NF-κB P-p65 Research Articles

Pathogenic role of NDUFA13 inactivation in spontaneous hepatitis in mice and the mechanism

To investigate the role of NDUFA13 inactivation in the pathogenesis of spontaneous hepatitis in mice and explore the possible mechanisms. Hepatocyte-specific NDUFA13 knockout (NDUFA13fl/-) mice were generated by intercrossing NDUFA13fl/fl and Alb-Cre mice based on Cre/loxP transgenic technology, and tail and liver DNA of the mice was genotyped by PCR analysis. Ten NDUFA13fl/- mice and 10 littermate control NDUFA13fl/fl mice were housed, and in each group, 5 mice were euthanized at the age of 4 weeks and the other 5 at two years for pathological examination of the liver tissues with HE staining. Immunohistochemistry was used to verify the expression levels of NDUFA13, NF-κB/p65, NF-κB/p-p65 and inflammasome NLRP3. The total intracellular ROS and mitochondrial ROS levels were measured with a ROS staining kit. The expressions of the inflammatory cell markers (CD45, MPO, and F4/80) and inflammatory cytokines (IL1β and IL33) in the liver were detected with immunohistochemistry and immunofluorescence assay. Liver-specific NDUFA13 heterozygous knockout mice were successfully constructed as verified by PCR results. HE staining revealed severe liver damage in both 4- week-old and 2-year-old NDUFA13fl/- mice as compared with their littermate controls. Immunohistochemistry showed a significant decrease of NDUFA13 expression in both 4-week-old and 2-year-old NDUFA13fl/- mice (P < 0.05). The expression levels of NF-κB signals p65, p-p65 and NLRP3 inflammasomes were all significantly increased in NDUFA13fl/- mice (P < 0.05). The total intracellular ROS and mitochondrial ROS levels in NDUFA13fl/- mice were also significantly increased. NDUFA13 knockout obviously promoted the expression of the inflammatory cell markers (CD45, MPO and F4/80) and the secretion of IL-1β and IL-33 in the liver tissue of the mice (P < 0.05). Hepatocytes-specific NDUFA13 ablation can trigger spontaneous hepatitis in mice possibly mediated by the activation of ROS/NF-κB/NLRP3 signaling.

Lactoferrin suppresses LPS-induced expression of HMGB1, microRNA 155, 146, and TLR4/MyD88/NF-кB pathway in RAW264.7 cells

Objective This current study evaluated the underlying mechanisms of LF against the inflammatory microRNAs (miRNAs), HMGB1 expression, and TLR4-MyD88-NF-кB pathway in LPS-activated murine RAW264.7 cells. Methods MTT assay was used to assess cell metabolism and the cell culture levels of the cytokines (TNF-α, IL-6) were evaluated by Enzyme-linked immunosorbent assay (ELISA). The expression of miRNAs was quantified by using qPCR and the expression of HMGB1, TLR4, MyD88, and phosphorylated NF-κB (P-p65) were determined with Western blot and qPCR, respectively. Results The results indicated that LF downregulates IL-6 and TNF-α expression. LF exhibited the degradation of P-p65 and reduced the production of HMGB1, TLR4, and MyD88 in LPS-induced inflammatory response. Importantly, in parallel with the suppression of cytokines and HMGB1-TLR4-MyD88-NF-кB pathway, LF could induce a decrease in inflammatory selected miRNAs, mmu-mir-155, and mmu-mir-146a expression. Conclusions Altogether, these findings provide LF as a prominent anti-inflammatory agent that could modulate HMGB1, mmu-mir-155, mmu-mir-146a, and TLR4/MyD88/NF-кB pathway.

The PKN1- TRAF1 signaling axis as a potential new target for chronic lymphocytic leukemia

ABSTRACT TRAF1 is a pro-survival adaptor molecule in TNFR superfamily (TNFRSF) signaling. TRAF1 is overexpressed in many B cell cancers including refractory chronic lymphocytic leukemia (CLL). Little has been done to assess the role of TRAF1 in human cancer. Here we show that the protein kinase C related kinase Protein Kinase N1 (PKN1) is required to protect TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling in lymphoma. We show that the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but minimally detected in unstimulated healthy donor B cells. Through a screen of 700 kinase inhibitors, we identified two inhibitors, OTSSP167, and XL-228, that inhibited PKN1 in the nanomolar range and induced dose-dependent loss of TRAF1 in RAJI cells. OTSSP167 or XL-228 treatment of primary patient CLL samples led to a reduction in TRAF1, pNF-κB p65, pS6, pERK, Mcl-1 and Bcl-2 proteins, and induction of activated caspase-3. OTSSP167 synergized with venetoclax in inducing CLL death, correlating with loss of TRAF1, Mcl-1, and Bcl-2. Although correlative, these findings suggest the PKN1-TRAF1 signaling axis as a potential new target for CLL. These findings also suggest the use of the orally available inhibitor OTSSP167 in combination treatment with venetoclax for TRAF1 overexpressing CLL.

