Objective To investigate the impact of Qizhi Jiangtang Capsule (QZJT) on renal damage in diabetic nephropathy (DN) mice via NOD like receptors family pyrin domain containing 3/caspase-1/ Gasdermin D (NLRP3/caspase-1/GSDMD) signaling pathway. Methods Mice were randomly allocated into six experimental groups: a normal control group (NC), a diabetic nephropathy model group (DN), a low-dose QZJT treatment group (L-QZJT), a high-dose QZJT treatment group (H-QZJT), a positive control group administered Shenqi Jiangtang Granules (SQJT), and an ML385 group (treated with an inhibitor of nuclear factor erythroid 2-related factor 2, Nrf2). Upon successful model induction, therapeutic interventions were commenced. Renal function impairment in the mice was evaluated through quantification of fasting blood glucose (FBG), 24-hour urinary albumin (UAlb), serum creatinine (SCr), blood urea nitrogen (BUN), and the kidney-to-body mass ratio (K/B). Renal tissue pathology was evaluated using HE and PAS staining. Serum levels of inflammatory cytokines IL-1β and IL-18 were quantified by ELISA. Levels of podocyte markers and proteins involved in relevant pathways were assessed using Western blot analysis. Results Compared with the NC group, FBG, 24 h UAlb, SCr, and BUN were increased in the DN group, and the K/B mass ratio was also increased. In contrast, compared with the DN group, FBG, 24 h UAlb, SCr, and BUN in both the low-dose (L-QZJT) and high-dose Quanzhou Jintang (H-QZJT) groups were decreased, and the K/B mass ratio was decreased as well. The therapeutic efficacy of H-QZJT was comparable to that of Shenqi Jiangtang Granules. QZJT ameliorated renal histopathological injury in DN mouse, increased the protein levels of Nephrin (a podocyte marker), and decreased the protein levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), pro-caspase-1, and GSDMD-N. After ML385 treatment, renal cells exhibited swelling and morphological changes, the inflammatory infiltrate area was enlarged, the protein levels of NLRP3, ASC, pro-caspase-1, and GSDMD-N were up-regulated, and the levels of IL-1β and IL-18 were increased. Conclusion QZJT may inhibit podocyte pyroptosis by acting on the Nrf2 to regulate the NLRP3/caspase-1/GSDMD pathway, thus improving renal damage in DN mouse.
Animals ; Diabetic Nephropathies/pathology* ; Podocytes/pathology* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Pyroptosis/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Caspase 1/genetics* ; Signal Transduction/drug effects* ; Mice ; Phosphate-Binding Proteins/genetics* ; Male ; Intracellular Signaling Peptides and Proteins/metabolism* ; Mice, Inbred C57BL ; Kidney/pathology* ; Gasdermins

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a high-molecular-weight protein complex in the cytoplasm, is composed of three core components: the sensor protein NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and the effector protein caspase-1. It plays a critical role in regulating host immune and inflammatory responses. Studies have shown that the NLRP3 inflammasome has increasingly become a focal point in tumor molecular biology field. A growing body of evidence indicates that the increased expression and activation of the NLRP3 inflammasome is closely associated with the pathogenesis of head and neck squamous cell carcinoma (HNSCC) and the tumor microenvironment (TME). It may promote tumor proliferation, invasion, migration, and other biological behaviors through various regulatory mechanisms while influencing tumor immune evasion and therapy resistance, which holds promise as a prognostic biomarker for patients. This review explores the current effect and mechanism of the NLRP3 inflammasome and its signaling pathways in head and neck cancer, providing insights into clinical targeted drug development and molecular immunotherapy.
Humans ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Inflammasomes/metabolism* ; Head and Neck Neoplasms/pathology* ; Squamous Cell Carcinoma of Head and Neck/metabolism* ; Tumor Microenvironment ; Signal Transduction ; Animals

