NF-κB Protein Expression Research Articles

Coumarin and eugenol ameliorate LPS-induced inflammation in RAW 264.7 cells via modulating the NLRP3 inflammasome pathway

Objective: To investigate the underlying mechanism of anti-inflammatory action of coumarin and eugenol in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Methods: RAW 264.7 cells were treated with 2.5 μg/mL of LPS, 50 μM of coumarin, and 50 μM eugenol for 24 h. The viability of the cells was assessed using MTT assay. The production of nitric oxide was determined using Griess reagent and DCFH-DA was used to measure the production of reactive oxygen species. The protein expression of NLRP3, IL-1β, NF-κB, and cyclooxygenase 2 was assessed using Western blot analysis. Results: Coumarin and eugenol showed anti-inflammatory effects against LPS-induced inflammatory response by ameliorating the expression of NLRP3 inflammasome and NF-κB, which further led to a subsequent reduction in IL-1β, nitric oxide, and reactive oxygen species. Conclusions: Coumarin and eugenol exert their anti-inflammatory activities by modulating the NLRP3 inflammasome pathway and NF-κB. These compounds may have promising therapeutic applications for the treatment of various inflammatory diseases.

Study on the effect of ginaton on reducing cerebral vasospasm and early brain injury after hemorrhagic stroke by inhibiting inflammatory response

Objective: The objective of this study was to investigate the effects and possible mechanisms of action of ginseng on cerebral vasospasm and early brain injury (EBI) following hemorrhagic stroke. Materials and Methods: Sprague-Dawley (SD) rats (n = 48) were randomly divided into sham operation (sham group), subarachnoid hemorrhage (SAH) model (SAH group), normal saline (NS group), and Ginaton (Extract of Ginkgo Biloba Leaves Drops) intervention (gin group) groups. MCP-1 mRNA and tumor necrosis factor levels were detected using reverse transcription–polymerase chain reaction. The relative expression of mRNA was detected by Western blotting. Results: (1) Compared with the sham group, the SAH, NS, and gin groups had different degrees of neurological dysfunction. Compared with the SAH and NS groups, the neurological deficits in the gin group were significantly improved (P < 0.05). (2) Compared with the sham group, the relative expression levels of MCP-1 mRNA in the SAH, NS, and gin groups were 5.1 ± 0.9, 3.4 ± 0.6, and 2.5 ± 0.4, respectively; the relative expression levels of mRNA were 13.3 ± 2.4, 11.2 ± 1.8, and 3.8 ± 0.6, respectively. (3) The apoptosis rates of brain tissue in the sham, SAH, NS, and gin groups were 4.8 ± 0.7, 54.2 ± 10.3, 50.1 ± 7.4, and 28.4 ± 4.5, respectively. (4) Western blot showed that the relative expression levels of toll-like receptor-4 (TLR-4) protein in the sham, SAH, NS, and gin groups were 0.29 ± 0.03, 0.87 ± 0.15, 0.65 ± 0.13, and 0.41 ± 0.17, respectively; the relative expression levels of B protein were 0.21 ± 0.04, 0.96 ± 0.14, 0.73 ± 0.18, and 0.30 ± 0.05, respectively. Gin treatment could inhibit TLR-4 and nuclear factor-κB (NF-κB) protein expression. Conclusions: Dona tablets may inhibit activation of the NF-κB signaling pathway, and SAH-induced inflammatory response, so as to reduce cerebral vasospasm and EBI.

Unacylated ghrelin attenuates acute liver injury and hyperlipidemia via its anti-inflammatory and anti-oxidative activities.

Liver injury and hyperlipidemia are major issues that have drawn more and more attention in recent years. The present study aimed to investigate the effects of unacylated ghrelin (UAG) on acute liver injury and hyperlipidemia in mice. UAG was injected intraperitoneally once a day for three days. Three hours after the last administration, acute liver injury was induced by intraperitoneal injection of carbon tetrachloride (CCl4), and acute hyperlipidemia was induced by intraperitoneal injection of poloxamer 407, respectively. Twenty-four hours later, samples were collected for serum biochemistry analysis, histopathological examination, and Western blotting. In acute liver injury mice, UAG significantly decreased liver index, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), reduced malondialdehyde (MDA) concentration and increased superoxide dismutase(SOD) in liver tissue. NF-kappa B (NF-κB) protein expression in the liver was down-regulated. In acute hyperlipidemia mice, UAG significantly decreased serum total cholesterol (TC), triglyceride (TG), ALT, and AST, as well as hepatic TG levels. Meanwhile, hepatic MDA decreased and SOD increased significantly. Moreover, UAG improved the pathological damage in the liver induced by CCl4 and poloxamer 407, respectively. Intraperitoneal injection of UAG exhibited hepatoprotective and lipid-lowering effects on acute liver injury and hyperlipidemia, which is attributed to its anti-inflammatory and anti-oxidant activities.

