In recent years, there has been a growing interest in the research on NOD-like receptor thermal protein domain associated protein 3(NLRP3) inflammasome inhibitors in the treatment of inflammatory diseases. The NLRP3 inflammasome is integral to the innate immune response, and its abnormal activation can lead to the release of pro-inflammatory cytokine, consequently facilitating the progression of various pathological conditions. Therefore, investigating the pharmacological inhibition pathway of the NLRP3 inflammasome represents a promising strategy for the treatment of inflammation-related diseases. Currently, the Food and Drug Administration(FDA) has not approved drugs targeting the NLRP3 inflammasome for clinical use due to concerns regarding liver toxicity and gastrointestinal side effects associated with chemical small molecule inhibitors in clinical trials. Natural small molecule compounds such as polyphenols, flavonoids, and alkaloids are ubiquitously found in animals, plants, and other natural substances exhibiting pharmacological activities. Their abundant sources, intricate and diverse structures, high biocompatibility, minimal adverse reactions, and superior biochemical potency in comparison to synthetic compounds have attracted the attention of extensive scholars. Currently, certain natural small molecule compounds have been demonstrated to impede the activation of the NLRP3 inflammasome via various action mechanisms, so they are viewed as the innovative, feasible, and minimally toxic therapeutic agents for inhibiting NLRP3 inflammasome activation in the treatment of both acute and chronic inflammatory diseases. Hence, this study systematically examined the effects and potential mechanisms of natural small molecule compounds derived from traditional Chinese medicine on the activation of NLRP3 inflammasomes at their initiation, assembly, and activation stages. The objection is to furnish theoretical support and practical guidance for the effective clinical application of these natural small molecule inhibitors.
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Inflammasomes/metabolism* ; Inflammation/drug therapy* ; Anti-Inflammatory Agents/therapeutic use* ; Humans ; Animals ; Disease Models, Animal ; Biological Products/therapeutic use* ; Drug Discovery ; Medicine, Chinese Traditional/methods*

This study aimed to explore the therapeutic mechanisms of Colquhounia Root Tablets(CRT) in treating diabetic kidney disease(DKD) by integrating biomolecular network mining with animal model verification. By analyzing clinical transcriptomics data, an interaction network was constructed between candidate targets of CRT and DKD-related genes. Based on the topological eigenvalues of network nodes, 101 core network targets of CRT against DKD were identified. These targets were found to be closely related to multiple pathways associated with type 2 diabetes, immune response, and metabolic reprogramming. Given that immune-inflammatory imbalance driven by metabolic reprogramming is one of the key pathogenic mechanisms of DKD, and that many core network targets of CRT are involved in this pathological process, receptor for advanced glycation end products(RAGE)-reactive oxygen species(ROS)-phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT)-nuclear factor-κB(NF-κB)-NOD-like receptor family pyrin domain containing 3(NLRP3) signaling axis was selected as a candidate target for in-depth research. Further, a rat model of DKD induced by a high-sugar, high-fat diet and streptozotocin was established to evaluate the pharmacological effects of CRT and verify the expression of related targets. The experimental results showed that CRT could effectively correct metabolic disturbances in DKD, restore immune-inflammatory balance, and improve renal function and its pathological changes by inhibiting the activation of the RAGE-ROS-PI3K-AKT-NF-κB-NLRP3 signaling axis. In conclusion, this study reveals that CRT alleviates the progression of DKD through dual regulation of metabolic reprogramming and immune-inflammatory responses, providing strong experimental evidence for its clinical application in DKD.