OBJECTIVE: To observe the effects of electroacupuncture at the pterygopalatine region on nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-mediated pyroptosis and inflammatory factors in rats with allergic rhinitis (AR). METHODS: Twenty-four SD rats were randomly divided into a blank group, a model group, an acupuncture group and an electroacupuncture group, 6 rats in each group. Except for the blank group, OVA-induced AR model was established in the remaining groups. In the electroacupuncture group, the rats were treated with electroacupuncture at the bilateral pterygopalatine region, with disperse-dense wave, in frequency of 2 Hz/100 Hz and current of 0.5-1 mA, 15 min each time, once every other day, for 3 times. In the acupuncture group, the rats were treated with acupuncture at bilateral pterygopalatine region simply, without electrical stimulation. The rhinitis symptom score was observed, the pathomorphology of the nasal mucosa was observed by HE staining; the serum levels of OVA-specific immunoglobulin E (OVA-sIgE), interleukin (IL)-4, IL-6 and IL-1β were detected by ELISA; the mRNA expression of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), cysteinyl aspartate specific proteinase-1 (caspase-1) and IL-18 in the nasal mucosa was detected by real-time PCR; the protein expression of NLRP3, ASC, caspase-1 and IL-18 in the nasal mucosa was detected by Western blot. RESULTS: Compared with the blank group, in the model group, the rhinitis symptom score was increased (P<0.01), the serum levels of OVA-sIgE, IL-4, IL-6 and IL-1β were increased (P<0.05), the nasal mucosa showed pathomorphology of inflammatory infiltration; the mRNA and protein expression of NLRP3, ASC, caspase-1 and IL-18 in the nasal mucosa was increased (P<0.05). Compared with the model group, in the electroacupuncture group, the rhinitis symptom score was reduced (P<0.01), the pathology of the nasal mucosa was improved; the serum levels of OVA-sIgE, IL-4, IL-6 and IL-1β were decreased (P<0.05); the mRNA and protein expression of NLRP3, ASC, caspase-1 and IL-18 in the nasal mucosa was decreased (P<0.05). CONCLUSION Electroacupuncture at the pterygopalatine region can exerting the anti-inflammatory effect by inhibiting NLRP3-mediated pyroptosis and inflammatory factor imbalance, thus alleviate rhinitis symptoms in AR rats.
Animals ; Electroacupuncture ; NLR Family, Pyrin Domain-Containing 3 Protein/immunology* ; Rats ; Rats, Sprague-Dawley ; Rhinitis, Allergic/physiopathology* ; Pyroptosis ; Male ; Acupuncture Points ; Humans ; Female ; Interleukin-1beta/genetics* ; Interleukin-18/immunology* ; Interleukin-6/genetics* ; Caspase 1/immunology*

OBJECTIVE: To observe the effect of Huayu Tongluo moxibustion on the NOD-like receptor protein 3 (NLRP3)/cysteine-aspartic acid protease-1 (Caspase-1)/gasdermin D (GSDMD) signaling pathway in rats with vascular dementia (VD), and to explore its mechanism in improving learning and memory ability and alleviating neuronal injury in the hippocampal CA1 region. METHODS: A total of 80 SPF-grade male Wistar rats were included. Three rats were excluded based on the Morris water maze test. From the remaining rats, 12 were randomly selected as the sham operation group. The rest were used to establish VD models via modified bilateral common carotid artery ligation. Thirty-six successfully modeled rats were randomly divided into a model group, a medication group, and a moxibustion group, with 12 rats in each group. The medication group was treated with nimodipine solution (12 mg/kg) via gavage. The moxibustion group was treated with Huayu Tongluo moxibustion. The suspended moxibustion was applied at Shenting (GV24) and Dazhui (GV14), and aconite cake-separated moxibustion was applied at Baihui (GV20), with each acupoint treated for 20 min. All treatments were administered once daily for 21 consecutive days. Before and after modeling, and after intervention, the Morris water maze test was used to assess cognitive function. After intervention, the activation and morphology of microglia in the hippocampal CA1 region were observed by immunofluorescence. Ultrastructure of hippocampal CA1 neurons was examined by transmission electron microscopy. Western blot was used to detect protein expression of NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, GSDMD, and interleukin-1β (IL-1β) in the hippocampal CA1 region. ELISA was used to detect the content of IL-6, IL-8, and tumor necrosis factor-α (TNF-α) in the hippocampal CA1 region. RESULTS: Compared with the sham operation group, the model group showed longer mean escape latency (P<0.01) and fewer platform crossings (P<0.01); the microglial processes in the hippocampal CA1 region were thickened, cytoplasm was hypertrophic, and relative fluorescence intensity of ionized calcium-binding adapter molecule 1 (IBA-1) was increased (P<0.05); the neuronal ultrastructure in the CA1 region was severely damaged, rough endoplasmic reticulum was swollen, mitochondria were deformed and swollen, some cristae were ruptured or dissolved, showing vacuolar changes; the protein expression of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β, as well as levels of IL-6, IL-8, and TNF-α were significantly elevated (P<0.001). Compared with the model group, both the medication group and the moxibustion group showed shortened mean escape latency (P<0.01) and increased platform crossings (P<0.01); the microglial processes were thinner, and IBA-1 fluorescence intensity was decreased (P<0.05); the neuronal ultrastructure in the CA1 region was partially improved; the protein expression of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β, and levels of IL-6, IL-8, and TNF-α were significantly reduced (P<0.001). Compared with the medication group, the moxibustion group showed shortened mean escape latency (P<0.05) and more platform crossings (P<0.05); the IBA-1 fluorescence intensity was decreased (P<0.05); the neuronal ultrastructure in the CA1 region was improved; the protein expression of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β, as well as levels of IL-6, IL-8, and TNF-α, were significantly lower (P<0.001). CONCLUSION The Huayu Tongluo moxibustion could enhance learning and memory abilities in VD rats, inhibit excessive activation of microglia, and alleviate neuronal injury in the hippocampal CA1 region. Its mechanism may involve modulation of the NLRP3/Caspase-1/GSDMD signaling pathway, reduction of inflammatory responses.
Animals ; Male ; Dementia, Vascular/physiopathology* ; Rats ; Signal Transduction ; Moxibustion ; Rats, Wistar ; CA1 Region, Hippocampal/injuries* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Caspase 1/genetics* ; Memory ; Humans ; Neurons/metabolism* ; Learning

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

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