Attenuating Renal Interstitial Fibrosis by Shenqi Pill via Reducing Inflammation Response Regulated by NF-κB Pathway In Vitro and In Vivo.

INTRODUCTION: One of the most significant clinical features of chronic  kidney disease is renal interstitial fibrosis (RIF). This study aimed  to investigate the role and mechanism of Shenqi Pill (SQP) on RIF. METHODS: RIF model was established by conducting unilateral  ureteral obstruction (UUO) surgery on rat or stimulating human  kidney-2 (HK-2) cell with transforming growth factor ß1 (TGFß1).  After modeling, the rats in the SQP low dose group (SQP-L), SQP  middle dose group (SQP-M) and SQP high dose group (SQP-H)  were treated with SQP at 1.5, 3 or 6 g/kg/d, and the cells in the  TGFß1+SQP-L/M/H were treated with 2.5%, 5%, 10% SQP-containing  serum. In in vivo assays, serum creatinine (SCr) and blood urea  nitrogen (BUN) content were measured, kidney histopathology  was evaluated., and α-smooth muscle actin (α-SMA) expression  was detected by immunohistochemistry. Interleukin-1ß (IL-1ß),  interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) content,  inhibitor of kappa B alpha (IKBα) and P65 phosphorylation were  assessed. Meanwhile, cell viability, inflammatory cytokines content,  α-SMA expression, IKBα and P65 phosphorylation were detected  in vitro experiment.  Results. SQP exhibited reno-protective effect by decreasing SCr  and BUN content, improving renal interstitial damage, blunting  fibronectin (FN) and α-SMA expression in RIF rats. Similarly, after  the treatment with SQP-containing serum, viability and α-SMA  expression were remarkably decreased in TGFß1-stimulated HK-2  cell. Furthermore, SQP markedly down-regulated IL-1ß, IL-6, and  TNF-α content, IKBα and RelA (P65) phosphorylation both in vivo and in vitro.  Conclusion. SQP has a reno-protective effect against RIF in vivo and in vitro, and the effect is partly linked to nuclear factor-kappa  B (NF-κB) pathway related inflammatory response, which indicates  that SQP may be a candidate drug for RIF. DOI: 10.52547/ijkd.7546.

[Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway].

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1ß, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(ß-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1ß, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and ß-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1ß, IL-6, CGRP, and NO in rat serum, increased VEGF and ß-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and ß-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1ß. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing ß-EP levels.

[Mongolian medicine Zhenbao Pill (Eridon Uril) inhibits inflammation and apoptosis in hippocampal neuronal cells of oxyglucose deprived/reoxygenated mice through activation of SIRT1/NF-κB pathway].

Objective To observe the effect of Mongolian medicine Zhenbao Pill (Eridon Uril, EU) on inflammation and apoptosis of HT22 mouse hippocampal neurons cells under oxygen glucose deprivation/reoxygenation (OGD/R), and to explore its potential mechanism. Methods Three-gas incubator and sugar-free anaerobic medium were used to construct a model of OGD/R-injured HT22 cells. OGD/R model cells were treated with EU (10, 20 and 40 µg/mL), and the optimal dose of 20 µg/mL was screened. The OGD/R injured cells treated with nicotinamide (NAM), inhibitor of SIRT1, combined with EU were set as EU combined with NAM group, and the OGD /R injured cells treated with dimethyl sulfoxide (DMSO) combined with EU were set as EU combined with DMSO group. CCK-8 assay and ELISA were used to detect cell activity and LDH leakage rate, respectively. Detection of the mRNA expression of tumor necrosis factor α (TNF-α), Interleukin 6 (IL-6), and IL-1ß was detected by real time fluorescence quantitative PCR in HT22 cells. The apoptosis was detected by flow cytometry. The protein expression of B-cell lymphoma 2 (Bcl2), Bcl2 related X protein (BAX), SIRT1, inhibitor of nuclear factor κB α (IκBα), and phosphorylated nuclear factor κB (p-NF-κB) were detected by Western blot analysis. Results Compared to the control group, HT22 cells in the OGD/R group showed significantly lower activity and higher LDH leakage rate, while (20, 40)µg/mL EU treatment significantly increased cell activity and lowered LDH leakage rate, with 20 µg/mL EU being the optimal dose. The mRNA expression of TNF-α, IL-6 and IL-1ß and the apoptosis rate of the cells in the OGD/R group were significantly higher than those in the control group. The protein levels of SIRT1, IκBα, and Bcl2 were significantly lower than that in the control group, while the protein levels of p-NF-κB, BAX were significantly higher than that in the control group. EU significantly inhibited the secretion of TNF-α, IL-6, IL-1ß and apoptosis in HT22 cells induced by OGD/R. Conclusion EU significantly reduces the inflammatory response and apoptosis in OGD/R-induced mouse hippocampal neurons, which is associated with the activation of SIRT1/NF-κB signal pathway.