Potent Suppression of Prostate Cancer Cell Growth and Eradication of Cancer Stem Cells by CD44-targeted Nanoliposome-quercetin Nanoparticles.

The bioavailability of quercetin, a natural compound, is hindered by low solubility, limited absorption, and restricted systemic availability. Therefore, encapsulating it in biocompatible nanoparticles presents a promising solution. This study aimed to target prostate cancer stem cells (CSCs) overexpressing CD44+ receptors as well as cancer cells, employing quercetin-loaded hyaluronic acid-modified nanoliposomes (LP-Quer-HA). Synthesized via a green ethanol injection method, these nanoliposomes had an average diameter of 134 nm and an impressive loading efficiency of 96.9%. Human prostate cancer cells were treated with either 10 μM of free quercetin or the same concentration delivered by LP-Quer-HA for 72 hours. Free quercetin reduced androgen-resistant PC3 cell viability by 16%, while LP-Quer-HA significantly increased cell death to 60%. It induced apoptosis, upregulating cytochrome c, Bax, caspases 3 and 8, and downregulating survivin and Bcl-2 expression. Compared to free quercetin, LP-Quer-HA upregulated E-cadherin expression while inhibiting cell migration and reducing the expression of fibronectin, N-cadherin, and MMP9. Treatment of PC3 cell tumor spheroids with LP-Quer-HA decreased the number of CD44 cells and expression of CD44, Oct3/4 and Wnt. Moreover, LP-Quer-HA inhibited p-ERK expression while increasing p38/MAPK and NF-κB protein expression. In androgen-sensitive LNCaP cells, LP-Quer-HA efficacy was notable, reducing cell viability from 10% to 52% compared to free quercetin. Utilizing HA-modified nanoliposomes as a quercetin delivery system enhanced its potency at lower concentrations, reducing the CD44+ cell population and effectively impeding prostate cancer cell proliferation and migration. These findings underscore the potential of quercetin-loaded cationic nanoliposomes as a robust therapeutic approach.

Regulatory effects of mangiferin on LPS-induced inflammatory responses and intestinal flora imbalance during sepsis.

Studies suggest that mangiferin (MAF) has good therapeutic effects on chronic bronchitis and hepatitis. Also, it is one of the antiviral ingredients in Anemarrhena asphodeloides Bunge. However, its effect on the LPS-induced inflammation and intestinal flora during sepsis remains unclear yet. In the present study, LPS-stimulated inflammation RAW264.7 cells and LPS-induced sepsis mice were used to evaluate the efficacy of MAF invitro and invivo. 16S rDNA sequencing was performed to analyze the characteristics of intestinal flora of the sepsis mice. It has been demonstrated that MAF (12.5 and 25 μg/mL) significantly inhibited protein expressions of TLR4, MyD88, NF-κB, and TNF-α in the LPS-treated cells and reduced the supernatant TNF-α and IL-6 levels. Invivo, MAF (20 mg/kg) markedly protected the sepsis mice and reduced the serum TNF-α and IL-6 levels. Also, MAF significantly downregulated the protein expressions of TLR4, NF-κB, and MyD88 in the livers. Importantly, MAF significantly attenuated the pathological injuries of the livers and small intestines. Further, MAF significantly increased proportion of Bacteroidota and decreased the proportions of Firmicutes, Desulfobacterota, Actinobacteriota, and Proteobacteria at phylum level, and it markedly reduced the proportions of Escherichia-Shigella, Pseudoalteromonas, Staphylococcus at genus level. Moreover, MAF affects some metabolism-related pathways such as citrate cycle (TCA cycle), lipoic acid metabolism, oxidative phosphorylation, bacterial chemotaxis, fatty acid biosynthesis, and peptidoglycan biosynthesis of the intestinal flora. Thus, it can be concluded that MAF as a treatment reduces the inflammatory responses invitro and invivo by inhibiting the TLR4/ MyD88/NF-κB pathway, and corrects intestinal flora imbalance during sepsis to some degree.

Isoliquiritigenin Inhibits the Growth of Colorectal Cancer Cells through the ESR2/PI3K/AKT Signalling Pathway.

