Effects of electroacupuncture with "intestinal disease prescription" on NLRP3 inflammasome and intestinal mucosal barrier in rats with acute ulcerative colitis. / 电针"è‚ ç— æ–¹"å¯¹æ€¥æ€§æºƒç–¡æ€§ç»“è‚ ç‚Žå¤§é¼ NLRP3ç‚Žæ€§å°ä½“å’Œè‚ é»è†œå±éšœçš„å½±å“.

OBJECTIVES: To observe the effects of electroacupuncture (EA) with "intestinal disease prescription" on the intestinal mucosal barrier and NLRP3 inflammasome in rats with dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC), and explore the underlying mechanism of EA with "intestinal disease prescription" for the treatment of UC. METHODS: Thirty-two healthy male SPF-grade SD rats were randomly divided into a blank group, a model group, a medication group, and an EA group, with 8 rats in each group. Except for the blank group, the UC model was established by administering 5% DSS solution for 7 days. After modeling, the rats in the medication group were treated with mesalazine suspension (200 mg/kg) by gavage, while the rats in the EA group were treated with acupuncture at bilateral "Tianshu" (ST 25), "Shangjuxu" (ST 37) and "Zhongwan" (CV 12), with the ipsilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37) connected to the electrodes of the EA instrument, using disperse-dense wave, with a frequency of 10 Hz/50 Hz, and each intervention lasted for 20 minutes. Both interventions were performed once daily for 3 days. The general conditions of rats were observed daily. After intervention, the disease activity index (DAI) score was calculated; colon tissue morphology was observed using HE staining; serum levels of pro-inflammatory cytokines (interleukin [IL]-18, IL-1ß) were measured by ELISA; protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in colon tissues was detected by Western blot; positive expression of zonula occludens-1 (ZO-1) and Occludin in colon tissues was examined by immunofluorescence. RESULTS: Compared with the blank group, the rats in the model group exhibited poor general conditions, slow body weight gain, shortened colon length (P<0.01), increased DAI score and spleen index (P<0.01), elevated serum IL-18 and IL-1ß levels, and increased protein expression of NLRP3, ASC, and Caspase-1 in colon tissues (P<0.01), along with decreased positive expression of ZO-1 and Occludin in colon tissues (P<0.01). Compared with the model group, the rats in the medication group and the EA group exhibited improved general conditions, accelerated body weight gain, increased colon length (P<0.05), reduced DAI scores and spleen indexes (P<0.05), decreased serum IL-18 and IL-1ß levels, and lower protein expression of NLRP3, ASC and Caspase-1 in colon tissues (P<0.05), as well as increased positive expression of ZO-1 and Occludin in colon tissues (P<0.05). There were no significant differences in the above indexes between the medication group and the EA group (P>0.05). Compared with the blank group, the rats in the model group exhibited disrupted colon mucosal morphology, disordered gland arrangement, and atrophy of crypts, along with significant inflammatory cell infiltration. Compared with the model group, the rats in both the medication group and the EA group showed relatively intact colon mucosal morphology, with restored and improved gland and crypt structures, and reduced inflammatory cell infiltration. CONCLUSIONS: EA with "intestinal disease prescription" has a significant therapeutic effect on DSS-induced UC, possibly by regulating the expression of NLRP3 inflammasome and proteins related to the intestinal mucosal barrier, thereby alleviating symptoms of ulcerative colitis.

[Role of NLRP3 inflammasome in prevention and treatment of cognitive impairment-related diseases and traditional Chinese medicine intervention: a review].

Alzheimer's disease(AD), vascular dementia(VD), and traumatic brain injury(TBI) are more common cognitive impairment diseases characterized by high disability and mortality rates, imposing a heavy burden on individuals and their families. Although AD, VD, and TBI have different specific mechanisms, their pathogenesis is closely related to the nucleotide-binding oligome-rization domain-like receptor protein 3(NLRP3). The NLRP3 inflammasome is involved in neuroinflammatory responses, mediating microglial polarization, regulating the reduction of amyloid ß-protein(Aß) deposition, neurofibrillary tangles(NFTs) formation, autophagy regulation, and maintaining brain homeostasis, and synaptic stability, thereby contributing to the development of AD, VD, and TBI. Previous studies have shown that traditional Chinese medicine(TCM) can alleviate neuroinflammation, promote microglial polarization towards the M2 phenotype, reduce Aß deposition and NFTs formation, regulate autophagy, and maintain brain homeostasis by intervening in NLRP3 inflammasome, hence exerting a role in preventing and treating cognitive impairment-related diseases, reducing psychological and economic pressure on patients, and improving their quality of life. Therefore, this article elucidated the role of NLRP3 inflammasome in AD, VS, and TBI, and provided a detailed summary of the latest research results on TCM intervention in NLRP3 inflammasome for the prevention and treatment of these diseases, aiming to inherit the essence of TCM and provide references and foundations for clinical prevention and treatment of cognitive impairment-related diseases with TCM. Meanwhile, this also offers insights and directions for further research in TCM for the prevention and treatment of cognitive impairment-related diseases.