Chinese medicine Jiangzhuo mixture regulates glucose and lipid metabolism in obese rats through TLR4/IκBα/NF-κB signaling pathway. / 降浊合剂通过调节TLR4/IκBα/NF-κBä¿¡å·é€šè·¯å½±å“è‚¥èƒ–å¤§é¼ ç³–è„‚ä»£è°¢.

OBJECTIVES: To explore the mechanism of Chinese medicine Jiangzhuo mixture regulating glucose and lipid metabolism in obese rats. METHODS: Thirty healthy male SD rats were randomly divided into normal control group, model control group, and Jiangzhuo mixture treatment group, with 10 rats in each group. The rats in the normal control group were fed with normal diet, the obesity model was induced by feeding high-fat diet in the model control group and the Jiangzhuo mixture treatment group, the rats in the treatment group were given with Jiangzhuo mixture 50 g/kg by gavage. After 8 weeks of intervention, the blood glucose (GLU), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were measured in the three groups. Quantitative reverse transcription PCR were used to detect the expression levels of PR domain containing 16 (PRDM16) and uncoupling protein 1 (UCP1) in white and brown adipose tissues of the rats in each group; Western blotting was used to detect the expression of PRDM16 in the white and brown adipose tissue of rats, and Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB) and inhibitor of NF-κB alpha (IκBα) in the white adipose tissue; immunohistochemistry was used to detect the expression of UCP1 protein in white and brown adipose tissues. RESULTS: Compared with the normal control group, the white fat weight (P<0.01), white fat coefficient (P<0.05) and Lee's coefficient (P<0.01) were significantly increased in the model control group; the contents of GLU, TC, TG and LDL-C were all increased, and the content of TG was significantly increased (P<0.05) in the model control group. The mRNA and protein expression levels of PRDM16 and UCP1 in white fat and brown fat were significantly decreased (P<0.05) in the model control group. Compared with the model control group, the white fat weight and white fat coefficient and Lee's coefficient were significantly reduced in the Jiangzhuo mixture treatment group (all P<0.01), the levels of GLU, TC, TG, and LDL-C in the the treatment group were all reduced, and the content of TG was reduced more obviously (P<0.01); expression levels of PRDM16 and UCP1 mRNA and protein were increased in brown and white adipose tissue. Compared with the normal control group, the expression levels of TLR4, phospho-IκBα and NF-κB-p65 proteins in white adipose tissue of the model control group were significantly increased (all P<0.01), while the expression levels of these proteins in the treatment group were significantly lower than those in the model control group (all P<0.05). CONCLUSIONS: Jiangzhuo mixture can alleviate high-fat diet-induced increase in body fat, abnormal expression of biochemical indexes and promote the expression of key proteins including UCP1 and PRDM16 in white and brown adipose tissues by regulating TLR4/IκBα/NF-κB signaling pathway.

[Effects of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination on inflammatory responses in atherosclerotic mice].

The study aims to observe the effects and explore the mechanisms of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination in the treatment of the inflammatory response of mice with atherosclerosis(AS) via the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Male ApoE~(-/-) mice were randomly assigned into a model group, a Buyang Huanwu Decoction group, an Astragali Radix-Angelicae Sinensis Radix combination group, and an atorvastatin group, and male C57BL/6J mice of the same weeks old were used as the control group. Other groups except the control group were given high-fat diets for 12 weeks to establish the AS model, and drugs were administrated by gavage. Aortic intimal hyperplasia thickness, blood lipid level, plasma inflammatory cytokine levels, M1/M2 macrophage markers, and expression levels of proteins in TLR4/MyD88/NF-κB pathway in the vessel wall were measured to evaluate the effects of drugs on AS lesions and inflammatory responses. The results showed that the AS model was successfully established with the ApoE~(-/-) mice fed with high-fat diets. Compared with the control group, the model group showed elevated plasma total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c) levels(P<0.05), thickened intima(P<0.01), and increased plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels(P<0.01). Moreover, the model group showed increased expression of vascular cell adhesion molecule-1(VCAM-1) and inducible nitric oxide synthase(iNOS)(P<0.01), inhibited expression of endothelial nitric oxide synthase(eNOS) and cluster of differentiation 206(CD206)(P<0.01), and up-regulated mRNA and protein levels of TLR4, MyD88, NF-κB inhibitor alpha(IκBα), and NF-κB in the vessel wall(P<0.05). Compared with the model group, Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination lowered the plasma TC and LDL-c levels(P<0.01), alleviated the intimal hyperplasia(P<0.01), and reduced the plasma TNF-α and IL-6 levels(P<0.05). Moreover, the two interventions promoted the expression of eNOS and CD206(P<0.05), inhibited the expression of VCAM-1 and iNOS(P<0.01), and down-regulated the mRNA and protein levels of TLR4, MyD88, IκBα, and NF-κB(P<0.05) in the vessel wall. This study indicated that Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination could delay the progression of AS, inhibit the polarization of vascular wall macrophages toward M1 type, and attenuate vascular inflammatory response by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in the vascular wall. Astragali Radix and Angelicae Sinensis Radix were the main pharmacological substances in Buyang Huanwu Decoction for alleviating the AS vascular inflammatory response.

Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish.

OBJECTIVE: To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. METHODS: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. RESULTS: The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01). CONCLUSION: Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.

[Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway].

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-ß1(TGF-ß1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-ß1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. AIM OF THIS STUDY: The present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. METHODS: The structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. RESULTS: Two new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. CONCLUSION: Two new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

Effects of Modified Glucosamine on the Chondrogenic Potential of Circulating Stem Cells under Experimental Inflammation.

Glucosamine (GlcN) is a glycosaminoglycan (GAGs) constituent in connective tissues. It is naturally produced by our body or consumed from diets. In the last decade, in vitro and in vivo trials have demonstrated that the administration of GlcN or its derivates has a protective effect on cartilage when the balance between catabolic and anabolic processes is disrupted and cells are no longer able to fully compensate for the loss of collagen and proteoglycans. To date, these benefits are still controversial because the mechanism of action of GlcN is not yet well clarified. In this study, we have characterized the biological activities of an amino acid (AA) derivate of GlcN, called DCF001, in the growth and chondrogenic induction of circulating multipotent stem cells (CMCs) after priming with tumor necrosis factor-alpha (TNFα), a pleiotropic cytokine commonly expressed in chronic inflammatory joint diseases. In the present work, stem cells were isolated from the human peripheral blood of healthy donors. After priming with TNFα (10 ng/mL) for 3 h, cultures were treated for 24 h with DCF001 (1 µg/mL) dissolved in a proliferative (PM) or chondrogenic (CM) medium. Cell proliferation was analyzed using a Corning® Cell Counter and trypan blue exclusion technique. To evaluate the potentialities of DCF001 in counteracting the inflammatory response to TNFα, we measured the amount of extracellular ATP (eATP) and the expression of adenosine-generating enzymes CD39/CD73, TNFα receptors, and NF-κB inhibitor IκBα using flow cytometry. Finally, total RNA was extracted to perform a gene expression study of some chondrogenic differentiation markers (COL2A1, RUNX2, and MMP13). Our analysis has shed light on the ability of DCF001 to (a) regulate the expression of CD39, CD73, and TNF receptors; (b) modulate eATP under differentiative induction; (c) enhance the inhibitory activity of IκBα, reducing its phosphorylation after TNFα stimulation; and (d) preserve the chondrogenic potentialities of stem cells. Although preliminary, these results suggest that DCF001 could be a valuable supplement for ameliorating the outcome of cartilage repair interventions, enhancing the efficacy of endogenous stem cells under inflammatory stimuli.

Geniposide ameliorates dextran sulfate sodium-induced ulcerative colitis via KEAP1-Nrf2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried fruit of Gardenia jasminoides Ellis (Zhizi in Chinese) is a traditional medicine used for thousands of years in China, Japan and Korea. Zhizi was recorded in Shennong Herbal, as a folk medicine, it reduces fever and treats gastrointestinal disturbance with antiphlogistic effects. Geniposide, an iridoid glycoside, is an important bioactive compound derived from Zhizi and possesses remarkable antioxidant and anti-inflammatory capacities. The pharmacological efficacy of Zhizi is highly related to the antioxidant and anti-inflammatory effects of geniposide. AIM OF THE STUDY: Ulcerative colitis (UC) is a common chronic gastrointestinal disease as a global public health threat. Redox imbalance is an essential factor in the progression and recurrence of UC. This study aimed to explore the therapeutic effect of geniposide on colitis and uncover the underlying mechanisms of geniposide-mediated antioxidant and anti-inflammatory activities. EXPERIMENTAL DESIGN: The study design involved investigating the novel mechanism by which geniposide ameliorates dextran sulfate sodium (DSS)-induced colitis in vivo and lipopolysaccharide (LPS)-challenged colonic epithelial cells in vitro. MATERIALS AND METHODS: The protective effect of geniposide against colitis was evaluated by histopathologic observation and biochemical analysis of colonic tissues in DSS-induced colitis mice. The antioxidant and anti-inflammatory effects of geniposide were evaluated in both DSS-induced colitis mice and LPS-challenged colonic epithelial cells. Immunoprecipitation, drug affinity responsive target stability (DARTS), and molecular docking were performed to identify the potential therapeutic target of geniposide and the potential binding sites and patterns. RESULTS: Geniposide ameliorated the symptoms of DSS-induced colitis and colonic barrier injury, inhibited pro-inflammatory cytokine expression, and suppressed activation of the NF-κB signaling in colonic tissues of DSS-challenged mice. Geniposide also ameliorated lipid peroxidation and restored redox homeostasis in DSS-treated colonic tissues. In addition, in vitro experiments also showed that geniposide exhibited significant anti-inflammatory and antioxidant activity, as evidenced by suppressed IκB-α and p65 phosphorylation and IκB-α degradation, and enhanced the phosphorylation and transcriptional activity of Nrf2 in LPS-treated Caco2 cells. ML385, a specific Nrf2 inhibitor, abolished the protective effect of geniposide against LPS-induced inflammation. Mechanistically, geniposide could bind to KEAP1, thereby disrupting the interaction between KEAP1 and Nrf2, preventing Nrf2 from degradation and activating the Nrf2/ARE signaling pathway, ultimately suppressing the onset of inflammation caused by redox imbalance. CONCLUSIONS: Geniposide ameliorates colitis by activation of Nrf2/ARE signaling, while preventing colonic redox imbalance and inflammatory damage, indicating that geniposide can be considered as a promising lead compound for the treatment of colitis.