Enhancement of DUSP14 (dual specificity phosphatase 14) limits osteoarthritis progression by alleviating chondrocyte injury, inflammation and metabolic homeostasis

ABSTRACT Osteoarthritis (OA) is a proverbial inflammatory degenerative joint disease associated with the acceleration of the aging process and is characterized by chondrocyte injury and articular cartilage degradation. Dual-specificity phosphatase 14 (Dusp14), a common member of the DUSP family, has been implicated in multiple inflammatory diseases and bone loss. Nevertheless, the function of DUSP14 in OA remains unclear. In the present study, down-regulation of DUSP14 was corroborated in anterior cruciate ligament transection (ACLT)-induced OA rats and interleukin-1β (IL-1β)-stimulated chondrocytes. Additionally, the gain of DUSP14 reversed IL-1β-induced inhibition of chondrocyte viability but attenuated cell apoptosis. Concomitantly, DUSP14 overexpression muted IL-1β-induced release of pro-inflammatory mediators NO and prostaglandin E2 (PGE2), as well as pro-inflammatory cytokine levels (IL-6 and TNF-α). Furthermore, up-regulation of DUSP14 overturned the effects of IL-1β on the inhibition of collagen II and aggrecan expression, and enhancement of A Disintegrin and Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS5) and matrix metalloproteinases (MMPs; MMP3 and MMP-13). Mechanistically, DUSP14 elevation increased the p-Adenosine 5ʹ-monophosphate-activated protein activated protein kinase(AMPK), inhibitor of NF-κB (IκB) expression and decreased p-p65 NF-κB expression, indicating that DUSP14 might restore the AMPK-IκB pathway to restrain NF-κB signaling under IL-1β exposure. Notably, blockage of AMPK signaling muted the protective efficacy of DUSP14 elevation against IL-1β-induced inflammatory injury and metabolism disturbance in chondrocytes. Interestingly, histological evaluation substantiated that DUSP14 injection alleviated cartilage degradation in OA rats. Together, DUSP14 may ameliorate OA progression by affecting chondrocyte injury, inflammatory response and cartilage metabolism homeostasis, implying a promising therapeutic strategy against OA.

TLR4-Associated IRF-7 and NFκB Signaling Act as a Molecular Link Between Androgen and Metformin Activities and Cytokine Synthesis in the PCOS Endometrium.

Low-grade chronic inflammation is commonly seen in polycystic ovary syndrome (PCOS) patients with elevated levels of inflammatory cytokines in the endometrium. This work aimed to increase the limited understanding of the mechanisms underlying cytokine synthesis and increased endometrial inflammation in PCOS patients. Endometrial biopsy samples were collected from non-PCOS (n = 17) and PCOS (n = 22) patients either during the proliferative phase of the menstrual cycle or with hyperplasia. Endometrial explants were prepared from PCOS patients and underwent pharmacological manipulation in vitro. The expression and localization of toll-like receptor 2 (TLR2)/4, key elements of innate immune signal transduction and nuclear factor κB (NFκB) signaling pathways, and multiple cytokines were comprehensively evaluated by Western blotting, immunohistochemistry, and immunofluorescence in endometrial tissues. We demonstrated the distribution of protein expression and localization associated with the significantly increased androgen receptor, TLR2, and TLR4-mediated activation of interferon regulatory factor-7 (IRF-7) and NFκB signaling, cytokine production, and endometrial inflammation in PCOS patients compared to non-PCOS patients with and without endometrial hyperplasia. In vitro experiments showed that 5-dihydrotestosterone (DHT) enhanced androgen receptor, TLR4, IRF-7, and p-NFκB p65 protein expression along with increased interferon α (IFNα) and IFNɣ abundance. The effects of DHT on IRF-7, p-NFκB p65, and IFN abundance were abolished by flutamide, an antiandrogen. Although 17β-estradiol (E2) decreased p-IRF-7 expression with little effect on TLR-mediated IRF7 and NFκB signaling or on cytokine protein levels, exposure to metformin alone or in combination with E2 suppressed interleukin-1 receptor-associated kinase 4 (IRAK4), p-IRF-7, IRF-7, IκB kinase α (IKKα), p-NFκB p65, IFNɣ, and tumor necrosis factor α protein expression. Cytokine synthesis and increased endometrial inflammation in PCOS patients are coupled to androgen-induced TLR4/IRF-7/NFκB signaling, which is inhibited by metformin treatment.