[Role of NLRP3 inflammasome in heart failure and traditional Chinese medicine intervention: a review].

Heart failure is characterized by high incidence and mortality rates, and the search for effective treatment strategies for heart failure and the improvement of clinical outcomes have always been important research directions. Imbalanced inflammation has been proven to be one of the critical pathological factors in heart failure, positively correlated with adverse events such as impaired cardiac function and myocardial fibrosis. In recent years, studies have confirmed that the activation of the NOD-like receptor thermal protein domain-associated protein 3(NLRP3) inflammasome plays a common regulatory role in the inflammation imbalance induced by various factors in heart failure. Moreover, certain traditional Chinese medicine(TCM) and active components can significantly inhibit the activation of the NLRP3 inflammasome, thereby improving heart failure. This article first overviewed the basic information about the NLRP3 inflammasome, summarized the regulatory mechanisms of the NLRP3 inflammasome in heart failure induced by various factors, introduced recent research progress on TCM and active components that inhibited the NLRP3 inflammasome to improve heart failure, aiming to provide references for innovative drug research in the field of integrated Chinese and western medicine for the prevention and treatment of heart failure.

Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1ß (IL-1ß) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Quanzhen Yiqi decoction attenuates inflammation in mice with smoking-induced COPD by activating the Nrf2/HO-1 pathway and inhibiting the NLRP3 inflammasome.

OBJECTIVE: Quanzhen Yiqi decoction (QZYQ) is a traditional Chinese medicine for treating chronic obstructive pulmonary disease. METHODS: Mice were exposed to cigarette smoke (CS) 6 days/week (40 cigarettes/day) for 24 weeks and then intragastrically administered QZYQ (4.72, 9.45, or 18.89 g/kg) or dexamethasone (DEX, 0.6 mg/kg) for 6 weeks. We examined the lung function and collected bronchoalveolar lavage fluid for inflammatory cell and cytokine quantification. The pathological lung changes, ROS and oxidative biomarkers were measured. We used immunohistochemistry and western blotting to evaluate the levels of Nrf2/HO-1, NLRP3/ASC/Caspase1/IL-1ß/IL-18. RESULTS: The CS group showed significant increases in the forced vital capacity, lung resistance, and chord compliance and a lower FEV50/FVC compared with the control, and QZYQ improved these changes. In addition, QZYQ effectively reduced emphysema, immune cell infiltration, and airway remodeling. QZYQ stimulated HO-1 expression and reduced oxidative stress through the Nrf2 pathway. QZYQ inhibited the production of NLRP3/ASC/Caspase-1 to inhibit IL-1ß and IL-18. CONCLUSION: Our study suggested that QZYQ can improve the function and histology of the lungs and reduce inflammatory cell recruitment. QZYQ inhibits ROS production and NLRP3 inflammasome activation by upregulating Nrf2 to reduce lung injury. The anti-inflammatory effects of QZYQ are similar to those of DEX.

Kai-Xin-San ameliorates Alzheimer's disease-related neuropathology and cognitive impairment in APP/PS1 mice via the mitochondrial autophagy-NLRP3 inflammasome pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Kai-Xin-San (KXS) is a classic famous prescription that has been utilized for centuries to address dementia. New investigations have shown that the anti-dementia effect of KXS is connected with improved neuroinflammation. Nevertheless, the underlying mechanism is not well elucidated. AIM OF THE STUDY: We propose to discover the ameliorative impact of KXS on Alzheimer's disease (AD) and its regulatory role on the mitochondrial autophagy-nod-like receptor protein 3 (NLRP3) inflammasome pathway. MATERIALS AND METHODS: The Y maze, Morris water maze, and new objection recognition tests were applied to ascertain the spatial learning and memory capacities of amyloid precursor protein/presenilin 1 (APP/PS1) mice after KXS-treatment. Meanwhile, the biochemical indexes of the hippocampus were detected by reagent kits. The pathological alterations and mitochondrial autophagy in the mice' hippocampus were detected utilizing hematoxylin and eosin (H&E), immunohistochemistry, immunofluorescence staining, and transmission electron microscopy. Besides, the PTEN-induced putative kinase 1 (PINK1)/Parkin and NLRP3 inflammasome pathways protein expressions were determined employing the immunoblot analysis. RESULTS: The results of behavioral tests showed that KXS significantly enhanced the AD mice' spatial learning and memory capacities. Furthermore, KXS reversed the biochemical index levels and reduced amyloid-ß protein deposition in AD mice brains. Besides, H&E staining showed that KXS remarkably ameliorated the neuronal damage in AD mice. Concurrently, the results of transmission electron microscopy suggest that KXS ameliorated the mitochondrial damage in microglia and promoted mitochondrial autophagy. Moreover, the immunofluorescence outcomes exhibited that KXS promoted the expression of protein 1 light chain 3B (LC3B) associated with microtubule and the generation of autophagic flux. Notably, the immunofluorescence co-localization results confirmed the presence of mitochondrial autophagy in microglia. Finally, KXS promoted the protein expressions of the PINK1/Parkin pathway and reduced the activation of NLRP3 inflammasome. Most importantly, these beneficial effects of KXS were attenuated by the mitochondrial autophagy inhibitor chloroquine. CONCLUSION: KXS ameliorates AD-related neuropathology and cognitive impairment in APP/PS1 mice by enhancing the mitochondrial autophagy and suppressing the NLRP3 inflammasome pathway.