Colorectal cancer (CRC) is one of the most common malignancies. Isoliquiritigenin (ISL), a flavonoid phytoestrogen, has shown anti-tumour activities against various cancers. However, its anti-CRC mechanism has not been clarified. In this study, the potential molecular mechanism of ISL against CRC was investigated through network pharmacological prediction and experimental validation. The results of the network prediction indicate that ESR2, PIK3CG and GSK3β might be the key targets of ISL against CRC, which was verified by molecular docking, and that its anti-tumour mechanisms might be related to the oestrogen and PI3K/AKT signalling pathway. The experimental results show that ISL reduced the viability of SW480 and HCT116 cells, induced apoptosis, blocked the cell cycle in the G2 phase in vitro, and suppressed xenograft tumour growth in vivo. In addition, ISL significantly down-regulated the protein expression of PIK3CG, AKT, p-AKT, p-GSK3β, CDK1, NF-κB and Bcl-2; up-regulated ESR2 and Bax; decreased the ratio of p-AKT/AKT and p-GSK3β/GSK3β; and increased the Bax/Bcl-2 ratio. This study indicates that ISL can inhibit the growth of CRC cells and induce apoptosis, which may be related to the up-regulation of ESR2 and inhibition of the PI3K/Akt signalling pathway.

Probiotics mitigated sepsis-related mortality through antioxidant and inflammatory-mediated mechanisms

The current therapies used to treat sepsis are ineffective and sepsis-related mortality is high. Therefore, it is important to find new treatment options that reduce mortality from that disease. In the present study, the effects of probiotic pretreatment in septic rats were studied on morality, hematological parameters, oxidative stress, antioxidant capacity, and inflammation. After preparation of rats and administration of probiotic VSL # 3, cecal ligation and puncture (CLP) surgery was used to induce sepsis. Then, the animals were divided into four groups: sham, sham + VSL # 3, septic, and septic + VSL # 3. Sepsis-related mortality, hematological parameters, and biochemical factors were assessed. Also, the expression of inflammatory cytokines IL-6, TNF-α and IL-10 in serum were studied using enzyme-linked immunosorbent assay (ELISA). Then, the NF-κB gene and protein expression in intestinal tissue was studied by RT-qPCR and western blot techniques, respectively. Daily pretreatment with VSL # 3 in septic rats resulted in a 20 % reduction in mortality over 10 days and a significant modulation in hematological parameters. Evaluation of oxidant and antioxidant indices showed a decrease in oxidative stress and an increase in antioxidant capacity in septic rats receiving VSL # 3. Also, decreased expressions of inflammatory cytokines IL-6, TNF-α, IL-10, and NF-κB were observed in septic rats receiving VSL # 3. In general, it can be concluded that the administration of probiotic VSL # 3, which resulted in the improved survival of septic rats and better antioxidant and anti-inflammatory conditions, can be considered as a new adjutant therapeutic approach in the treatment of sepsis.

Liuwei Dihuang Pills alleviates postmenopausal osteoporosis and fatigue in rats by inhibiting the epigenetic regulatory molecule BRD4 pathway

To explore the role of epigenetic signal molecule bromodomain protein 4 (BRD4) in mediating the therapeutic effect of Liuwei Dihuang (LWDH) Pills on postmenopausal osteoporosis (PMOP) and fatigue. Thirty rat models of PMOP induced by bilateral ovariectomy were randomized equally into two groups for treatment with normal saline (model group) or LWDH Pills (385.7 mg/kg), with another 15 sham-operated rats as the sham operation group. After 12 weeks of treatment, femoral samples were taken to determine the bone density and BRD4 protein expression. The weight-bearing exhaustive swimming time of the rat models was recorded, and serum levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured using enzyme-linked immunosorbent assay. In cultured primary osteoblasts the changes in the expressions of BRD4, MAPK and NF-κB proteins were detected by immunofluorescence staining following treatment with LWDH Pills. The rat models of PMOP showed significantly up-regulated expression of BRD4 protein in the femoral tissue (P < 0.05), which was obviously lowered by treatment with LWDH Pills. The rats treated with LWDH Pills also showed significant improvement of fatigue. Immunofluorescent staining of the osteoblasts showed that treatment with LWDH Pills significantly decreased the protein expressions of BRD4, MAPK and NF-κB. Analysis of the GSE56116 dataset revealed that that patients with kidney-yin deficiency had significantly higher BRD4 expression than those in the kidney-yang-deficiency group and non-kidney-deficiency group (P < 0.05). The upregulation of BRD4 expression involved multiple signaling pathways including neural ligand receptor response, cytoskeleton rearrangement, cytokine interaction, and granulocyte colony-stimulating factor chemotaxis pathways. LWDH can alleviate PMOP and fatigue by decreasing BRD4 signaling pathway, suggesting that potential of BRD4 as a promising therapeutic target for PMOP.