GSK3β: A plausible molecular target in the cytokinemodulating effect of exogenous insulin in a murine model of malarial infection.

Malaria is a life-threatening disease caused by the Plasmodium sp. parasite. Infection results in heightened pro-inflammatory response which contributes to the pathophysiology of the disease. To mitigate the overwhelming cytokine response, host-directed therapy is a plausible approach. Glycogen synthase kinase-3β (GSK3β), a serine/threonine kinase plays a pivotal role in the regulation of inflammatory response during pathogenic infections. The present study was conducted to investigate the chemo-suppressive and cytokine-modulating effects of insulin administration in malaria-infected mice and the involvement of GSK3β. Intraperitoneal administrations of 0.3 and 0.5 U/kg body weight insulin each for four consecutive days into Plasmodium berghei NK65 (PbN)-infected mice resulted in chemo-suppression exceeding 60% and improved median survival time of infected mice (20.5 days and 19 days respectively compared to 15.5 days for non-treated control). Western analysis revealed that pGSK3β (Ser9) intensity in brain samples from insulin-treated (0.3 and 0.5 U/kg body weight) infected mice each were 0.6 and 2.2 times respectively than that in control. In liver samples, pGSK3β (Ser9) intensity from insulin-treated infected mice were significantly higher (4.8 and 16.1 fold for 0.3 and 0.5 U/kg bw respectively) than that in control. Insulin administration decreased both brain and liver pNF-κB p65 (Ser536) intensities (to 0.8 and 0.6 times for 0.3 U/kg bw insulin; and to 0.2 and 0.1 times for 0.5 U/kg bw insulin respectively compared to control). Insulin treatment (0.5 U/kg bw) also significantly decreased the serum levels of pro-inflammatory cytokines (TNF-α (3.3 times) and IFN-γ (4.9 times)) whilst significantly increasing the levels of anti-inflammatory cytokines (IL-4 (4.9 fold) and IL-10 (2.1 fold)) in PbN-infected mice. Results from this study demonstrated that the cytokinemodulating effects of insulin at least in part involve inhibition of GSK3β and consequent inhibition of the activation of NF-κB p65 suggesting insulin as a potential adjunctive therapeutic for malaria.

Panax Notoginseng Saponins Inhibits Ventricular Remodeling after Myocardial Infarction in Rats Through Regulating ATF3/MAP2K3/p38 MAPK and NF κ B Pathway.

To explore whether Panax notoginseng saponins (PNS) exhibits heart protective effect in myocardial infarction (MI) rats and to identify the potential signaling pathways involved. MI rats induced by ligating the left anterior descending (LAD) coronary artery were assigned to sham coronary artery ligation or coronary artery ligation. Totally 36 Sprague-Dawley rats were randomly divided into sham group (distilled water, n=9), MI group (distilled water, n=9), PNS group (PNS, 40 mg/kg daily, n=9) and fosinopril group (FIP, 1.2 mg/kg daily, n=9) according to a random number table. The left ventricular morphology and function were conducted by echocardiography. Histological alterations were evaluated by the stainings of HE and Masson. The serum levels of C reactive protein (CRP), tumor necrosis factor α (TNF-α), growth differentiation factor-15 (GDF-15) and the ratio of metalloproteinase-9 (MMP-9) and tissue inhibitor of MMP-9 (TIMP-1) were determined by ELISA. The levels of activating transcription factor 3 (ATF3), mitogen-activated protein kinase kinase 3 (MAP2K3), p38 mitogen-activated protein kinase (p38 MAPK), phosphorylation of p38 MAPK (p-p38 MAPK), transforming growth factor-β (TGF-β1), collagen I, nuclear factor kappa B p65 (NFκB p65), phosphorylation of NFκB p65 (p-NFκB p65), and phosphorylation of inhibitory kappa Bα (p-Iκ Bα) in hearts were measured by Western blot and immunohistochemical staining, respectively. PNS improved cardiac function and fibrosis in MI rats (P<0.05). The serum levels of CRP, TNF-α, GDF-15 and the ratio of MMP9/TIMP1 were reversed by PNS in MI rats. The expressions of TGF-β1, collagen I, MAP2K3, p38 MAPK, p-p38 MAPK, NFκB p65, p-NFκB p65, and p-IκBα were down-regulated, while ATF3 increased with the treatment of PNS (P<0.05). PNS may improve cardiac function and fibrosis in MI rats via regulating ATF3/MAP2K3/p38 MAPK and NFκB signaling pathways. These results suggest the potential of PNS in preventing the development of ventricular remodeling in MI rats.