Exocarpium Citri Grandis ameliorates LPS-induced acute lung injury by suppressing inflammation, NLRP3 inflammasome, and ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Exocarpium Citri Grandis (ECG), the epicarp of C. grandis 'Tomentosa' which is also known as Hua-Ju-Hong in China, has been widely used for thousands of years to treat inflammatory lung disorders such as asthma, and cough as well as dispelling phlegm. However, its underlying pharmacological mechanisms in acute lung injury (ALI) remain unclear. AIM OF THE STUDY: To explore the therapeutic effect of ECG on ALI and reveal the potential mechanisms based on experimental techniques in vivo and in vitro. MATERIALS AND METHODS: Lipopolysaccharides (LPS) induced ALI in mice and induced RAW 264.7 cell inflammatory model were established to investigate the pharmacodynamics of ECG. ELISA kits, commercial kits, Western Blot, qPCR, Hematoxylin and Eosin (H&E) staining, immunohistochemistry, and immunofluorescence technologies were used to evaluate the pharmacological mechanisms of ECG in ameliorating ALI. RESULTS: ECG significantly attenuated pulmonary edema in LPS-stimulated mice and decreased the levels of IL1ß, IL6, and TNF-α in serum and BALF, reduced MDA and iron concentration as well as increased SOD and GSH levels in lung tissues, and also decreased the ROS level in BALF and Lung tissue. Further pharmacological mechanism studies showed that ECG significantly inhibited mRNA expression of inflammatory signaling factors and chemokines, and down-regulated the expression of TLR4, MyD88, NF-κB p65, NF-κB p-p65 (S536), COX2, iNOS, Txnip, NLRP3, ASC, Caspase-1, JAK1, p-JAK1 (Y1022), JAK2, STAT1, p-STAT1 (S727), STAT3, p-STAT3 (Y705), STAT4, p-STAT4 (Y693), and Keap1, and also up-regulated the expression of Trx-1, Nrf2, HO-1, NQO1, GPX4, PCBP1, and SLC40A1. In the LPS-induced RAW264.7 cell inflammatory model, ECG showed similar results to animal experiments. CONCLUSIONS: Our results showed that ECG alleviated ALI by inhibiting TLR4/MyD88/NF-κB p65 and JAK/STAT signaling pathway-mediated inflammatory response, Txnip/NLRP3 signaling pathway-mediated inflammasome activation, and regulating Nrf2/GPX4 axis-mediated ferroptosis. Our findings provide an experimental basis for the application of ECG.

Ginsenoside Rg1 alleviates chronic inflammation-induced neuronal ferroptosis and cognitive impairments via regulation of AIM2 - Nrf2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng is a valuable herb in traditional Chinese medicine. Modern research has shown that it has various benefits, including tonifying vital energy, nourishing and strengthening the body, calming the mind, improving cognitive function, regulating fluids, and returning blood pressure, etc. Rg1 is a primary active component of ginseng. It protects hippocampal neurons, improves synaptic plasticity, enhances cognitive function, and boosts immunity. Furthermore, it exhibits anti-aging and anti-fatigue properties and holds great potential for preventing and managing neurodegenerative diseases (NDDs). AIM OF THE STUDY: The objective of this study was to examine the role of Rg1 in treating chronic inflammatory NDDs and its molecular mechanisms. MATERIALS AND METHODS: In vivo, we investigated the protective effects of Rg1 against chronic neuroinflammation and cognitive deficits in mice induced by 200 µg/kg lipopolysaccharide (LPS) for 21 days using behavioral tests, pathological sections, Western blot, qPCR and immunostaining. In vitro experiments involved the stimulation of HT22 cells with 10 µg/ml of LPS, verification of the therapeutic effect of Rg1, and elucidation of its potential mechanism of action using H2DCFDA staining, BODIPY™ 581/591 C11, JC-1 staining, Western blot, and immunostaining. RESULTS: Firstly, it was found that Rg1 significantly improved chronic LPS-induced behavioral and cognitive dysfunction in mice. Further studies showed that Rg1 significantly attenuated LPS-induced neuronal damage by reducing levels of IL-6, IL-1ß and ROS, and inhibiting AIM2 inflammasome. Furthermore, chronic LPS exposure induced the onset of neuronal ferroptosis by increasing the lipid peroxidation product MDA and regulating the ferroptosis-associated proteins Gpx4, xCT, FSP1, DMT1 and TfR, which were reversed by Rg1 treatment. Additionally, Rg1 was found to activate Nrf2 and its downstream antioxidant enzymes, such as HO1 and NQO1, both in vivo and in vitro. In vitro studies also showed that the Nrf2 inhibitor ML385 could inhibit the anti-inflammatory, antioxidant, and anti-ferroptosis effects of Rg1. CONCLUSIONS: This study demonstrated that Rg1 administration ameliorated chronic LPS-induced cognitive deficits and neuronal ferroptosis in mice by inhibiting neuroinflammation and oxidative stress. The underlying mechanisms may be related to the inhibition of AIM2 inflammasome and activation of Nrf2 signaling. These findings provide valuable insights into the treatment of chronic neuroinflammation and associated NDDs.