Probiotic-fermented Portulaca oleracea L. alleviated DNFB-induced atopic dermatitis by inhibiting the NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Probiotic fermentation is a mild and safe biological method to boost the performance of herbs. Portulaca oleracea L. (PO), with folklore records of purgative, anti-dermatological and anti-epidemic effects, has been demonstrated to possess anti-inflammatory, immunomodulatory, and antioxidant properties. However, the potential of PO for the treatment of atopic dermatitis (AD) has not been sufficiently explored. AIM OF STUDY: This study aimed to evaluate the therapeutic benefits of PO and fermented Portulaca oleracea L. (FPO) and explore their intrinsic mechanisms. METHODS: By utilizing 2,4-dinitrofluorobenzene-induced AD mice as a model, the histopathology of the lesions was observed using H&E and toluidine blue staining methods; the levels of immunoglobulin E (Ig E), histamine (HIS), and thymic stromal lymphopoietin (TSLP) in serum were measured using ELISA, whereas, the expression of inflammatory cytokines in skin lesion was measured using ELISA and immunohistochemistry experiments. The expression of tumor necrosis factor-α (TNF-α), IKKα, NF-κB mRNA was measured using qPCR; and the expression of TNF-α、p-IKKα, p-IκBα, p-NF-κB was measured using western blotting. RESULTS: Both 20 mg/mL PO and FPO alleviated mast cell infiltration and lesion pathology, reduced serum levels of Ig E, HIS and TSLP, down-regulated the expression of AD-related inflammatory cytokines, such as, TNF-α, interferon-γ, and interleukin-4, and increased filaggrin expression. Furthermore, they inhibited the expression of TNF-α, IKKα, and NF-κB genes and TNF-α, p-IKKα, p-NF-κB and p-IκBα proteins associated with the NF-κB signaling pathway. CONCLUSIONS: PO and FPO has a positive therapeutic potential on AD, indicating that it may be employed as alternative therapies for AD.

Isochlorogenic acid A alleviates dextran sulfate sodium-induced ulcerative colitis in mice through STAT3/NF-кB pathway.

Isochlorogenic acid A (ICGA-A) is a dicaffeoylquinic acid widely found in various medicinal plants or vegetables, such as Lonicerae japonicae Flos and chicory, and multiple properties of ICGA-A have been reported. However, the therapeutic effect of ICGA-A on colitis is not clear, and thus were investigated in our present study, as well as the underlying mechanisms. Here we found that ICGA-A alleviated clinical symptoms of dextran sodium sulfate (DSS) induced colitis model mice, including disease activity index (DAI) and histological damage. In addition, DSS-induced inflammation was significantly attenuated in mice given ICGA-A supplementation. ICGA-A reduced the fraction of neutrophils in peripheral blood and the infiltration of neutrophils and macrophages in colon tissue, and reduced pro-inflammatory cytokine production and tight junctions in mouse models. Furthermore, ICGA-A down-regulated expression of STAT3 and up-regulated the protein level of IκBα. Our in vitro studies confirmed that ICGA-A inhibited the mRNA expression of pro-inflammatory cytokines. ICGA-A blocked the phosphorylation of STAT3, p65, and IκBα, suppressed the expression STAT3 and p65. In addition, the present study also demonstrated that ICGA-A had no obvious toxicity on normal cells and organs. Taken together, we conclude that ICGA-A mitigates experimental ulcerative colitis (UC) at least in part by inhibiting the STAT3/NF-кB signaling pathways. Hence, ICGA-A may be a promising and effective drug for treating UC.

[Mechanism of Buyang Huanwu Decoction glycosides against atherosclerotic inflammation through NF-κB signaling pathway].