ZnO NPs delay the recovery of psoriasis-like skin lesions through promoting nuclear translocation of p-NFκB p65 and cysteine deficiency in keratinocytes

BackgroundThis study aimed to evaluate the safety of applying zinc oxide nanoparticles (ZnO NPs) to pathological skin. The majority of previous studies confirmed the safety of applying ZnO NPs to normal skin. However, we know very little about the risks of using sunscreen, cosmetics and topical drugs containing ZnO NPs for individuals with skin diseases. ResultsZnO NPs passed through gaps between keratinocytes and entered stratum basale of epidermis and dermis in imiquimod-induced psoriasis-like skin lesions. Application of a ZnO NP-containing suspension for 3 connective days delayed the healing of the epidermal barrier; increased the expression levels of inflammatory cytokines; promoted keratinocyte apoptosis and disturbed redox homeostasis. In TNF-α-stimulated HaCaT cells, QNZ and JSH-23 (NFκB inhibitors) blocked ZnO NP-induced inflammation. JSH-23 and NAC (a precursor of cysteine) inhibited ZnO NP-induced nuclear translocation of p-NFκB p65, cysteine deficiency and apoptosis. Additionally, ZnO NPs decreased CD98 level in main pathway and failed to activate transsulfuration pathway in cysteine biosynthesis. ConclusionsZnO NPs can enter psoriasis-like skin lesions and promote inflammation and keratinocyte apoptosis through nuclear translocation of p-NFκB p65 and cysteine deficiency. This work reminds the public that ZnO NPs have harmful effects on the recovery of inflammatory skin diseases.

Antioxidant and Anti-Inflammatory Activities of Six Flavonoids from Smilax glabra Roxb.

This study aimed to isolate, prepare and identify the main flavonoids from a standardized Smilax glabra flavonoids extract (SGF) using preparative HPLC, MS, 1H NMR and 13C NMR, determine the contents of these flavonoids using UPLC, then compare their pharmacological activities in vitro. We obtained six flavonoids from SGF: astilbin (18.10%), neoastilbin (11.04%), isoastilbin (5.03%), neoisoastilbin (4.09%), engeletin (2.58%) and (−)-epicatechin (1.77%). The antioxidant activity of six flavonoids were evaluated by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS+) radical scavenging activity and ferric reducing antioxidant power (FRAP). In addition, the anti-inflammatory activity of six flavonoids were evaluated by determining the production of cytokines (IL-1β, IL-6), nitric oxide (NO) using enzyme linked immunosorbent assay and the NF-κB p65 expression using Western blotting in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that (−)-epicatechin, astilbin, neoastilbin, isoastilbin and neoisoastilbin had strong antioxidant activities, not only in DPPH and ABTS+ radicals scavenging capacities, but in FRAP system. Furthermore, all the six flavonoids could significantly inhibit the secretion of IL-1β, IL-6, NO (p < 0.01) and the protein expression of NF-κB p-p65 (p < 0.01) in LPS-stimulated RAW264.7 cells. This study preliminarily verified the antioxidant and anti-inflammatory activities of six flavonoids in S. glabra.

Initiation of Acupoint Molecular Mechanisms for Manual Acupuncture Analgesia-Nuclear Factor κB Signaling Pathway.

To identify the prominent molecular signaling in acupoints and explore their roles in initiating the analgesia effect of manual acupuncture (MA). A three-step study was conducted, the experiment 1 was a genome-wide analysis of the tissue at acupoint Zusanli (ST 36), including 12 Wistar rats which were divided into control, control+MA1, and control+MA7 groups. In the experiment 2, the paw withdrawal latency (PWL), immunohistochemistry and Western blot analysis of phospho-nuclear factor kappa B (NFκB) p65 (p-p65), phospho-NFκB p50 (p-p50) at ST 36 were performed on rats of saline, saline+MA, and complete Freund's adjuvant (CFA)+MA groups (n=6). In experiment 3, 24 rats were divided into saline+DMSO, CFA+DMSO, CFA+DMSO+MA, and CFA+BAY 11-7082+MA groups, the PWL and immunofluorescence assay of NFκB p65 at ST 36 was conducted. (1) The gene: inhibitor of NFκB (Nfkbia), interleukin-1β (Il1b), interleukin-6 (Il6), chemokine c-x-c motif ligand 1 (Cxcl1), monocyte chemoattractant protein-1 (MCP-1/Ccl2) expressions in the control+MA7 group were significantly increased (P<0.05 or P<0.01), and the expression of NFκB p65 (Rela), NFκB p50 (Nfkb1) were increased in the control+MA7 group (P<0.05). (2) CFA+MA groups showed increased PWL from day 1 to 7 (P<0.01 vs. CFA), and the Western blot results were consistent with immunohistochemistry, the expression of NFκB p-p65 and NFκB p-p50 were significantly increased in the MA-related groups compared with control and CFA groups (P<0.05). (3) Compared with the CFA+DMSO+MA group, the PWL of the CFA+ BAY 11-7082+MA group decreased significantly and continued until day 5 and 7 (P<0.05 and P<0.01, respectively), and the NFκB p65 expression of CFA+BAY 11-7082+MA was significantly reduced compared with CFA+DMSO+MA (P<0.01). Local NFκB signaling cascade in acupoint caused by MA is an important step in initiating the analgesic effect, which would provide new evidence for the initiation of MA-effect and improve the understanding of the scientific basis of acupuncture analgesia.