Aloe-emodin alleviates cerebral ischemia-reperfusion injury by regulating microglial polarization and pyroptosis through inhibition of NLRP3 inflammasome activation.

BACKGROUND: Microglial activation plays a crucial role in injury and repair after cerebral ischemia, and microglial pyroptosis exacerbates ischemic injury. NOD-like receptor protein 3 (NLRP3) inflammasome activation has an important role in microglial polarization and pyroptosis. Aloe-emodin (AE) is a natural anthraquinone compound originated from rhubarb and aloe. It exerts antioxidative and anti-apoptotic effects during cerebral ischemia/reperfusion (I/R) injury. However, whether AE affects microglial polarization, pyroptosis, and NLRP3 inflammasome activation remains unknown. PURPOSE: This study aimed to explore the effects of AE on microglial polarization, pyroptosis, and NLRP3 inflammasome activation in the cerebral infarction area after I/R. METHODS: The transient middle cerebral artery occlusion (tMCAO) and oxygen-glucose deprivation/re-oxygenation (OGD/R) methods were used to create cerebral I/R models in vivo and in vitro, respectively. Neurological scores and triphenyl tetrazolium chloride and Nissl staining were used to assess the neuroprotective effects of AE. Immunofluorescence staining, quantitative polymerase chain reaction and western blot were applied to detect NLRP3 inflammasome activation and microglial polarization and pyroptosis levels after tMCAO or OGD/R. Cell viability and levels of interleukin (IL)-18 and IL-1ß were measured. Finally, MCC950 (an NLRP3-specific inhibitor) was used to evaluate whether AE affected microglial polarization and pyroptosis by regulating the activation of the NLRP3 inflammasome. RESULTS: AE improved neurological function scores and reduced the infarct area, brain edema rate, and Nissl-positive cell rate following I/R injury. It also showed a protective effect on BV-2 cells after OGD/R. AE inhibited microglial pyroptosis and induced M1 to M2 phenotype transformation and suppressed microglial NLRP3 inflammasome activation after tMCAO or OGD/R. The combined administration of AE and MCC950 had a synergistic effect on the inhibition of tMCAO- or OGD/R-induced NLRP3 inflammasome activation, which subsequently suppressed microglial pyroptosis and induced microglial phenotype transformation. CONCLUSION: AE exerts neuroprotective effects by regulating microglial polarization and pyroptosis through the inhibition of NLRP3 inflammasome activation after tMCAO or OGD/R. These findings provide new evidence of the molecular mechanisms underlying the neuroprotective effects of AE and may support the exploration of novel therapeutic strategies for cerebral ischemia.

A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1ß production in tissue-resident macrophages.

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1ß (IL-1ß), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1ß production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1ß production, indicating that IRE1α is required for CT- or CTB-induced IL-1ß production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

Gegen Qinlian decoction ameliorates TNBS-induced ulcerative colitis by regulating Th2/Th1 and Tregs/Th17 cells balance, inhibiting NLRP3 inflammasome activation and reshaping gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.

Study on the therapeutic mechanism of HJ granules in a rat model of urinary tract infection caused by Escherichia coli.