This study aims to explore the effect of Buyang Huanwu Decoction glycosides on the inflammatory response of apolipoprotein E~(-/-)(ApoE~(-/-)) mice and RAW264.7 cells through nuclear factor kappa-B(NF-κB) signaling pathway. In the in vivo experiment, ApoE~(-/-) mice were fed with high-fat diets for 12 weeks to induce the animal model of atherosclerosis, and 75 µg·mL~(-1) oxidized low-density lipoprotein(Ox-LDL) incubated RAW264.7 cells for 24 h to establish the atherosclerosis cell model. Automatic biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay(ELISA), Western blot, and droplet digital polymerase chain reaction(PCR) were used to determine the blood lipid levels, aortic intimal thickness, inflammatory factor content, NF-κB pathway-related proteins, and mRNA expression levels, and evaluate arterial atherosclerotic lesions and anti-atherosclerotic mechanisms of the drug. The model of atherosclerosis was successfully established in ApoE~(-/-) mice after 12 weeks of feeding with high-fat diets. In the model group, the plasma levels of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-C) were increased(P<0.01), the intima of the blood vessels was thickened, the levels of inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were increased, and the protein and mRNA expressions of NF-κB and inhibitor of NF-κB(IκBα) were significantly increased as compared with the control group. Compared with the model group, the high-dose Buyang Huanwu Decoction glycoside group decreased the plasma levels of TC, TG, and LDL-C, reduced the plaque area and thickness and the content of inflammatory factor TNF-α, and inhibited the protein and mRNA expressions of NF-κB and IκBα, with the effect same as Buyang Huanwu Decoction. In the in vivo experiment, 75 µg·mL~(-1) Ox-LDL stimulated RAW264.7 cells for 24 h to successfully establish a foam cell model. As compared with the control group, the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα in the model group increased. Compared with the model group, the middle-dose and high-dose Buyang Huanwu Decoction glycoside groups decreased the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα. The above results show that the glycosides are the main effective substances of Buyang Huanwu Decoction against atherosclerosis, which inhibit the NF-κB pathway and reduce the inflammatory response, thus playing the role against atherosclerotic inflammation same as Buyang Huanwu Decoction.

Toll-like receptor 4 is a key regulator of asthma exacerbation caused by aluminum oxide nanoparticles via regulation of NF-κB phosphorylation.

Aluminum oxide nanoparticles (Al2O3 NPs) have recently been reported to cause an inflammatory response in the lungs, and studies are being conducted on their adverse effects, especially in patients with underlying lung diseases such as asthma. However, the underlying mechanism of asthma aggravation caused by Al2O3 NPs remains unclear. This study investigated whether Al2O3 NPs exacerbate ovalbumin (OVA)-induced asthma and focused on the correlation between toll-like receptor 4 (TLR4) signaling and Al2O3 NP-induced asthma exacerbation. Al2O3 NP exposure in asthmatic mice resulted in increased inflammatory cell counts in the lungs, airway hyperresponsiveness, and increased levels of inflammatory cytokines compared with only OVA-induced mice, and excessive secretion of mucus was observed in the airways. Moreover, Al2O3 NP exposure in OVA-induced mice increased the expression levels of TLR4, phospho-nuclear transcription factor-kappa B (p-NFκB), myeloid differentiation factor 88 (MyD88), and phospho-NF kappa B inhibitor alpha (p-IκBα). Furthermore, in the lungs of TLR4 knockout mice exposed to Al2O3 NPs and in a human airway epithelial cell line with down regulated TLR4, the expression levels of MyD88, p-NFκB, and p-IκBα were decreased, and asthma-related allergic responses were reduced. Therefore, we demonstrated that TLR4 is important for aggravation of asthma induced by Al2O3 NPs, and this study provides useful information regarding as yet undiscovered novel target signaling.

Cornuside, by regulating the AGEs-RAGE-IκBα-ERK1/2 signaling pathway, ameliorates cognitive impairment associated with brain aging.

Anti-Alzheimer's disease (AD) drugs can only change the symptoms of cognitive impairment in a short time but cannot prevent or completely cure AD. Thus, a more effective drug is urgently needed. Cornuside is extracted from Corni Fructus, a traditional Chinese medicine that plays an important role in treating dementia and other age-related diseases. Thus, the study aimed to explore the effects and mechanisms of Cornuside on the D-galactose (D-Gal) induced aging mice accompanied by cognitive decline. Initially, we found that Cornuside improved the learning and memory abilities of D-Gal-treated mice in behavioral experiments. Pharmacological experiments indicated that Cornuside acted on anti-oxidant and anti-inflammatory effects. Cornuside also reversed acetylcholin esterase (AChE) activity. Meanwhile, pathology tests showed that Cornuside had a protective effect on neuron damage. Cornuside increased the expression of brain-derived neurotrophic factor (BDNF), and down-regulated the expression of receptor for advanced glycosylation end products (RAGE), ionized calcium binding adapter molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP) respectively. Further studies claimed that Cornuside had important effects on the expression of IκBα and extracellular signal-regulated kinases 1/2 (ERK1/2). These effects might be achieved through regulating the AGEs-RAGE-IκBα-ERK1/2 signaling pathway, among which, ERK1/2 might be the key protein. The study provides direct preclinical evidence for the research of Cornuside, which may become an excellent candidate drug for the treatment of aging-related AD.

