterol, low-density lipoprotein cholesterol, liver function parameters, and fasting blood glucose levels. Liver lipid accumulation significantly increased, along with elevated mRNA and protein expression of TLR4, MyD88, and NF-κB p65 in the liver. After Rb_1 treatment, the above-mentioned parameters in the intervention groups showed significant reversals. In conclusion, Rb_1 can improve obesity and obesity-related hepatic steatosis in mice while regulating abnormal lipid and glucose meta-bolism. Mechanistically, Rb_1 may improve liver steatosis in db/db obese mice by modulating the TLR4/MyD88/NF-κB signaling pathway.

Tanshinone IIA Promoted Autophagy and Inhibited Inflammation to Alleviate Podocyte Injury in Diabetic Nephropathy.

Tanshinone IIA (Tan-IIA) is widely used in patients with diabetic nephropathy (DN), but its protective effect on podocytes in DN has not been well studied. In this study, the effects of Tan-IIA on autophagy and inflammation of glomerular podocytes in DN were observed in vivo and in vitro, and the underlying mechanisms were investigated. Irbesartan, an angiotensin II receptor blocker, is a representative medication for the clinical treatment of DN. So irbesartan was chosen as a positive control drug. Eight-week-old male db/db mice were randomly divided into a DN group, an irbesartan group, and three groups receiving different doses of Tan-IIA. The control group consisted of the db/m littermate mice. Blood, urine, and kidney samples were taken from the mice after 12 weeks of continuous administration. Renal protection of Tan-IIA was evaluated using enzyme-linked immunosorbent assay kits, haematoxylin and eosin staining, transmission electron microscopy, Western blotting, and immunohistochemistry. In vitro, the protective effect of Tan-IIA on podocytes was explored using MPC5 cells cultured with high glucose. Tan-IIA significantly improved renal pathological injury and relieved the renal dysfunction in DN. Compared with the DN group, Tan-IIA could up-regulate the expression of Synaptopodin, Podocin, LC3II/I and Beclin-1 (p < 0.05), and down-regulate the expression of p62, F4/80, NF-κB p65, IL-1β, TNF-α and IL-6 (p < 0.05) both in vivo and in vitro, suggesting that Tan-IIA treatment alleviated podocyte injury by promoting autophagy and inhibiting inflammation during DN. The levels of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR in Tan-IIA group were lower than those in DN group (p < 0.05), indicating that Tan-IIA inhibited the PI3K/Akt/mTOR signalling pathway in podocytes, which was a key pathway in regulating both autophagy and inflammation. Tan-IIA prevented podocyte injury in DN by fostering autophagy and inhibiting inflammation, at least in part via inhibition of the PI3K/Akt/mTOR signalling pathway.