Animals ; Diabetic Nephropathies/metabolism* ; Receptor for Advanced Glycation End Products/genetics* ; NF-kappa B/genetics* ; Signal Transduction/drug effects* ; Rats ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Phosphatidylinositol 3-Kinases/genetics* ; Reactive Oxygen Species/metabolism* ; Humans ; Plant Roots/chemistry* ; Rats, Sprague-Dawley ; Tablets/administration & dosage*

This study aims to investigate the effect of Congrong San(CRS) on endoplasmic reticulum stress-induced neuroinflammation in the rat model of Aβ_(1-42)-induced Alzheimer's disease(AD). Sixty male Sprague-Dawley rats(2 months old) were randomized into blank(CON), model(MOD), low-dose Congrong San(L-CRS), medium-dose Congrong San(M-CRS), high-dose Congrong San(H-CRS), and memantine hydrochloride(MJG) groups. The Morris water maze test was carried out to examine the learning and memory abilities of rats in each group. Hematoxylin-eosin staining and Nissl staining were employed to observe the morphology and number of CA1 neurons in the hippocampus of rats in each group. The morphology and structure of the endoplasmic reticulum in the hippocampus were observed by transmission electron microscopy. The immunofluorescence assay was employed to detect the expression of 78 kDa glucose-regulated protein(GRP78) in the hippocampus. Western blot was employed to determine the expression of apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate-specific proteinase(caspase-1), interleukin-18(IL-18), interleukin-1β(IL-1β), GRP78, and pathway proteins including protein kinase RNA-like endoplasmic reticulum kinase(PERK), phosphorylated PERK(p-PERK), C/EBP homologous protein(CHOP), and NOD-like receptor pyrin domain-containing protein 3(NLRP3) in the rat hippocampus. Compared with the MOD group, the M-CRS and H-CRS groups showed improved learning and memory abilities, reduced neuron losses in the hippocampus, alleviated endoplasmic reticulum stress, inhibited PERK-CHOP-NLRP3 pathway, and lowered levels of IL-1β, IL-6, and tumor necrosis factor-alpha(TNF-α). The results suggest that CRS can alleviate cognitive impairment and hippocampal neuron damage and reduce neuroinflammation in AD rats by alleviating endoplasmic reticulum stress to inhibit the activation of NLRP3 inflammasomes.
Animals ; Endoplasmic Reticulum Stress/drug effects* ; Male ; Alzheimer Disease/psychology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Rats, Sprague-Dawley ; Rats ; Inflammasomes/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Cognitive Dysfunction/metabolism* ; Disease Models, Animal ; Hippocampus/drug effects* ; Humans ; Neuroinflammatory Diseases/drug therapy*

This study aims to investigate the pathogenesis of precancerous lesions of gastric cancer(PLGC) and explore the potential molecular mechanism of Weifuchun Capsules(WFC) in treating PLGC via the nuclear factor-κB(NF-κB)/NOD-like receptor protein 3(NLRP3) inflammasome signaling pathway. Ninety male SPF-grade Wistar rats were randomized into a normal feeding group and a modeling group. The normal feeding group received a regular diet, while the modeling group was subjected to the disease-syndrome combined modeling of PLGC. Specifically, the rats had free access to the water containing 120 μg·mL~(-1) N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and received a diet containing 0.05% ranitidine in an irregular feeding pattern(alternations between fasting and overfeeding). After 15 weeks, the rats in the normal feeding group were randomized into control, control-NF-κB activator betulinic acid(C-BA), and control-NF-κB inhibitor pyrrolidine dithiocarbamaten(C-PDTC) groups. Meanwhile, the rats in the modeling group continuously underwent the modeling procedure and were randomized into model, WFC, model-NF-κB activator(M-BA), and model-NF-κB inhibitor(M-PDTC) groups. The model group and control group were given aseptic water by intragastric administration, once a day. WFC was given at a dose(432 mg·kg~(-1)) 6 times the equivalent dose for adults(body weight: 60 kg) by gavage, once a day. The rats in the C-BA and M-BA groups were administrated with BA by intraperitoneal injection at a dose of 10 mg·kg~(-1), twice a week. The rats in the C-PDTC and M-PDTC groups were administrated with PDTC by intraperitoneal injection at a dose of 50 mg·kg~(-1), twice a week. The interventions were carried out for 4 weeks. Histopathological changes of the gastric mucosa were observed and scored by hematoxylin-eosin(HE) and alcian blue-periodic acid Sthiff(AB-PAS) staining. The levels of inflammatory cytokines including interleukin(IL)-1β, IL-6, IL-18, tumor necrosis factor-alpha(TNF-α), and IL-10 in the gastric