The dichloromethane extract of Callicarpa longissima rich in diterpenoid phenols exerts anti-inflammatory effect via inhibiting the TLR4/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Callicarpa longissima is a typical Yao ethnomedicine that has been used to treat arthritis in China. Our previous study found that the dichloromethane extract (DCME) of C. longissima showed anti-inflammatory activity in vitro. However, the anti-inflammatory mechanism and detailed chemical composition of DCME remain unclear, which lead to the original interest of this study. AIM OF THE STUDY: The study aimed to evaluate the anti-inflammatory properties of the DCME from C. longissima and further explore the accurate chemical components responsible for this active extract. MATERIALS AND METHODS: The anti-inflammatory activity of DCME in vivo was tested with carrageenan-induced mice paw edema model. Its anti-inflammatory mechanism was explored with LPS-stimulated RAW264.7 macrophages model. The compounds in DCME were isolated by repeated column chromatography and their structures were identified on the basis of nuclear magnetic resonance spectroscopy. The anti-inflammatory activities of the isolates in vitro were also tested by suppressing releases of inflammatory mediators (NO, IL-6 and TNF-α) in RAW264.7 macrophages model. In addition, the molecular docking analysis, which evaluated the potential interaction between the compounds and Toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB), was performed. RESULTS: DCME effectively alleviated the mice paw edema induced by carrageenan. In LPS-stimulated RAW264.7 cells, DCME significantly decreased the production of interleukin (IL)-6 and tumor necrosis factor α (TNF-α) via inhibiting their mRNA transcription, down-regulated the expression of TLR4 and myeloid differentiation factor 88, inhibited the phosphorylation of alpha inhibitor of NF-κB (IκBα), NF-κB p65, and degradation of IκBα. Twelve diterpenoid phenols were identified from DCME, and they not only showed different inhibitory effects on the production of NO, IL-6 and TNF-α in LPS-stimulated RAW264.7 cells, but also could bind to TLR4 and NF-κB as analyzed by molecular docking. CONCLUSIONS: Taken together, DCME from C. longissima could inhibit inflammatory response both in vitro and in vivo, which is mainly attributed to the synergistic effect of abundant diterpenoid phenols through inhibiting the TLR4/NF-κB signaling pathway, and might be a promising agent for the treatment of inflammatory diseases.

ALP Inhibitors Inhibit Inflammatory Responses and Osteoblast Differentiation in hVIC via AKT-ERK Pathways.

Objective: The aim of this study was to explore the calcification process of aortic valve interstitial cells and its potential association with osteogenic differentiation and alkaline phosphatase (ALP) activity. Methods: The study patients were divided into 3 groups: the control group, the osteogenic induction medium (OM) group and the OM+ALP inhibitor group. Cell calcification was measured by alizarin red S staining and alizarin red S dye released by extracellular matrix (ECM) was quantified by spectrophotometry. Immunohistochemical staining was performed on valve tissues of patients harboring calcified and non-calcified aortic valve disease. Expression of bone morphogenetic protein (BMP), runt related transcription factor 2 (RUNX2), osteocalcin and osteopontin (OPN), was evaluated using immunohistochemistry¸and expression of osteogenic specific markers (BMP, RUNX2 and OPN) was detected using Wesern blot analysis. RNA sequencing was analyzed to further study the exact mechanism of ALP inhibitors in terms of inhibiting the osteogenic differentiation of valvular interstitial cells (VIC). The mRNA levels of tumor necrosis factor alpha (TNF-α), Toll-like receptor 4 (TLR4) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3), were detected using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In addition, Western blot analysis was performed to evaluate the expression of phosphorylated extracellular regulated protein kinases (ERK), nuclear factor κ B inhibitor α (IκBα) and protein kinase B (AKT) in protein. Results: Alizarin red staining was positive in the OM and OM+ALP inhibitor groups, and calcified nodules were formed in VIC, which showed a significant difference compared with the control group (P < .05). The semi-quantitative level of calcification in the OM group was higher than in the control group (P < .05), and the semi-quantitative level of calcification in the OM+ALP inhibitor group was lower than in the OM group (P < .05). ALP staining intensity, ALP activity and messenger RNA (mRNA) levels of BMP, RUNX2, osteocalcin, OPN, ERK, IκBα, AKT, TNF-α, Toll-like receptor 4 (TLR4) and NLRP3 inflammasome (NLRP3) in the OM group were higher than in the control group (P < .05). ALP staining intensity, ALP activity and mRNA expressions of BMP, RUNX2, osteocalcin, OPN, phosphorylated ERK, IκBα, AKT, TNF-α and NLRP3 in the OM+ALP inhibitor group were lower than in the OM group (P < .05). Compared with the control group, 723 genes were upregulated and 248 genes were downregulated in the OM group. Compared with the OM group, 352 genes were upregulated and 586 genes were downregulated in the OM+ALP inhibitor group. Conclusion: We suggest that ALP inhibitors have potential in terms of inhibiting the inflammatory response and osteoblast differentiation of human VIC (hVIC) via the TLR4, AKT, ERK and NLRP3 pathways.