Inhibition of transient receptor potential vanilloid 1 (TRPV1) channel regulates chikungunya virus infection in macrophages.

Chikungunya virus (CHIKV), a virus that induces pathogenic inflammatory host immune responses, is re-emerging worldwide, and there are currently no established antiviral control measures. Transient receptor potential vanilloid 1 (TRPV1), a non-selective Ca2+-permeable ion channel, has been found to regulate various host inflammatory responses including several viral infections. Immune responses to CHIKV infection in host macrophages have been reported recently. However, the possible involvement of TRPV1 during CHIKV infection in host macrophages has not been studied. Here, we investigated the possible role of TRPV1 in CHIKV infection of the macrophage cell line RAW 264.7. It was found that CHIKV infection upregulates TRPV1 expression in macrophages. To confirm this observation, the TRPV1-specific modulators 5'-iodoresiniferatoxin (5'-IRTX, a TRPV1 antagonist) and resiniferatoxin (RTX, a TRPV1 agonist) were used. Our results indicated that TRPV1 inhibition leads to a reduction in CHIKV infection, whereas TRPV1 activation significantly enhances CHIKV infection. Using a plaque assay and a time-of-addition assay, it was observed that functional modulation of TRPV1 affects the early stages of the viral lifecycle in RAW 264.7 cells. Moreover, CHIKV infection was found to induce of pNF-κB (p65) expression and nuclear localization. However, both activation and inhibition of TRPV1 were found to enhance the expression and nuclear localization of pNF-κB (p65) and production of pro-inflammatory TNF and IL-6 during CHIKV infection. In addition, it was demonstrated by Ca2+ imaging that TRPV1 regulates Ca2+ influx during CHIKV infection. Hence, the current findings highlight a potentially important regulatory role of TRPV1 during CHIKV infection in macrophages. This study might also have broad implications in the context of other viral infections as well.

Sodium butyrate attenuated iE-DAP induced inflammatory response in the mammary glands of dairy goats fed high-concentrate diet.

Long-term high-concentrate (HC) diet feeding increased bacterial endotoxins, which translocated into the mammary glands of dairy goats and induced inflammatory response. γ-d-Glutamyl-meso-diaminopimelic acid (iE-DAP), bacterial peptidoglycan component, triggered inflammatory response through activating nucleotide oligomerization domain protein 1 (NOD1) signaling pathway. While dietary supplemented with sodium butyrate (SB) relieved inflammatory response and improved animal health and production. To investigate the effects and the mechanisms of action of SB on the inflammatory response in the mammary glands of dairy goats fed HC diet, 12 Saanen dairy goats were randomly assigned into HC group and SB regulated (BHC) group. The results showed that SB supplementation attenuated ruminal pH decrease caused by HC diet in dairy goats resulting in a decrease of proinflammatory cytokines and iE-DAP plasma concentration and the mRNA expression of NOD1 and other inflammation-related genes. The protein levels of NOD1, NF-κB p65 and NF-κB pp65 were decreased by the SB supplementation. The expression of histone deacetylase 3 (HDAC3) was also inhibited by the SB supplementation. Meanwhile, the chromatin compaction ratios and DNA methylation levels of NOD1 and receptor-interacting protein 2 (RIP2) of BHC group were upregulated. Collectively, the SB supplementation mitigated the inflammatory response in the mammary glands of dairy goats during HC-induced subacute ruminal acidosis (SARA) by inhibiting the activation of the NOD1/NF-κB signaling pathway through the decrease of the iE-DAP concentration in the rumen fluid and plasma and HDAC3 expression. DNA methylation and chromatin remodeling also contributed to the anti-inflammatory effect of SB. © 2020 Society of Chemical Industry.