β-carotene inhibits MAPKs signaling pathways on rat colonic epithelial cells to attenuate TNF-α-induced intestinal inflammation and injury.

This experiment successfully isolated the rat colonic epithelial cells and established a TNF-α-induced intestinal inflammation model. Western Blot was used to detect the related protein expression levels of the MAPKs signaling pathway. QPCR technology was used to detect the expression of aquaporins, intestinal mucosal repair factor, and inflammatory factors. The results show that 25 μM β-carotene pretreatment at 24 h can inhibit MAPKs signaling pathway activated by TNF-α, change the relative mRNA expression of inflammatory cytokines, intestinal mucosal repair factors, and aquaporins, and the phosphorylated protein expression of p38, ERK, and NF-κB were attenuated to reduce inflammatory damage. After inhibiting p38 and ERK, the effect of β-carotene was reduced significantly (P < 0.05). In conclusion, β-carotene can alleviate the abnormal expression of aquaporins caused by inflammation through the MAPKs signaling pathway. This is for β-carotene as a functional nutrient that provides new insights.

The enteroprotective effect of nifuroxazide against methotrexate-induced intestinal injury involves co-activation of PPAR-γ, SIRT1, Nrf2, and suppression of NF-κB and JAK1/STAT3 signals

Methotrexate (MTX) has long manifested therapeutic efficacy in several neoplastic and autoimmune disorders. However, MTX-associated intestinal toxicity restricts the continuation of treatment. Nifuroxazide (NIF) is an oral antibiotic approved for gastrointestinal infections as an effective antidiarrheal agent with a high safety profile. The current study was designed to explore the potential efficacy of NIF in alleviating intestinal toxicity associated with MTX chemotherapy with the elucidation of the proposed molecular mechanisms. Rats were administered NIF (50 mg/kg; p.o.) for ten days. On day five, a single i.p. injection of MTX (20 mg/kg) was given to induce intestinal intoxication. At the end of the experiment, duodenal tissue samples were isolated for biochemical, Western blotting, immunohistochemical (IHC), and histopathological analysis via H&E, PSA, and Alcian blue stains. NIF showed antioxidant enteroprotective effects against MTX intestinal intoxication through enhanced expression of the redox-sensitive signals of PPAR-γ, SIRT1, and Nrf2 estimated by IHC. Moreover, NIF down-regulated the pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), NF-κB protein expression, and the phosphorylation of JAK1/STAT3 proteins, leading to mitigation of intestinal inflammation. In accordance, the histological investigation revealed that NIF ameliorated the intestinal pathological changes, preserved the goblet cells, and reduced the inflammatory cells infiltration. Therefore, NIF could be a promising candidate for adjunctive therapy with MTX to mitigate the associated intestinal injury and increase its tolerability.

Effect of Wu Zhi San supplementation in LPS-induced intestinal inflammation and barrier damage in broilers.

Intestinal inflammation and barrier damage can inhibit the absorption and transportation of nutrients in the small intestine, and lead to various chronic diseases. Wu Zhi San (WZS) is a traditional Chinese formula composed of Schisandrae, Anemarrhenae, Lonicerae, and Glycyrrhizae that was made to cure intestinal inflammation and barrier damage in broilers. To evaluate the protective effect of WZS on intestinal inflammation and barrier damage of broilers under lipopolysaccharide (LPS) stress, a total of 200 one-day-old broilers were randomly divided into five groups, namely, the CON group, LPS group, and three WZS groups (WZS-H, WZS-M, and WZS-L). The groups were designed for stress phase I (days 15, 17, 19, and 21) and stress phase II (days 29, 31, 33, and 35). The protective effect of WZS on the intestinal tract was evaluated by measuring the levels of serum myeloperoxidase (MPO), diamine oxidase (DAO), super oxide dismutase (SOD), and serum D-lactate (D-LA) and the expression of inflammatory factors in jejunum. The results showed that the diet supplemented with WZS could significantly reduce serum MPO, DAO, and D-LA levels and jejunal CD in broilers (p < 0.05), increase serum SOD levels and jejunal VH (p < 0.05), significantly downregulate the expression of NF-κB, TLR4, MyD88, and inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10), and upregulate Claudin-1, Occludin-1, and ZO-1 in broiler jejunum mucosa (p < 0.05). On the other hand, WZS could significantly reduce the protein expression of NF-κB (p65) in broiler jejunum (p < 0.05). These results indicate that supplementing WZS in the diet can reduce intestinal inflammation and alleviate intestinal barrier damage, and by inhibiting the NF-κB/TLR4/MyD88 signaling pathway, supplementation with WZS intervenes in LPS-induced stress injury in broilers.