tissue were determined by enzyme-linked immunosorbent assay(ELISA). The expression levels of proteins associated with the NF-κB/NLRP3 inflammasome in the gastric mucosa were determined by Western blot. The positive expression areas of proteins related to NF-κB/NLRP3 inflammasome in the gastric mucosa were measured by immunohistochemistry. The results showed that compared with the control group, the model, C-BA, and M-BA groups showed significantly risen scores of mucosal inflammation, degree of inflammatory activity, gland atrophy, and intestinal metaplasia, and the model and M-BA groups showed significanly risen scores of dysplasia. Compared with the model group, the WFC group demonstrated significantly declined scores of mucosal inflammation and degree of inflammatory activity, as well as declined scores of intestinal metaplasia and dysplasia. Compared with the control group, the model and C-BA groups showed significantly elevated levels of IL-1β, IL-6, IL-18, and TNF-α in the gastric tissue, and the model group showed significantly elevated level of IL-10. In addition, the model and C-BA groups showed significantly up-regulated expression of NF-κB p65, NLRP3, cysteine-aspartic acid protease 1(caspase-1), and apoptosis-associated speck-like protein containing a CARD(ASC) in the gastric mucosa and increased positive expression areas of NF-κB p65, NLRP3, and ASC. Compared with the model group, the WFC group showed significantly decreased levels of IL-1β, IL-6, IL-18, TNF-α, and IL-10 in the gastric tissue, and the M-PDTC group showed significantly lowered levels of IL-1β, IL-18, and TNF-α in the gastric mucosa. Both WFC and M-PDTC groups demonstrated significantly down-regulated expression levels of NF-κB p65, phosphorylated NF-κB p65(p-NF-κB p65), NLRP3, and caspase-1 in the gastric mucosa, along with significant decreases in the positive expression areas of NF-κB p65, NLRP3, and ASC. In conclusion, the pathogenesis of PLGC is closely related to the activation of the NF-κB/NLRP3 inflammasome signaling pathway. WFC can alleviate mucosal inflammation, inhibit glandular atrophy, partially reverse intestinal metaplasia, and reduce dysplasia to delay the process of inflammation-cancer transformation, and meanwhile it can effectively lower the levels of inflammatory cytokines and down-regulate the expression of pathway-related proteins in the stomach. Therefore, WFC may treat PLGC by inhibiting the NF-κB/NLRP3 inflammasome signaling pathway.
Animals ; Male ; NF-kappa B/genetics* ; Rats ; Rats, Wistar ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Stomach Neoplasms/pathology* ; Inflammasomes/genetics* ; Humans ; Precancerous Conditions/metabolism* ; Capsules

Gastric cancer is one of the most common malignant tumors in the digestive tract, which has the characteristics of high morbidity and mortality. However, gastric cancer is not achieved overnight but is gradually developing through the interaction of many factors. Therefore, actively delaying or blocking the "inflammation-cancer" transformation in gastric mucosa is the key to treatment. Nod-like receptor protein 3(NLRP3) inflammasome is a multi-protein signal complex and one of the important innate immune signal receptors. Inflammation plays an important role in the occurrence and development of gastric cancer, and continuous inflammation mediation will trigger the transformation from inflammation to cancer. Therefore, the significance of NLRP3 inflammasome to gastric mucosa lies in the transformation between inflammation and cancer. Traditional Chinese medicine(TCM) has the functions of multi-components, multi-targets, and few adverse reactions. A large number of studies show that TCM and related monomers have significant effects in treating liver, kidney, and immune diseases through mediating NLRP3 inflammasome, but there is less research on the "inflammation-cancer" transformation in gastric mucosa. By combing the NLRP3-related nuclear factor-κB transcription factor(NF-κB), hypoxia inducible factor-1α(HIF-1α), phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt), and other signal pathways, this paper clarified their mechanisms in the "inflammation-cancer" transformation in gastric mucosa, delayed the process of "inflammation-cancer" transformation in gastric mucosa through four aspects: energy metabolism, pyroptosis, immune response, and vascular endothelial growth factor, and prevented and treated "inflammation-cancer" transformation in gastric mucosa from three aspects: TCM monomer, TCM compound prescription, and other therapies, so as to provide ideas for the subsequent treatment of "inflammation-cancer" transformation in gastric mucosa with TCM.