ETHNOPHARMACOLOGICAL RELEVANCE: Urinary tract infections (UTIs) are globally prevalent infectious diseases, predominantly caused by uropathogenic Escherichia coli (UPEC). The misuse of antibiotics has led to the emergence of several drug-resistant strains. Traditional Chinese Medicine (TCM) has its own advantages in the treatment of UTIs. HJ granules is a herbal formula used for the treatment of UTIs. However, its mechanism of action is not clear. AIM OF THE STUDY: The aim of this study was to investigate the therapeutic efficacy and mechanism of action of HJ granules in a rat model of UTI caused by Escherichia coli (E coli) CFT073. MATERIALS AND METHODS: SD rats were selected to establish a rat UTI model by injecting UPEC strain CFT073 into the bladder using the transurethral placement method. HJ granules were administered to rats after modelling and the efficacy of HJ granule was investigated by measuring urinary decanalogue, inflammatory factors in bladder tissue and pathological changes in the bladder after 3d of administration. Expression of sonic hedgehog (SHH), NOD-like receptor thermoprotein domain 3 (NLRP3), apoptosis-associated speck-like protein (ASC) and activation of cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by western blotting and immunofluorescence staining in rat bladder tissue. NLRP3, ASC and caspase-1, a cysteine-containing aspartic protein, were expressed and activated. RESULTS: The results showed that infection of rats with UPEC resulted in increased pH and erythrocytes in bladder irrigation fluid; increased expression of IL-1ß, IL-6 and SHH and decreased expression of IL-10 in bladder tissue; and significant upregulation of the expression of both SHH and NLRP3 inflammasom and significant activation of NLRP3 inflammasom. HJ granules significantly increased the concentration of IL-10 in the bladder, inhibited the expression of SHH and NLRP3 inflammasom in bladder tissue, and suppressed the activation of NLRP3 inflammasom, thereby reducing inflammatory lesions in bladder tissue. CONCLUSION: HJ granules may improve bladder injury and treat UTIs by inhibiting the expression and activation of NLRP3 inflammasom.

Mechanism of Nicotiflorin in San-Ye-Qing rhizome for anti-inflammatory effect in ulcerative colitis.

BACKGROUND: The incidence of ulcerative colitis (UC) is on the rise globally and the development of drugs targeting UC is urgent. Finding the target of action of natural products is important for drug discovery, elucidation of drug action mechanism, and disease mechanism. San-Ye-Qing (SYQ), is an ancient herbal medicine, but whether the powder of its rhizome has pharmacological effects against UC and its mechanism of action are not clear. PURPOSE: To evaluate the therapeutic effectiveness of rhizome powder of SYQ in treating UC, and conduct an isolation and characterization of the chemical constituents of the powder. Further, screen the most potent compounds among them and determine the potential mechanism for treating UC. METHODS: In vivo, the therapeutic effect of SYQ's rhizome powder on UC was assessed by mice's body weight, DAI score, colon length, tissue MPO activity, serum inflammatory markers, etc. Additionally, HPLC was used to isolate and identify the specific chemical components of SYQ's rhizome powder. Then, the most effective compounds and their therapeutic targets were analysed and screened in SYQ rhizome powder using network pharmacology, combined with CCK-8 assay, NO release assay and molecular docking assay, in conjunction with CETSA, DARTS, SPR and enzyme activity assay. Finally, the biological effects of the key compound on the targets were validated using Western blot and ELISA. RESULTS: In vivo, SYQ rhizome powder effectively restored mice's body weight, lowered DAI and pathological score, downregulated the expression of inflammatory biomarkers, and restored colon length, as well as the colonic epithelial and mucus barriers. Afterward, 9 compounds were isolated and identified from the powder of the rhizomes of SYQ by HPLC. Nicotiflorin is the primary compound in SYQ with the highest concentration. According to both CCK-8 and NO release tests, Nicotiflorin is also the most efficacious compound. Combined with network pharmacological prediction, molecular docking analysis, CETSA, DARTS, SPR and enzyme activity assay, Nicotiflorin may ultimately suppress inflammation by targeting p65 and inhibiting the NF-κB pathway, thereby attenuating the activation of NLRP3 inflammasome. To verify this conclusion, Western blot and ELISA experiments were conducted. CONCLUSIONS: Our results suggest that the extract from SYQ rhizomes has therapeutic properties for UC. Its active ingredient Nicotiflorin exerted potent anti-UC effects by binding to p65 and inhibiting the activation of NF-κB and NLRP3 inflammasomes.

Total paeony glycoside relieves neuroinflammation to exert antidepressant effect via the interplay between NLRP3 inflammasome, pyroptosis and autophagy.