Exploring the Mechanism of Chang-An-Kang on Active Ulcerative Colitis Rats Based on NF-κB/STAT3 Signaling Pathway

Objectives The objective of this study was to investigate the therapeutic effect and mechanism of Chang-An-Kang on ulcerative colitis rats. Materials and Methods The rat model of ulcerative colitis induced by 6% glacial acetic acid was established. The corresponding therapeutic drugs were given 24 h after the model was established. The pathological morphology of colon tissue was observed after 21 days of continuous administration. The levels of interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α) were measured by ELISA kit. The expression levels of NF-κB and STAT3 proteins in colon tissue were detected by IHC. Results The results showed that the changes in the pathological and biochemical indexes of the rat colon tissue indicate that the model was successfully established. Chang-An-Kang could significantly reduce rat colon tissue damage, decrease the content of IL-6, IL-7, and TNF-α, and increase the content of IL-10. The NF-κB and STAT3 protein expression levels were decreased. Conclusion By increasing the content of anti-inflammatory factor IL-10, decreasing the content of pro-inflammatory factors IL-6 and TNF-α, and inhibiting the expression of NF-κB and STAT3 proteins, Chang-An-Kang can reduce inflammatory infiltration. It has an obvious therapeutic effect on acetic acid ulcerative colitis in rats.

Investigating the effect of intramyocardial injection of DNAzyme-containing nisosome of NF-κB gene on the level of cardiac tissue NF-κB protein expression in cardiac ischemia-reperfusion of male rats

Investigating the effect of intramyocardial injection of DNAzyme-containing nisosome of NF-κB gene on the level of cardiac tissue NF-κB protein expression in cardiac ischemia-reperfusion of male rats

L-Carnitine augments probenecid anti-inflammatory effect in monoiodoacetate-induced knee osteoarthritis in rats: involvement of miRNA-373/P2X7/NLRP3/NF-κB milieu

Osteoarthritis (OA) is a degenerative joint disease, whereas the underlying molecular trails involved in its pathogenesis are not fully elucidated. Hence, the current study aimed to investigate the role of miRNA-373/P2X7/NLRP3/NF-κB trajectory in its pathogenesis as well as the possible anti-inflammatory effects of probenecid and l-carnitine in ameliorating osteoarthritis via modulating this pathway. In the current study, male Sprague Dawley rats were used and monoiodoacetate (MIA)-induced knee osteoarthritis model was adopted. Probenecid and/or L-carnitine treatments for 14 days succeeded in reducing OA knee size and reestablishing motor coordination and joint mobility assessed by rotarod testing. Moreover, different treatments suppressed the elevated serum levels of IL-1β, IL-18, IL-6, and TNF-α via tackling the miRNA-373/P2X7/NLRP3/NF-κB, witnessed as reductions in protein expressions of P2X7, NLRP3, cleaved caspase-1 and NF-κB. These were accompanied by increases in procaspase-1 and IκB protein expression and in miRNA-373 gene expression OA knee to various extents. In addition, different regimens reversed the abnormalities observed in the H and E as well as Safranin O-Fast green OA knees stained sections. Probenecid or l-carnitine solely showed comparable results on the aforementioned parameters, whereas the combination therapy had the most prominent effect on ameliorating the aforementioned parameters. In conclusion, l-carnitine augmented the probenecid’s anti-inflammatory effect to attenuate MIA-induced osteoarthritis in rats by provoking the miRNA-373 level and inhibiting the P2X7/NLRP3/NF-κB milieu, leading to the suppression of serum inflammatory cytokines: IL-1β, IL-18, IL-6, and TNF-α. These findings suggest the possibility of using probenecid and l-carnitine as a useful therapeutic option for treatment of osteoarthritis.Graphical abstract

Cyclophosphamide reduces gene transcriptional activity and embryo in vitro development by inhibiting NF-κB expression through decreasing AcH4K12