Humans ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Inflammasomes/metabolism* ; Gastric Mucosa/metabolism* ; Stomach Neoplasms/pathology* ; Animals ; Drugs, Chinese Herbal/pharmacology* ; Medicine, Chinese Traditional ; Inflammation/drug therapy* ; Signal Transduction/drug effects*

This study aims to investigate the protective effects and mechanisms of polysaccharide extract PCP1 from Polygonatum cyrtonema in ameliorating cerebral ischemia-reperfusion(I/R) injury in rats through modulation of the Toll-like receptor 4(TLR4)/NOD-like receptor protein 3(NLRP3) signaling pathway. In vivo, SD rats were randomly divided into the sham group, model group, PCP1 group, nimodipine(NMDP) group, and TLR4 signaling inhibitor(TAK-242) group. A middle cerebral artery occlusion/reperfusion(MCAO/R) model was established, and neurological deficit scores and infarct size were evaluated 24 hours after reperfusion. Hematoxylin-eosin(HE) and Nissl staining were used to observe pathological changes in ischemic brain tissue. Transmission electron microscopy(TEM) assessed ultrastructural damage in cortical neurons. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-18(IL-18), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), and nitric oxide(NO) in serum. Immunofluorescence was used to analyze the expression of TLR4 and NLRP3 proteins. In vitro, a BV2 microglial cell oxygen-glucose deprivation/reperfusion(OGD/R) model was established, and cells were divided into the control, OGD/R, PCP1, TAK-242, and PCP1 + TLR4 activator lipopolysaccharide(LPS) groups. The CCK-8 assay evaluated BV2 cell viability, and ELISA determined NO release. Western blot was used to analyze the expression of TLR4, NLRP3, and downstream pathway-related proteins. The results indicated that, compared with the model group, PCP1 significantly reduced neurological deficit scores, infarct size, ischemic tissue pathology, cortical cell damage, and the levels of inflammatory factors IL-1β, IL-6, IL-18, TNF-α, and NO(P<0.01). It also elevated IL-10 levels(P<0.01) and decreased the expression of TLR4 and NLRP3 proteins(P<0.05, P<0.01). Moreover, in vitro results showed that, compared with the OGD/R group, PCP1 significantly improved BV2 cell viability(P<0.05, P<0.01), reduced cell NO levels induced by OGD/R(P<0.01), and inhibited the expression of TLR4-related inflammatory pathway proteins, including TLR4, myeloid differentiation factor 88(MyD88), tumor necrosis factor receptor-associated factor 6(TRAF6), phosphorylated nuclear factor-kappaB dimer RelA(p-p65)/nuclear factor-kappaB dimer RelA(p65), NLRP3, cleaved-caspase-1, apoptosis-associated speck-like protein(ASC), GSDMD-N, IL-1β, and IL-18(P<0.05, P<0.01). The protective effects of PCP1 were reversed by LPS stimulation. In conclusion, PCP1 ameliorates cerebral I/R injury by modulating the TLR4/NLRP3 signaling pathway, exerting anti-inflammatory and anti-pyroptotic effects.
Animals ; Toll-Like Receptor 4/genetics* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Rats, Sprague-Dawley ; Rats ; Reperfusion Injury/genetics* ; Male ; Signal Transduction/drug effects* ; Polysaccharides/isolation & purification* ; Polygonatum/chemistry* ; Brain Ischemia/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Humans

This study investigated the effects and underlying mechanisms of vanillic acid(VA) against cardiac fibrosis(CF) induced by isoproterenol(ISO) in mice. Male C57BL/6J mice were randomly divided into control group, VA group(100 mg·kg~(-1), ig), ISO group(10 mg·kg~(-1), sc), ISO + VA group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig), ISO + dynamin-related protein 1(Drp1) inhibitor(Mdivi-1) group(10 mg·kg~(-1), sc + 50 mg·kg~(-1), ip), and ISO + VA + Mdivi-1 group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig + 50 mg·kg~(-1), ip). The treatment groups received the corresponding medications once daily for 14 consecutive days. On the day after the last administration, cardiac functions were evaluated, and serum and cardiac tissue samples were collected. These samples were analyzed for serum aspartate aminotransferase(AST), lactate dehydrogenase(LDH), creatine kinase-MB(CK-MB), cardiac troponin I(cTnI), reactive oxygen species(ROS), interleukin(IL)-1β, IL-4, IL-6, IL-10, IL-18, and tumor necrosis factor-α(TNF-α) levels, as well as cardiac tissue catalase(CAT), glutathione(GSH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC) activities, and cytochrome C levels in mitochondria and cytoplasm. Hematoxylin-eosin, Masson, uranium acetate and lead citrate staining were used to observe morphological and mitochondrial ultrastructural changes in the cardiac tissues, and myocardial injury area and collagen volume fraction were calculated. Flow cytometry was applied to detect the relative content and M1/M2 polarization of cardiac macrophages. The mRNA expression levels of macrophage polarization markers [CD86, CD206, arginase 1(Arg-1), inducible nitric oxide synthase(iNOS)], CF markers [type Ⅰ collagen(Coll Ⅰ), Coll Ⅲ, α-smooth muscle actin(α-SMA)], and cytokines(IL-1β, IL-4, IL-6, IL-10, IL-18, TNF-α) in cardiac tissues were determined by quantitative real-time PCR. Western blot was used to detect the protein expression levels of Coll Ⅰ, Coll Ⅲ, α-SMA, Drp1, p-Drp1, voltage-dependent anion channel(VDAC), hexokinase 1(HK1), NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein(ASC), caspase-1, cleaved-caspase-1, gasdermin D(GSDMD), cleaved N-terminal gasdermin D(GSDMD-N), IL-1β, IL-18, B-cell lymphoma-2(Bcl-2), B-cell lymphoma-xl(Bcl-xl), Bcl-2-associated death promoter(Bad), Bcl-2-associated X protein(Bax), apoptotic protease activating factor-1(Apaf-1), pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, poly(ADP-ribose) polymerase-1(PARP-1), and cleaved-PARP-1 in cardiac tissues. The results showed that VA significantly improved cardiac function in mice with CF, reduced myocardial injury area and cardiac index, and decreased serum levels of AST, CK-MB, cTnI, LDH, ROS, IL-1β, IL-6, IL-18, and TNF-α. VA also lowered MDA and MPO levels, mRNA expressions of IL-1β, IL-6, IL-18, and TNF-α, and mRNA and protein expressions of Coll Ⅰ, Coll Ⅲ, and α-SMA in cardiac tissues, and increased serum levels of IL-4 and IL-10, cardiac tissue levels of CAT, GSH, SOD, and T-AOC, and mRNA expressions of IL-4 and IL-10. Additionally, VA ameliorated cardiac pathological damage, inhibited myocardial cell apoptosis, inflammatory infiltration, and collagen fiber deposition, reduced collagen volume fraction, and alleviated mitochondrial damage. VA decreased the ratio of F4/80~+CD86~+ M1 cells and the mRNA expressions of CD86 and iNOS in cardiac tissue, and increased the ratio of F4/80~+CD206~+ M2 cells and the mRNA expressions of CD206 and Arg-1. VA also reduced protein expressions of p-Drp1, VDAC, NLRP3, ASC, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-1β, IL-18, Bad, Bax, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP-1, and cytoplasmic cytochrome C, and increased the expressions of HK1, Bcl-2, Bcl-xl, pro-caspase-3, pro-caspase-9 proteins, as well as the Bcl-2/Bax and Bcl-xl/Bad ratios and mitochondrial cytochrome C content. These results indicate that VA has a significant ameliorative effect on ISO-induced CF in mice, alleviates ISO-induced oxidative damage and inflammatory response, and its mechanism may be closely related to the inhibition of Drp1/HK1/NLRP3 and mitochondrial apoptosis signaling pathways, suppression of myocardial cell inflammatory infiltration and collagen fiber deposition, reduction of collagen volume fraction and CollⅠ, Coll Ⅲ, and α-SMA expressions, thus mitigating CF.
Animals ; Isoproterenol/adverse effects* ; Male ; Mice ; Signal Transduction/drug effects* ; Vanillic Acid/administration & dosage* ; Dynamins/genetics* ; Mice, Inbred C57BL ; Fibrosis/genetics* ; Apoptosis/drug effects* ; Mitochondria/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Myocardium/metabolism* ; Humans

This study aims to explore the mechanism through which Yishen Jiangtang Decoction(YSJTD) regulates endoplasmic reticulum stress(ERS)-mediated NOD-like receptor thermal protein domain associated protein 3(NLRP3) inflammasome to improve diabetic nephropathy(DN) in db/db mice. Thirty db/db mice were randomly divided into the model group, YSJTD group, ERS inhibitor 4-phenylbutyric acid(4-PBA) group, with 10 mice in each group. Additionally, 10 db/m mice were selected as the control group. The YSJTD group was orally administered YSJTD at a dose of 0.01 mL·g~(-1), the 4-PBA group was orally administered 4-PBA at a dose of 0.5 mg·g~(-1), and the control and model groups were given an equal volume of carboxylmethyl cellulose sodium. The treatments were administered once daily for 8 weeks. Food intake, water consumption, and body weight were recorded every 2 weeks. After the intervention, fasting blood glucose(FBG), glycosylated hemoglobin(HbA1c), urine microalbumin(U-mALB), 24-hour urine volume, serum creatinine(Scr), and blood urea nitrogen(BUN) were measured. Inflammatory markers interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected using the enzyme-linked immunosorbent assay(ELISA). Renal pathology was assessed through hematoxylin-eosin(HE), periodic acid-Schiff(PAS), and Masson staining, and transmission electron microscopy(TEM). Western blot was used to detect the expression levels of glucose-regulated protein 78(GRP78), C/EBP homologous protein(CHOP), NLRP3, apoptosis-associated speck-like protein containing CARD(ASC), cysteinyl aspartate-specific proteinase(caspase-1), and gasdermin D(GSDMD) in kidney tissues. The results showed that compared to the control group, the model group exhibited poor general condition, increased weight and food and water intake, and significantly higher levels of FBG, HbA1c, U-mALB, kidney index, 24-hour urine volume, IL-1β, and IL-18. Compared to the model group, the YSJTD and 4-PBA groups showed improved general condition, increased body weight, decreased