MiR-708 affords protective efficacy in anoxia/reoxygenation-stimulated cardiomyocytes by blocking the TLR4 signaling via targeting HMGB1

Ischemic heart disease is a proverbial and common cardiovascular disease, and constitutes a leading cause of disability and mortality globally. Myocardial ischemic/reperfusion (MI/R) injury is a highly orchestrated phenomenon that involves the excessive activation of high mobility group box 1 (HMGB1) signaling. In the present study, we sought to investigate the function of miR-708 in MI/R injury due to the predicted binding to HMGB1. Intriguingly, down-regulation of miR-708 and up-regulation of HMGB1 were observed in MI/R rat model and H9c2 cardiomyocytes exposed to hypoxia/reoxygenation (H/R) conditions. Dual luciferase reporter assays substantiated that HMGB1 was a direct target of miR-708. Moreover, miR-708 overexpression suppressed the mRNA and protein expression of HMGB1. Noticeably, elevation of miR-708 antagonized H/R-induced inhibition in cell viability; whilst, increased cell apoptosis evoked by H/R was restrained after miR-708 up-regulation. Simultaneously, miR-708 elevation suppressed H/R exposure-increased lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) generation, but elevated the activity of anti-oxidative stress superoxide dismutase (SOD). Additionally, H/R-increased production of pro-inflammatory cytokine TNF-α and IL-6 was offset following miR-708 overexpression. Moreover, enhancement of miR-708 inhibited H/R-evoked activation of the HMGB1-TLR4-NF-κB pathway by inhibiting the protein levels of HMGB1, TLR4 and p-p65 NF-κB. Specially, restoring this pathway offset the protective effects of miR-708 on H/R-induced cardiomyocyte injury. Together, these data indicate that miR-708 may protect against H/R-induced cardiomyocyte damage by directing targeting HMGB1 signaling, implying a promising therapeutic agent against ischemic heart disease including myocardial infarction.

NLRC5 alleviated OGD/R-induced PC12-cell injury by inhibiting activation of the TLR4/MyD88/NF-κB pathway.

ObjectiveTo assess the role of NOD-like receptor C5 (NLRC5; a major NLRC family protein that regulates immunity, inflammation and tissue fibrosis), in cerebral ischemia-reperfusion injury, characterized by inflammation and oxidative damage.MethodsBlood NLRC5 levels were assessed in neonates with cerebral ischemia and in healthy controls. A stable PC12 cell line was established that overexpressed or knocked down NLRC5. Inflammatory responses, apoptosis rate and oxidative damage in PC12 cells under oxygen-glucose deprivation/reperfusion (OGD/R) conditions were evaluated using enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and reactive oxygen species (ROS) assay.ResultsBlood NLRC5 levels were suppressed in neonates with cerebral ischemia. ELISAs showed that NLRC5 suppressed levels of tumour necrosis factor-α, interleukin (IL)-6, IL-1β, ROS and superoxide dismutase in OGD/R-treated PC12 cells. Furthermore, NLRC5 overexpression was associated with reduced apoptosis rate in PC12 cells treated by OGD/R. Overexpression of NLRC5 also inhibited levels of toll-like receptor (TLR)4, myeloid differentiation primary response protein MyD88 (MyD88) and phosphorylated nuclear factor kappa B-transcription factor p65 (NF-κB p-p65) in PC12 cells, and decreased nuclear levels of NF-κB p-p65.ConclusionNLRC5 alleviated inflammatory responses, oxidative damage and apoptosis in PC12 cells under OGD/R conditions by suppressing activation of the TLR4/MyD88/NF-κB pathway.

Ligustilide modulates oxidative stress, apoptosis, and immunity to avoid pathological damages in bleomycin induced pulmonary fibrosis rats via inactivating TLR4/MyD88/NF-KB P65.

BackgroundPulmonary fibrosis (PF) is a fatal disease with increasing incidence. Ligustilide (LIG) has been shown to inhibit oxidative stress, apoptosis, and inflammation. Here we investigated the possible effect of LIG on bleomycin-induced PF in Sprague-Dawley rats.MethodsPF rats were set up through a single endotracheal injection of bleomycin (5 mg/kg). Then rats were treated with 20, 40, and 80 mg/kg LIG for four weeks, and the effects were estimated.ResultsOverall, LIG significantly improved ventilation and reduced hyperplasia, and treatment of LIG reduced fibrosis as indicated by Masson staining and reduced expression of transforming growth factor-beta (TGF-β), Fibronectin, and alpha-smooth muscle actin (α-SMA). Oxidative stress was induced with bleomycin while inhibited with LIG, as showed with rebalanced serum lactate dehydrogenase (LDH), and tissue superoxide dismutase (SOD), glutathione peroxidase (GSH) and malondialdehyde (MDA). Apoptosis was further inhibited with LIG, as shown with Terminal dUTP nick-end labeling (TUNEL) staining and expression of Caspase-3, Caspase-9, Bax, and Bcl-2. Th1/Th2 balance was also rebuilt as evaluated with CD4 and IFNγ/IL-4 labeled flow cytometry of peripheral blood mononuclear cells (PBMCs) and expression of inducible nitric oxide synthase (iNOS) and IL-10 in the serum and lung. Protein expression of Toll-like receptor 4 (TLR4), HSP60-TLR4-myeloid differentiation factor 88 (Myd88) and nuclear factor-kappa B (NF-κB) p-P65/P65 was significantly reduced with LIG treatment. All the effects of LIG exhibited in a dose-dependent way.ConclusionsLIG improved bleomycin-induced PF with improved ventilation, reduced fibroblast, reduced oxidative stress and apoptosis, and rebalanced Th1/Th2 immunity, through TLR4/MyD88/NF-κB P65 signaling.