Cyclophosphamide (CTX), a widely used chemotherapeutic agent for cancer treatment, has been associated with long-term toxicity and detrimental effects on oocytes and ovaries, resulting in female reproductive dysfunction. This study aimed to investigate the potential impact of CTX on in vitro maturation (IVM) injury of porcine oocytes and subsequent embryonic development, as well as its effects on epigenetic modification and gene activation during early embryonic development. The results demonstrated that CTX treatment caused aberrant spindle structure and mitochondrial dysfunction during oocyte maturation, inducing DNA damage and early apoptosis, which consequently disrupted meiotic maturation. Indeed, CTX significantly reduced the in vitro developmental capacity of porcine embryos, and induced DNA damage and apoptosis in in vitro fertilization (IVF) blastocysts. Importantly, CTX induced abnormal histone modification of AcH4K12 in early porcine embryos. Moreover, addition of LBH589 before zygotic genome activation (ZGA) effectively increased AcH4K12 levels and restored the protein expression of NF-κB, which can effectively enhance the in vitro developmental potential of IVF embryos. The DNA damage and apoptosis induced by CTX compromised the quality of the blastocysts, which were recovered by supplementation with LBH589. This restoration was accompanied by down-regulation of BAX mRNA expression and up-regulation of BCL2, POU5F1, SOX2 and SOD1 mRNA expression. These findings indicated that CTX caused abnormal histone modification of AcH4K12 in early porcine embryos and reduced the protein expression of NF-κB, a key regulator of early embryo development, which may block subsequent ZGA processes.

Structural analysis of a soluble polysaccharide GSPA-0.3 from the root of Panax ginseng C. A. Meyer and its adjuvant activity with mechanism investigation

A novel polysaccharide (GSPA-0.3) was isolated and purified from the root of cultivated Panax ginseng C. A. Meyer, and its structure, adjuvant activities, and mechanisms for inducing the maturation of mouse dendritic 2.4 cells (DC2.4) were extensively studied. Fraction GSPA-0.3, mainly composed by the galacturonic acid, galactose, arabinose, glucose, rhamnose, mannose, and xylose, had a molecular weight of 62,722 Da. The main chain of GSPA-0.3 was composed of →3)-α-L-Rhap-(1→, →4)-α-D-GalpA-(1→, and →3, 4)-α-D-GalpA-(1→. Branched chains comprised α-L-Araf-(1→3, 5)-α-L-Araf-(1→5)-α-L-Araf-(1→, α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→, β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Galp-(1→, and α-D-GalpA-(1→ units connected to the C3 position of →3, 4)-α-D-GalpA-(1→. In vivo, GSPA-0.3 was found to stimulate the production of IgG, IgG1, and IgG2a; increase the splenocyte proliferation index; and promote the expression of GATA-3, T-bet, IFN-γ, and IL-4 in H1N1 vaccine-immunized mice. Moreover, GSPA-0.3 significantly increased the levels of neutralizing antibodies in the mice, and its adjuvant activity was found to be superior to aluminum adjuvant (Alum adjuvant). Mechanistic investigations showed that GSPA-0.3 activated the TLR4-dependent pathway by upregulating the expressions of TLR4, MyD88, TRAF-6, and NF-κB proteins and gens. The results presented herein suggested that GSPA-0.3 could significantly promote the efficacy of the H1N1 vaccine by modulating Th1/Th2 response via the TLR4-MyD88-NF-κB signaling pathway.

Activin suppresses the expression of inflammatory genes and signaling proteins in human leukemia monocytic THP-1 cells.

Activin regulates inflammation, cell proliferation, immune response, wound repair, and endocrine function. In this study, we investigated the effect of activin on inflammatory genes in THP-1 cells and the involvement of NF-κB, AKT, and mitogen-activated protein kinase (MAPK) signaling. Cell viability was determined using a colorimetric assay with the MTS/PES solution. The mRNA levels were analyzed using reverse transcription-quantitative polymerase chain reaction. The expression of NF-κB, AKT, and MAPK signaling proteins was measured using immunoblot analysis. Activin A did not affect THP-1 cell viability at concentrations below 50 ng/ml. Activin decreased the mRNA expression of cytokines (interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), toll-like receptor 4 (TLR4), and matrix metallo-proteinases (MMP)-9 proteins but did not affect IL-8 expression. Activin increased the expression of TLR2 and MMP-2. In addition, activin inhibited the phosphorylation of NF-κB p65, AKT, and MAPK (c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK) signaling proteins. Our results suggest that activin may be involved in anti-inflammation by inhibiting inflammatory gene expression and regulating NF-κB and AKT/MAPK signaling.