food intake, and lower levels of FBG, U-mALB, kidney index, 24-hour urine volume, and IL-1β. Specifically, the YSJTD group showed a significant reduction in IL-18 levels compared to the model group, while the 4-PBA group exhibited decreased water intake and HbA1c levels compared to the model group. Although there was a decreasing trend in water intake and HbA1c in the YSJTD group, the differences were not statistically significant. No significant differences were observed in BUN, Scr, and kidney weight among the groups. Renal pathology revealed that the model group exhibited more severe renal damage compared to the control group. Kidney sections from the model group showed diffuse mesangial proliferation in the glomeruli, tubular edema, tubular dilation, significant inflammatory cell infiltration in the interstitium, and increased glycogen staining and blue collagen deposition in the basement membrane. In contrast, the YSJTD and 4-PBA groups showed varying degrees of improvement in renal damage, glycogen staining, and collagen deposition, with the YSJTD group showing more significant improvements. TEM analysis indicated that the model group had extensive cytoplasmic edema, homogeneous thickening of the basement membrane, fewer foot processes, and widening of fused foot processes. In the YSJTD and 4-PBA groups, cytoplasmic swelling of renal tissues was reduced, the basement membrane remained intact and uniform, and foot process fusion improved.Western blot results indicated that compared to the control group, the model group showed upregulation of GRP78, CHOP, GSDMD, NLRP3, ASC, and caspase-1 expression. In contrast, both the YSJTD and 4-PBA groups showed downregulation of these markers compared to the model group. These findings suggest that YSJTD exerts a protective effect against DN by alleviating NLRP3 inflammasome activation through the inhibition of ERS, thereby improving the inflammatory response in db/db DN mice.
Animals ; Endoplasmic Reticulum Stress/drug effects* ; Diabetic Nephropathies/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Inflammasomes/drug effects* ; Male ; Kidney/pathology* ; Endoplasmic Reticulum Chaperone BiP ; Humans ; Interleukin-18/genetics* ; Mice, Inbred C57BL

This study aims to evaluate the therapeutic effect of Alpiniae Oxyphyllae Fructus-Saposhnikoviae Radix(AOF-SR) in a diabetic kidney disease(DKD) mouse model, explore its potential mechanism in regulating the NOD-like receptor protein 3(NLRP3) inflammasome via phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway, and provide new theoretical support for traditional Chinese medicine(TCM) intervention in DKD. Using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), the active ingredients and potential targets of AOF-SR were screened and its molecular mechanisms were investigated through molecular docking, molecular dynamics simulations, and experimental validation. The db/db mice were randomly divided into four groups: model group, low-dose AOF-SR group, high-dose AOF-SR group, and canagliflozin group. The db/m mice served as normal group. After one week of acclimatization, the mice underwent drug intervention. Starting from one week after treatment, body weight, blood glucose levels, and 24-hour urinary protein(24hUP) were measured every two weeks. After 13 weeks of administration, tissue collection and indicator detection were performed. Blood glucose, 24hUP, urinary microalbumin(mAlb), serum creatinine(Scr), and blood urea nitrogen(BUN) levels were determined. Pathological changes in kidney tissue were observed using hematoxylin-eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of serum IL-1β, IL-18, and caspase-1, while RT-qPCR was employed to measure the mRNA expression levels of IL-1β, IL-18, caspase-1, and NLRP3. Western blot was used to assess the protein expression levels of NLRP3, PI3K, p-Akt, Akt, p-mTOR, and mTOR. Network pharmacology analysis indicated that wogonin, pinocembrin, hancinol, and kaempferol were the core compounds for drug treatment of the disease. Molecular docking and molecular dynamics simulations showed that core compounds, particularly wogonin, could specifically bind to PIK3R1, thereby regulating the PI3K/Akt/mTOR pathway. The experimental results indicated that both low and high doses of AOF-SR and canagliflozin significantly reduced blood glucose, 24hUP, mAlb, Scr, and BUN levels in db/db mice, while improving kidney pathological damage and inflammatory cell infiltration. Moreover, the treatments reduced the mRNA expression levels of caspase-1, IL-1β, and IL-18 in the kidneys of db/db mice, as well as the secretion of these factors in the serum. The drugs also inhibited the mRNA and protein expression levels of NLRP3 in the kidneys of db/db mice and decreased the protein levels of PI3K, p-Akt/Akt, and p-mTOR/mTOR. In conclusion, AOF-SR may improve kidney inflammation in DKD mice by regulating the PI3K/Akt/mTOR signaling pathway and inhibiting NLRP3 inflammasome activation.