Liver injury in septic mice were suppressed by a camptothecin-bile acid conjugate via inhibiting NF-κB signaling pathway

Background and objectivesSepsis is a life-threatening organ dysfunction syndrome arising from uncontrolled inflammatory responses. Liver injury is a crucial factor for the prognosis of sepsis. Camptothecins (CPTs) have been reported to suppress the inflammatory response induced by sepsis. G2, a CPT-bile acid conjugate, has been demonstrated the property of liver targeting in our previous research. This study aimed to research the effects of G2 on liver injury induced by cecal ligation and puncture (CLP). MethodsC57BL/6 mice were subjected to CLP surgery, and effects of G2 on liver damage and survival rates of CLP-induced mice were evaluated. To detect the related markers of hepatic injury or neutrophil infiltration, inflammatory cytokines and protein levels, hematoxylin-eosin staining assay, corresponding Detection Kits assay, ELISA and Western blot analysis were performed. ResultsIntraperitoneal administration of G2 reduced liver injury and enhanced the survival rates in mice with sepsis. Treatment with G2 decreased the levels of hepatic injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of mice induced by CLP. The hepatic level of neutrophil infiltration marker myeloperoxidase (MPO) was reduced in G2 administration group. And the levels of serum inflammatory cytokines, including Tumor Necrosis Factor-α (TNFα), Interleukin-6 (IL-6) and IL-1β, were decreased by G2. Furthermore, the results of Western blot analysis indicated that G2 suppressed the up-regulation of NF-κB p-P65 and p-IκBα. It suggested that G2 suppressed the activation of NF-κB signaling pathway. ConclusionG2 alleviated sepsis-induced liver injury via inhibiting the NF-κB signaling pathway.

Semen Brassicae reduces thoracic aortic remodeling, inflammation, and oxidative damage in spontaneously hypertensive rats

BackgroundThis study aimed to evaluate the role of Semen Brassicae, a common Traditional Chinese Medicine, in the treatment of hypertension. MethodsSpontaneously hypertensive rats (SHRs) were divided into five groups and were gavaged with either distilled water, water-decocted solution from Semen Brassicae (0.5, 1 or 2 g/kg), or nifedipine (2.7 mg/kg). Normal rats gavaged with distilled water were used as a control. Systolic (SBP) and diastolic blood pressure (DBP) were measured using a non-invasive method. After 8 weeks of administration, all animals were anesthetized. Abdominal aortic serum was collected to measure serum factors; the thoracic aorta was collected for hematoxylin and eosin staining and western blot analysis. ResultsBoth SBP and DBP were significantly decreased after Semen Brassicae treatment. Endothelin-1 and angiotensin II levels in abdominal aortic serum, as well as the levels of inflammatory factors interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha, were significantly decreased after Semen Brassicae treatment. The wall thickness of the thoracic aorta was significantly reduced after Semen Brassicae treatment. Nitric oxide level and the activity of superoxide dismutase and glutathione peroxidase were significantly increased, and malondialdehyde level was significantly decreased in the abdominal aortic serum after Semen Brassicae treatment. Semen Brassicae treatment increased the levels of peroxisome proliferator-activated receptor gamma and IκB-α and decreased the levels of intercellular adhesion molecule 1, monocyte chemoattractant protein-1, von Willebrand factor, p-IκB-α and p-p65 NF-κB. ConclusionsIn conclusion, water-decocted solution from Semen Brassicae can decrease blood pressure, improve vascular remodeling, and attenuate oxidative stress and inflammation in SHRs.