BACKGROUND: Depression is a common mental illness characterised by abnormal and depressed emotions. Total paeony glycoside (TPG) is a naturally active saponin extracted from the traditional Chinese medicine Radix Paeoniae rubra. However, the antidepressant and neuroinflammatory effects of TPG have not been thoroughly studied. PURPOSE: To study the therapeutic potential of TGP in depression caused by neuronal injury and neuroinflammation and to explore the mechanism of TGP and the relationship between the NLRP3 inflammasome, pyroptosis, and autophagy. STUDY DESIGN: A chronic unpredictable mild stress (CUMS)-induced depression model and a cell model of corticosterone (CORT)-induced hippocampal neuron injury were established to evaluate the therapeutic effects of TPG. METHODS: The composition of TPG was analysed using high-performance liquid chromatography and mass spectrometry. The effects of TPG and fluoxetine on depression-like behaviour, neuronal injury, neuroinflammation, pyroptosis, and mitochondrial autophagy in the mice models were evaluated. RESULTS: TGP alleviated depression-like behaviours in mice and inhibited hippocampal neuronal apoptosis. The secretion of inflammatory cytokines was significantly reduced in CORT-induced hippocampal neuron cells and in the serum of a mouse model of CUMS-induced depression. In addition, TGP treatment reduced the levels of NLRP3 family pyrin structural domains, including NLRP3, pro-caspase-1, caspase-1, and IL-1ß, and the pyroptosis related proteins such as GSDMD-N. Importantly, TPG attenuated mitochondrial dysfunction, promoted the clearance of damaged mitochondria, and the activation of mitochondrial autophagy, which reduced ROS accumulation and NLRP3 inflammasome activation. An in-depth study observed that the regulatory effect of TPG on autophagy was attenuated by the autophagy inhibitor 3-methyladenine (3-MA) in vitro and in vivo. However, administration of the caspase-1 inhibitor Belnacasan (VX-765) successfully inhibited pyroptosis and showed a synergistic therapeutic effect with TPG. CONCLUSION: These results indicate that TPG can repair neuronal damage by activating autophagy, restoring mitochondrial function, and reducing inflammation-mediated pyroptosis, thereby playing an important role in the alleviation of neuroinflammation and depression. This study suggests new potential drugs and treatment strategies for neuroinflammation-related diseases and depression.

Xijiao Dihuang decoction combined with Yinqiao powder promotes autophagy-dependent ROS decrease to inhibit ROS/NLRP3/pyroptosis regulation axis in influenza virus infection.

BACKGROUND: Influenza viral pneumonia is a common complication after influenza virus infection. Xijiao Dihuang Decoction combined with Yinqiao Powder (XDY) is effective on improving influenza viral pneumonia. PURPOSE: This study further explores the anti-inflammatory mechanism of XDY in the treatment of influenza viral pneumonia. STUDY DESIGN: The effects of XDY on inflammation, autophagy, NACHT-LRR-PYD-containing protein 3 (NLRP3) inflammasome and pyroptosis were assessed in the mice with influenza viral pneumonia. In addition, the mouse macrophage cell line (J774A.1) infected with influenza virus was adopted to decode the in vitro effects of XDY on autophagy, reactive oxygen species (ROS), NLRP3 inflammasome and pyroptosis. We analyzed the XDY-induced autophagy, especially the mitophagy-related ROS clearance, and the subsequent inhibition of ROS/NLRP3 inflammasome/pyroptosis signaling in the infected macrophages by different assays based on quantitative polymerase chain reaction, western blot, flow cytometry, immunofluorescence and enzyme-linked immunosorbent assay. RESULTS: In vivo, XDY could effectively improve the lung inflammatory response in the mice with influenza virus pneumonia, due to an intact autophagy flux-promoting effect and the inhibiting roles on NLRP3 inflammasome and pyroptosis. Notably, in vitro, compared with the infected macrophages treated by the NLRP3 inflammasome agonist (Monosodium urate) or the mitochondrial-targeted antioxidant agent, the XDY-dependent treating could inhibit pyroptosis by negatively regulating the signaling axis of ROS/NLRP3 inflammasome/pyroptosis in the influenza virus-infected macrophages. More interestingly, XDY could promote an intact autophagy flux, inducing mitophagy eliminating the damaged mitochondria to reduce the intracellular ROS accumulation, and thus decrease the oxidative stress in the infected macrophages. Especially, the inhibitor of autophagy inition, 3-Methyladenine, could reverse the inhibitory effect of XDY on ROS-NLRP3 inflammasome-mediated pyroptosis, indicating an XDY-promoted mitophagy-dependent ROS scavenging. CONCLUSION: XDY can promote an intact autophagy flux to eliminate damaged mitochondria, namely mitophagy, which reduces the intracellular ROS accumulation contributing to NLRP3 inflammasome activation, restricting pyroptosis and eventually alleviating the influenza virus-induced inflammatory lesions. The obtained results provide new insights into the mechanism of action of XDY in alleviating influenza virus pneumonia, especially the roles of XDY in anti-oxidation, anti-inflammation and anti-pyroptosis, with potential therapeutic targets for future application in integrative medicine.

Dehydrocostus lactone alleviates irinotecan-induced intestinal mucositis by blocking TLR4/MD2 complex formation.