Effects of electroacupuncture at "Jiaji" (EX-B 2) combined with neurodynamic mobilization on gastrocnemius muscle atrophy and expression of NF-κB and MuRF1 in rabbits after sciatic nerve injury.

To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) combined with neurodynamic mobilization (NM) on the cross-sectional area of the gastrocnemius muscle fibers after sciatic nerve injury in rabbits, and the expression of nuclear factor κB (NF-κB) and muscle-specific ring-finger protein 1 (MuRF1). A total of 180 common-grade New Zealand rabbits (half male and half female) were randomly divided into five groups, i.e. a normal control group, a model control group, a NM group, an EA group and a combined intervention group, 36 rabbits in each group. Except in the normal control group, clipping method was used to prepare the model of sciatic nerve injury in the rest groups. On the 3rd day of successful modeling, NM was delivered in the NM group. In the EA group, EA was exerted at bilateral "Jiaji" (EX-B 2) of L4 to L6, stimulated with disperse-dense wave and the frequency of 2 Hz/100 Hz. In the combined intervention group, after EA delivered at bilateral "Jiaji" (EX-B 2) of L4 to L6 , NM was operated. The intervention in each group was delivered once daily, for 6 days a week, and lasted 1, 2 or 4 weeks according to the collection time of sample tissue. After 1, 2 and 4 weeks of intervention, in each group, the toe tension reflex score and the modified Tarlov test score were observed; the morphology of the gastrocnemius muscle was observed by HE staining and the cross-sectional area of muscular fiber was measured; using Western blot method, the expression of NF-κB and MuRF1 of the gastrocnemius muscle was detected. After 1, 2 and 4 weeks of intervention, the toe tension reflex scores and the modified Tarlov scores in the model control group were lower than those of the normal control group (P<0.05), and these two scores in the NM group, the EA group and the combined intervention group were all higher than those of the model control group (P<0.05); the scores in the combined intervention group were higher than those in the EA group and the NM group (P<0.05). The gastrocnemius fibers were well arranged and the myocyte morphology was normal in the normal control group. In the model control group, the gastrocnemius fibers were disarranged, the myocytes were irregular in morphology and the inflammatory cells were infiltrated in the local. In the NM group, the EA group and the combined intervention group, the muscle fibers were regularly arranged when compared with the model control group. After 1, 2 and 4 weeks of intervention, the cross-sectional areas of the gastrocnemius muscle fibers in the model control group were smaller than those of the normal control group (P<0.05). The cross-sectional areas in the NM group, the EA group and the combined intervention group were larger than those of the model control group (P<0.05), and the cross-sectional areas in the combined intervention group were larger than those in the NM group and the EA group (P<0.05). After intervention for 1, 2 and 4 weeks, the protein expressions of NF-κB and MuRF1 in the gastrocnemius muscle were higher in the model control group in comparison of those in the normal control group (P<0.05). In the NM group, the EA group and the combined intervention group, the expressions of NF-κB after intervention for 1, 2 and 4 weeks and the expressions of MuRF1 after 2 and 4 weeks of intervention were lower when compared with those in the model control group (P<0.05). In the combined intervention group, the protein expressions of NF-κB after intervention for 1, 2 and 4 weeks and the expressions of MuRF1 after 2 and 4 weeks of intervention were decreased when compared with those in the NM group and the EA group (P<0.05). Electroacupuncture at "Jiaji" (EX-B 2) combined with NM may increase the muscle strength and sciatic function and alleviate gastrocnemius muscle atrophy in the rabbits with sciatic nerve injury. The underlying mechanism is related to the inhibition of NF-κB and MuRF1 expression.

Effects of SGLT2 inhibition on cardiac remodeling and heart failure development in rats with aortic stenosis