Design and development of spirulina polysaccharide-loaded nanoemulsions with improved the antitumor effects of paclitaxel

Aims: In this study, we prepared spirulina polysaccharides into spirulina polysaccharide-loaded nanoemulsions (SPS-NEs), and determined the antitumor effect of SPS-NEs, when combined with paclitaxel (PTX).Methods: SPS-NEs were prepared by a phase transformation method. The Characterisation and stability of SPS-NEs was measured. The antitumor effect of SPS-NEs combined with PTX was determined by S180 cells or RAW 264.7 macrophages and S180 tumour-bearing mice.Results: SPS-NEs were spherical and stable, the particle size of SPS-NEs was 84.6 ± 3.31 nm, PDI = 0.235 ± 0.02. PTX + SPS-NEs exhibited a much greater toxicity against RAW 264.7 cells than PTX. PTX + SPS-NEs increased the release of NO, IL-6 and TNF-α, and the expression of p-p65 NF-κB, p-I-κB, TLR4. In addition, PTX + SPS-NEs significantly inhibited tumour growth by 72.82% and increased the secretion of serum IL-2, TNF-α and IFN-γ.Conclusions: SPS-NEs can regulate immunity through TLR4/NF-κB signalling pathways, which enhances the anti-tumour effect of PTX.

IL-38 alleviates the inflammatory response and the degeneration of nucleus pulposus cells via inhibition of the NF-κB signaling pathway in vitro

Previous studies have suggested that the inflammatory response contributes to the onset of intervertebral disc degeneration (IVDD). Interleukin (IL)-38, a newly discovered cytokine of the IL-1 family, has been demonstrated to play an anti-inflammatory role in autoimmune diseases, such as Crohn’s disease, rheumatoid arthritis and psoriasis. However, whether IL-38 participates in the pathogenesis of IVDD remains unknown. In this study, human disc tissues from IVDD patients and rat disc tissues from an IVDD model were collected to measure the expression of IL-38 in the IVDD groups and the control groups by western blot and immunohistochemical staining. To further determine the role of IL-38 in IVDD, human nucleus pulposus cells (HNPCs) were stimulated with TNF-α to generate an in vitro model of inflammation to mimic the local inflammatory environment of the lumbar disc. The inflammatory response and HNPC degeneration markers were measured after stimulation with TNF-α and IL-38. IL-38 was upregulated in both the human and rat degenerated disc tissues compared with the control tissues. In vitro, IL-38 significantly decreased the TNF-α-induced expression of IL-1β, IL-6, COX-2, MMP-13 and ADAMTS-5 in the HNPCs, and IL-38 also alleviated the TNF-α-induced reductions in type II collagen and aggrecan. Moreover, IL-38 inhibited the activation of the NF-κB signaling pathway in the HNPC-based model of inflammation by reducing the expression level of the NF-κB P-P65 protein. In conclusion, IL-38 could alleviate the inflammatory response and HNPC degeneration in vitro via the inhibition of the NF-κB signaling pathway. These results suggest that IL-38 may be a new strategy for the treatment of IVDD.

Chanling Gao Attenuates Bone Cancer Pain in Rats by the IKKβ/NF-κB Signaling Pathway.

Cancer pain is one of the most common and serious symptoms of cancer patients. At present, the agents used for the prevention or treatment of cancer pain do not act with optimal safety and efficacy. The nuclear factor kappa B (NF-κB) signaling pathway and its downstream inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) play an important regulatory role in the developmental process of cancer pain. IKKβ is a key molecule of the IκB (IKK) kinase that propagates cellular responses to inflammation. Previous studies have shown that phosphorylation and degradation of the IκBα protein promotes the activation of NF-κB and the expression of TNF-α, IL-1β, and IL-6, participating in the formation and development of cancer pain. Chanling Gao (CLG) is a compound preparation of traditional Chinese medicine. It contains specific functions, namely nourishing Yin, activating blood circulation and relieving pain and dysfunction syndrome. It is used in the treatment of a variety of pain disorders including cancer-induced bone pain (CIBP), which has a certain relief effect. However, its mechanism of action still remains unclear. In the present study, a rat model of tibia CIBP was successfully established using the Walker 256 breast cancer cell line. The IKKβ/NF-κB signaling pathway and its related factors TNF-α, IL-1β, and IL-6 were used as the entry points to explore the effect of CLG on CIBP and their possible mechanisms of action. The results indicated that CLG improved the body mass of the CIBP rat model and increased the pain threshold in rats. CLG significantly inhibited the degradation of IκBα and the levels of p-IκBα, p-IKKβ, and p-p65 NF-κB proteins in the spinal cord of CIBP rats, inhibiting the contents of TNF-α, IL-1β, and IL-6. Therefore, we conclude that the analgesic effect of CLG in this rat model of CIBP may be related to the inhibition of the IKKβ/NF-κB signaling pathway and the reduction of synthesis and release of TNF-α, IL-1β, and IL-6.