BACKGROUND: Irinotecan (CPT-11) is used as chemotherapeutic drug for treatment of colorectal cancer. However, without satisfactory treatments, its gastrointestinal toxicities such as diarrhea and intestinal inflammation severely restrained its clinical application. Roots of Aucklandia lappa Decne. are used as traditional Chinese medicine to relieve gastrointestinal dysfunction and dehydrocostus lactone (DHL) is one of its main active components. Nevertheless, the efficacy and mechanism of DHL against intestinal mucositis remains unclear. PURPOSE: The present study aimed to investigate the protective effects of DHL on CPT-11-induced intestinal mucositis and its underlying mechanisms. METHODS: The protective effect of DHL was investigated in CPT-11-induced mice and lipopolysaccharide (LPS)+CPT-11 induced THP-1 macrophages. Body weight, diarrhea score, survival rate, colon length, and histopathological changes in mice colon and jejunum were analyzed to evaluate the protective effect of DHL in vivo. And DHL on reducing inflammatory response and regulating TLR4/NF-κB/NLRP3 pathway in vivo and in vitro were explored. Moreover, DHL on the interaction between TLR4 and MD2 was investigated. And silencing TLR4 targeted by siRNA was performed to validate the mechanisms of DHL on regulating the inflammation. RESULTS: DHL prevented CPT-11-induced intestinal damage, represented by reducing weight loss, diarrhea score, mortality rate and the shortening of the colon. Histological analysis confirmed that DHL prevented intestinal epithelial injury and improved the intestinal barrier function in CPT-11 induced mice. Besides, DHL significantly downregulated the level of inflammatory cytokines by inhibiting TLR4/NF-κB/NLRP3 signaling pathway in CPT-11-induced mice and LPS+CPT-11-induced THP-1 macrophages. In addition, DHL blocked TLR4/MD2 complex formation. Molecular docking combined with SIP and DARTS assay showed that DHL could bind to TLR4/MD2 and occludes the hydrophobic pocket of MD2. Furthermore, Silencing TLR4 abrogated the effect of DHL on LPS+CPT-11 induced inflammatory response in THP-1 macrophages. Additionally, DHL ameliorate the CPT-11-induced intestinal mucositis without affecting the anti-tumor efficacy of CPT-11 in the tumor xenograft mice. CONCLUSION: This study found that DHL exhibited the anti-inflammatory effects in CPT-11-induced intestinal mucositis by inhibiting the formation of TLR4/MD2 complex and then regulation of NF-κB/NLRP3 signaling pathway. DHL is potentially served as a novel strategy of combined medication with CPT-11.

Sodium butyrate alleviates experimental autoimmune prostatitis by inhibiting oxidative stress and NLRP3 inflammasome activation via the Nrf2/HO-1 pathway.

BACKGROUND: Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) leads to severe discomfort in males and loss of sperm quality. Current therapeutic options have failed to achieve satisfactory results. Sodium butyrate (NaB) plays a beneficial role in reducing inflammation, increasing antioxidant capacities, and improving organ dysfunction; additionally NaB has good safety prospects and great potential for clinical application. The purpose of the current research was to study the effect of NaB on CP/CPPS and the underlying mechanisms using a mouse model of experimental autoimmune prostatitis (EAP) mice. METHODS: The EAP mouse model was successfully established by subcutaneously injecting a mixture of prostate antigen and complete Freund's adjuvant. Then, EAP mice received daily intraperitoneal injections of NaB (100, 200, or 400 mg/kg/day) for 16 days, from Days 26 to 42. We then explored anti-inflammatory potential mechanisms of NaB by studying the effects of Nrf2 inhibitor ML385 and HO-1 inhibitor zinc protoporphyrin on prostate inflammation and pelvic pain using this model. On Day 42, hematoxylin-eosin staining and dihydroethidium staining were used to evaluate the histological changes and oxidative stress levels of prostate tissues. Chronic pelvic pain was assessed by applying Von Frey filaments to the lower abdomen. The levels of inflammation-related cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor were detected by enzyme-linked immunosorbent assay. The regulation of Nrf2/HO-1 signaling pathway and the expression of NLRP3 inflammasome-related protein in EAP mice were detected by western blot analysis assay. RESULTS: Compared with the EAP group, chronic pain development, histological manifestations, and cytokine levels showed that NaB reduced the severity of EAP. NaB treatment could inhibit NLRP3 inflammasome activation. Mechanism studies showed that NaB intervention could alleviate oxidative stress in EAP mice through Nrf2/HO-1 signal pathway. Nrf2/HO-1 pathway inhibitors can inhibit NaB -mediated oxidative stress. The inhibitory effect of NaB on the activation of NLRP3 inflammasome and anti-inflammatory effect can also be blocked by Nrf2/HO-1 pathway. CONCLUSIONS: NaB treatment can alleviates prostatic inflammation and pelvic pain associated with EAP by inhibiting oxidative stress and NLRP3 inflammasome activation via the Nrf2/HO-1 pathway. NaB has the potential as an effective agent in the treatment of EAP.