Animals ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Signal Transduction/drug effects* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Diabetic Nephropathies/metabolism* ; Inflammasomes/drug effects* ; Male ; Drugs, Chinese Herbal/chemistry* ; Humans ; Mice, Inbred C57BL

This paper investigated the inhibitory effect of carbonized Scutellariae Radix(Cb-SR) on pyroptosis in endometrial epithelial cells of mice with endometritis and its correlation with the IKBKE/NLRP3 signaling axis. Mice model of endometritis was established by using an intrauterine injection of 10 μL polysaccharides(LPS, 5 mg·mL~(-1)), and the mice were randomly divided into model group(LPS), low-dose group of Cb-SR(L-Cb-SR, 0.55 g·kg~(-1)), medium-dose group of Cb-SR(M-Cb-SR, 1.10 g·kg~(-1)), high-dose group of Cb-SR(H-Cb-SR, 2.20 g·kg~(-1)), crude Scutellariae Radix group(Cr-SR, 1.63 g·kg~(-1)), and Fuke Qianjin Capsule group(FQC, 0.30 g·kg~(-1)), with 10 mice in each group. Ten healthy female mice were selected and injected with PBS of equal volume into the bilateral uterus, and they were set as the sham group. The mice in the drug treatment groups were given the corresponding doses of Cb-SR, Cr-SR, FQC, or physiological saline of equal volume by gavage twice a day for seven days. Thirty minutes after the last administration, each mouse was euthanized by cervical dislocation. Hematoxylin-eosin(HE) staining and transmission electron microscopy were applied to observe the histopathological morphology of the uterine tissue. Immunohistochemistry was used to detect the expression of CD38 and CD138. Myeloperoxidase(MPO) values in neutrophils were measured by the kit; Enzyme-linked immunosorbent assay(ELISA) was used to measure the secretion of interleukin-18(IL-18), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α). Immunofluorescence and Western blot were used to analyze the expression of the proteins related to the IKBKE/NLRP3 signaling axis. Mouse endometrial epithelial cells(MEECs) were separated and purified from the uterine tissue of pregnant female mice through in vitro experiments and injured by LPS for 24 h, and then they were cultured with Cb-SR-containing serum. The anti-endometritis effect of Cb-SR was investigated by CCK-8 assay, scanning electron microscopy, and Western blot. The results showed that Cb-SR significantly reduced MPO values, attenuated uterine tissue damage, inhibited the expression of CD38 and CD138, decreased the levels of IL-1β, IL-18, and TNF-α, and inhibited the expression of proteins associated with IKBKE/NLRP3 signaling axis in mice with endometritis. In addition, Cb-SR-containing serum reduced swelling of MEECs organelles induced by LPS, decreased the expression of inflammatory factors, and suppressed the expression of IKBKE/NLRP3 signaling axis-related proteins. These results suggest that Cb-SR can inhibit endometrial epithelial cell pyroptosis in endometritis by suppressing the IKBKE/NLRP3 signaling axis.
Animals ; Female ; Mice ; Pyroptosis/drug effects* ; Signal Transduction/drug effects* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Drugs, Chinese Herbal/chemistry* ; Endometritis/chemically induced* ; Lipopolysaccharides/adverse effects* ; Scutellaria baicalensis/chemistry* ; Humans ; Epithelial Cells/drug effects*