23-O-acetylshengmanol-3-O-α-L-arabinoside alleviates lipopolysaccharide-induced acute lung injury through inhibiting IκB/NF-κB and MAPK/AP-1 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Cimicifuga foetida L. is a well-established traditional Chinese medicine with heat-clearing and detoxifying effects and has good therapeutic effect on oral mucosal ulcer and pharyngitis. The rhizome of this herb is rich in triterpenoid glycosides, including 23-O-acetylshengmanol-3-o-α-L-arabinoside (DA). AIM OF THE STUDY: Whether and how DA attenuates acute lung injury (ALI) are unclear. Accordingly, we focused on its anti-inflammatory effects and underlying molecular mechanisms in lipopolysaccharide (LPS)-stimulated ALI mice and RAW264.7 cells. MATERIALS AND METHODS: The model of ALI mice was established by exposed intratracheal instillation of LPS. Lung pathological changes were evaluated by hematoxylin and eosin staining. Pulmonary function was assessed by whole-body plethysmography. Total protein content in bronchoalveolar lavage fluid (BALF) was detected by bicinchoninic acid method. Wet/dry lung ratio was used to evaluate the degree of pulmonary edema in mice. The levels of pro-inflammatory mediators were measured using enzyme-linked immunosorbent assay. The relative expression of pro-inflammatory gene mRNA was examined by RT-qPCR. The expression of inflammatory-related proteins was detected by Western blot. RAW264.7 cells were used to test the anti-inflammatory effects of DA in vitro. Cytotoxicity was assessed using a MTT assay. Nitric oxide production was measured by Griess assay. The production and expression of inflammatory mediators and the protein levels of inflammatory signaling molecules in the NF-κB and MAPK pathways were measured. Furthermore, immunofluorescence staining was used to analyze the expression of p-IκBα, p-ERK, and p-p38 in lung macrophages and the nuclear translocation of NF-κB p65 and AP-1 in cells. RESULTS: DA evidently alleviated histopathological changes and ameliorated pulmonary edema. Moreover, DA could reduce excessive inflammatory reaction in lung tissue as manifested by the reduction of proinflammatory mediators (IL-1ß, IL-6, TNF-α, MCP-1, iNOS, and COX-2) in BALF, serum, and lung tissues. Further, DA inhibited the activation of the NLRP3/caspase-1 pathway in the lung. DA reduced the production and expression of the proinflammatory mediators above in RAW264.7 cells. Mechanistically, DA remarkably blocked the nuclear translocation of NF-κB p65, suppressed IκBα phosphorylation, and markedly reduced the nuclear translocation of AP-1 and the phosphorylation of ERK and p38. CONCLUSIONS: The findings demonstrated that DA exerts anti-inflammatory effects in LPS-stimulated ALI mice and macrophages by downregulating the NLRP3/caspase-1 signaling pathway in lung tissue and the IκB/NF-κB and MAPKs/AP-1 pathways in macrophages, suggesting that DA may be promising in ALI treatment.

Effect of Berberine on Activation of TLR4-NFκB Signaling Pathway and NLRP3 Inflammasome in Patients with Gout.