Abstract Introduction Aortic stenosis prevalence has been increasing as population ages. Currently, no medical therapies influence the natural history of aortic stenosis. Sodium glucose co-transporter 2 inhibitors (SGLT2i) have reduced heart failure hospitalization and cardiovascular mortality in heart failure patients with both reduced and preserved ejection fraction. The aim of this study was to evaluate the effects of SGLT2 inhibitor empagliflozin (EMPA) on cardiac remodeling and heart failure development in aortic stenosis rats. Methods Eighteen weeks after supra-aortic banding surgery, male Wistar rats were divided in four groups: Sham (n=15); sham + EMPA (Sham-EMPA, n=16); aortic stenosis (AS, n=27); and aortic stenosis + EMPA (AS-EMPA, n=27). Empagliflozin (10 mg/kg/day) was added to rat chow for 8 weeks. Echocardiogram was performed to evaluate cardiac structure and function. Myocardial collagen content was quantified by histology; activity of antioxidant enzymes and enzymes involved in energy metabolism was assessed by spectrophotometry. Protein expression was quantified by Western blot, and matrix metalloproteinase (MMP) activity by zymography. Statistical analysis: ANOVA and Bonferroni or Kruskal-Wallis and Dunn (p&amp;lt;0.05). Results AS-EMPA group had a lower frequency of anatomopathological heart failure features, a lower left ventricular (LV) dilatation (LV diastolic diameter: Sham 8.39±0.57; Sham-EMPA 8.25±0.38; AS 9.80±0.53; AS-EMPA 9.08±0.87 mm; LV systolic diameter: Sham 4.24±0.68; Sham-EMPA 4.19±0.57; AS 5.72±0.74; AS-EMPA 5.09±1.08 mm) and LV mass (Sham 0.85±0.11; Sham-EMPA 0.82±0.07; AS 2.19±0.27; AS-EMPA 1.76±0.34 g), and better diastolic function (isovolumetric relaxation time: Sham 28.6±2.28; Sham-EMPA 29.8±3.04; AS 18.9±2.59; AS-EMPA 22.9±5.02 ms) than AS. Systolic function was impaired in both AS and AS-EMPA compared to their respective controls and did not differ between AS-EMPA and AS. AS and AS-EMPA had larger myocyte diameter, higher interleukin-6 protein expression and lower catalase activity than Sham and Sham-EMPA, respectively. AS and AS-EMPA groups had lower enzyme activity from energy metabolism than their respective controls. AS presented higher lipid hydroperoxide concentration, collagen III and p65 NF-kB protein expression, and lower glutathione peroxidase activity than Sham. AS-EMPA had higher collagen I expression and inactive MMP-2 than Sham-EMPA. AS-EMPA presented higher glutathione peroxidase and intermediary active MMP-2, and lower interstitial collagen fraction (Sham 2.68±0.59; Sham-EMPA 3.16±0.33; AS 5.68±0.97; AS-EMPA 3.55±0.70 %) than AS. Conclusion Empagliflozin improves cardiac remodeling, diastolic function and myocardial oxidative stress, and reduces heart failure feature frequency and interstitial collagen fraction in aortic stenosis rats.

Mechanism of DaiTongXiao in the treatment of gouty arthritis through the NLRP3 signaling pathway

Ethnopharmacological relevanceDaiTongXiao (DTX) is a traditional Chinese Dai folk formulation utilized for gouty arthritis treatment, with substantial evidence supporting its anti-inflammatory properties. The NLRP3 inflammasome disorder is tightly linked to the development of many inflammatory diseases. Aim of the studyTo elucidate the therapeutic efficacy of DTX in gouty arthritis and reveal its potential underlying mechanism. Materials and methodsThe primary active constituents in DTX were determined through ultraviolet spectrophotometry and gas chromatography. Rats underwent induction with monosodium urate (MSU), followed by treatment of J774A.1 cells with adenosine triphosphate (ATP) activation and lipopolysaccharide (LPS) induction and the subsequent culture in Dulbecco’s modified Eagle’s medium. The degree of foot joint swelling in rats was assessed, and ankle joints were evaluated through H&E staining. Enzyme-linked immunosorbent assay was performed to measure the levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α in both serum and cells. Reverse transcription–polymerase chain reaction (RT-PCR) was performed to determine the relative mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 macrophages. The expression of NLRP3, ASC, Caspase-1, and NF-κB was examined by western blotting. ResultsDTX could alleviate MSU-induced joint swelling in rats, as evidenced by a reduction in joint inflammation. Moreover, DTX effectively enhanced the survival rate of J774A.1 cells following LPS induction and ATP activation. Furthermore, DTX significantly reduced IL-1β, IL-6, IL-8, and TNF-α levels in both cell culture medium and rat serum. RT-PCR results revealed that DTX notably downregulated the mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 cells. Additionally, DTX downregulated NLRP3, ASC, NF-κB, and Caspase-1 expression in the joint tissue. ConclusionsDTX exerts a significant anti–gouty arthritis effect, with its mechanism being tightly linked to the NLRP3 inflammatory signaling pathway. This pathway may be modulated by inhibiting IL-1β differentiation and maturation by downregulating NLRP3, ASC, Caspase-1, and NF-κB protein expression. This, in turn, leads to a reduction in the release of IL-6, IL-8, and TNF-α, ultimately impeding gouty arthritis progression.