Effect of Pulsatilla decoction on vulvovaginal candidiasis in mice. Evidences for its mechanisms of action.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common infection that affects the female reproductive tract. Pulsatilla decoction (PD), a traditional Chinese herbal medicine, is a classic and effective prescription for VVC. However, its mechanism of action remains unclear. PURPOSE: This study aimed to evaluate the efficacy and potential mechanism of action of the n-butanol extract of Pulsatilla decoction (BEPD) in VVC treatment. METHODS: High performance liquid chromatography (HPLC) was used to detect the main active ingredients in BEPD. A VVC-mouse model was constructed using an estrogen-dependent method to evaluate the efficacy of BEPD in VVC treatment. Fungal burden and morphology in the vaginal cavity were comprehensively assessed. Candida albicans-induced inflammation was examined in vivo and in vitro. The effects of BEPD on the Protein kinase Cδ (PKCδ) /NLR family CARD domain-containing protein 4 (NLRC4)/Interleukin-1 receptor antagonist (IL-1Ra) axis were analyzed using by immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and reverse transcription-quantitative polymerase chain reaction (qRT-PCR). RESULTS: BEPD inhibited fungal growth in the vagina of VVC mice, preserved the integrity of the vaginal mucosa, and suppressed inflammatory responses. Most importantly, BEPD activated the "silent" PKCδ/NLRC4/IL-1Ra axis and negatively regulated NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, thereby exerting a therapeutic efficacy on VVC. CONCLUSIONS: BEPD effects on mice with VVC were dose-dependent. BEPD protects against VVC by inhibiting inflammatory response and NLRP3 inflammasome via the activation of the PKCδ/NLRC4/IL-1Ra axis. This study revealed the pharmacological mechanism of BEPD in VVC treatment and provided further evidence for the application of BEPD in VVC treatment.

Ginsenosides from Panax ginseng as potential therapeutic candidates for the treatment of inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by a chronic inflammation of the intestine, which significantly affects patients' quality of life. As a perennial plant with the homology of medicine and food, Panax ginseng is known for its substantial anti-inflammatory effects in various inflammatory disorders. Ginsenosides, the main bioactive compounds of P. ginseng, are recognized for their efficacy in ameliorating inflammation. PURPOSE: Over the past decade, approximately 150 studies have investigated the effects of P. ginseng and ginsenosides on IBD treatment and new issues have arisen. However, there has yet to be a comprehensive review assessing the potential roles of ginsenosides in IBD therapy. METHOD: This manuscript strictly adheres to the PRISMA guidelines, thereby guaranteeing systematic synthesis of data. The research articles referenced were sourced from major scientific databases, including Google Scholar, PubMed, and Web of Science. The search strategy employed keywords such as "ginsenoside", "IBD", "colitis", "UC", "inflammation", "gut microbiota", and "intestinal barrier". For image creation, Figdraw 2.0 was methodically employed. RESULTS: Treatment with various ginsenosides markedly alleviated clinical IBD symptoms. These compounds have been observed to restore intestinal epithelia, modulate cellular immunity, regulate gut microbiota, and suppress inflammatory signaling pathways. CONCLUSION: An increasing body of research supports the potential of ginsenosides in treating IBD. Ginsenosides have emerged as promising therapeutic agents for IBD, attributed to their remarkable efficacy, safety, and absence of side effects. Nevertheless, their limited bioavailability presents a substantial challenge. Thus, efforts to enhance the bioavailability of ginsenosides represent a crucial and promising direction for future IBD research.

Yanggan Jiangmei Formula alleviates hepatic inflammation and lipid accumulation in non-alcoholic steatohepatitis by inhibiting the NF-κB/NLRP3 signaling pathway.

The role of NF-κB and the NLRP3 inflammasome in the chronic inflammatory microenvironment of non-alcoholic steatohepatitis (NASH) has been posited as crucial. The Yanggan Jiangmei Formula (YGJMF) has shown promise in ameliorating hepatic steatosis in NASH patients, yet its pharmacological mechanisms remain largely unexplored. This study was conducted to investigate the efficacy of YGJMF in NASH and to elucidate its pharmacological underpinnings. To simulate NASH both in vivo and in vitro, high-fat-diet (HFD) rats and HepG2 cells stimulated with free fatty acids (FFAs) were utilized. The severity of liver injury and lipid deposition was assessed using serum indicators, histopathological staining, micro-magnetic resonance imaging (MRI), and the liver-to-muscle signal intensity ratio (SIRL/M). Furthermore, a combination of enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunofluorescence, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting analyses was employed to investigate the NF-κB/NLRP3 signaling pathway and associated cytokine levels. The results from liver pathology, MRI assessments, and biochemical tests in rat models demonstrated YGJMF's significant effectiveness in reducing liver damage and lipid accumulation. Additionally, YGJMF markedly reduced hepatocyte inflammation by downregulating inflammatory cytokines in both liver tissue and serum. Furthermore, YGJMF was found to disrupt NF-κB activation, consequently inhibiting the assembly of the NLRP3 inflammasome in both the in vitro and in vivo models. The preliminary findings of this study suggest that YGJMF may alleviate hepatic steatosis and inhibit the NF-κB/NLRP3 signaling pathway, thereby exerting anti-inflammatory effects in NASH.