OBJECTIVE: To determine the effects of berberine (BBR) on the activation of toll-like receptor 4 (TLR4), nuclear factor (NF)κB (NF-κB) signaling and NLRP3 inflammasome in patients with gout. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from 24 acute (AP) and 41 non-acute (NAP) phases of primary gout patients, respectively, as well as 30 healthy controls (HC). TLR4, NF-κB (p65), NLRP3, apoptosis-associated specklike protein containing a CARD (PYCARD), cysteinyl aspartate specific proteinase-1 (CASP1), and interleukin-1ß (IL-1ß) mRNA expression levels in PBMCs were measured by quantitative reverse transcriptase polymerase chain reaction. The protein levels of TLR4, myeloid differentiation factor 88 (MyD88), NF-κB (p50/65), inhibitor of kappa B kinase α/ß (IKKα/ß), NF-κB inhibitor α (IKBα), phospho-IKKα/ß (p-IKKα/ß), NLRP3, PYCARD, and CASP1 were monitored by Western blotting. Serum IL-1ß protein level was measured using enzyme-linked immunosorbent assay (ELISA). In addition, PBMCs from HC and macrophages derived from a spontaneously immortalized monocyte-like cell line (THP-1) were stimulated using monosodium urate (MSU, 100 µg/mL), 0.1% dimethyl sulfoxide, 25 µmol/L BBR, and 10, 25, and 50 µmol/L BBR+100 µg/mL MSU for different time periods. The protein levels of IL-1ß and IL-18 in cell culture supernatants was measured by ELISA, and the protein expressions of TLR4, MyD88, NF-κB (p50/p65), IKKα/ß, I κBß, p-IKKα/ß, NLRP3, PYCARD, and CASP1 in macrophages were analyzed by Western blotting. RESULTS: (1) TLR4, NF-κB (p65), PYCARD, CASP1, and IL-1ß mRNA levels in PBMCs were significantly higher in the AP group than in the HC group (P<0.05). The NLRP3 mRNA expression levels in PBMCs were found to be significantly lower in the AP and NAP groups than in the HC group (P<0.05, P<0.01). (2) The protein levels of TLR4, IKKß, MyD88, NF-κB, p-IKKα/ß, PYCARD, and CASP1 in PBMCs were significantly higher, and those of IκBα, IKKα, and NLRP3 were found to be significantly lower in the AP group than in the HC group (P<0.05 or P<0.01). (3) The serum IL-1ß protein levels were significantly higher in the AP and NAP groups than in the HC group (P<0.01). (4) The IL-1ß protein level was significantly lower in the culture supernatants of the PBMCs stimulated with MSU for 3 and 6 h in the 25 and 50 µmoL/L BBR groups compared with that in the MSU group (P<0.01). (5) The protein levels of IL-1ß and IL-18 were also significantly lower in the culture supernatants of macrophages stimulated with MSU for 3 and 6 h in BBR groups compared with those in the MSU group (P<0.01). (6) The protein levels of TLR4, MyD88, NF-κB (p50, p65, p105), IKKα/ß, p-IκBα, p-IKKα/ß, PYCARD, and CASP1 were significantly differed between the macrophages stimulated with MSU for 0.5 and 6 h in BBR groups compared with those in the MSU group (P<0.05 or P<0.01). CONCLUSIONS: Activation of TLR4-NFκB signaling and NLRP3 inflammasome by MSU crystals drives the progression of gout inflammation. BBR ameliorates gouty inflammation, which is mechanistically associated with its regulation of TLR4-NF-κB signaling and NLRP3 inflammasome expression.

1,25-Dihydroxyvitamin D3 Negatively Regulates the Inflammatory Response to Porcine Epidemic Diarrhea Virus Infection by Inhibiting NF-κB and JAK/STAT Signaling Pathway in IPEC-J2 Porcine Epithelial Cells.

Porcine epidemic diarrhea virus (PEDV) infection causes watery diarrhea and vomiting in piglets. The pathogenesis of PEDV infection is related to intestinal inflammation. It is known that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has potent anti-inflammatory activity, but it is unknown whether 1,25(OH)2D3 can inhibit the PEDV-induced inflammatory response and the underlying mechanism. We used transcriptome analysis, gene and protein expression, RNA interference and overexpression, and other techniques to study the anti-inflammatory effects of 1,25(OH)2D3 on PEDV infection in IPEC-J2 cells. The results showed that interleukin 19 (IL-19) and C-C motif chemokine ligand 20 (CCL20) gene expression were enhanced with the increase in PEDV infection time in IPEC-J2 cells. Interestingly, 1,25(OH)2D3 supplementation obviously inhibited IL-19 and CCL20 expression induced by PEDV. Meanwhile, we also found that 1,25(OH)2D3 reduced p-NF-κB, p-STAT1, and p-STAT3 protein levels induced by PEDV at 24 h post-infection. IκBα and SOCS3, NF-κB, and STAT inhibitor respectively, were increased by 1,25(OH)2D3 supplementation upon PEDV infection. In addition, 1,25(OH)2D3 supplementation inhibited ISG15 and MxA expression induced by PEDV. Although 1,25(OH)2D3 suppressed the JAK/STAT signal pathway and antiviral gene expression, it had no significant effects on PEDV replication and IFN-α-induced antiviral effects. In addition, when the vitamin D receptor (VDR) was silenced by siRNA, the anti-inflammatory effect of 1,25(OH)2D3 was inhibited. Meanwhile, the overexpression of VDR significantly downregulated IL-19 and CCL20 expression induced by PEDV infection. Together, our results provide powerful evidence that 1,25(OH)2D3 could alleviate PEDV-induced inflammation by regulating the NF-κB and JAK/STAT signaling pathways through VDR. These results suggest that vitamin D could contribute to inhibiting intestinal inflammation and alleviating intestinal damage in PEDV-infected piglets, which offers new approaches for the development of nutritional strategies to prevent PEDV infection in piglets.