NF κB Research Articles

Akirin2 plays an important role in protecting Megalobrama amblycephala from Aeromonas hydrophila infection

Akirins are highly conserved, small, nuclear proteins that have been recognized as key regulators of multiple gene expression patterns and play important roles in immune responses. Previous studies on Akirins have mainly focused on the model species Drosophila and mice, while there have been few studies on fish. In the present study, two Akirin genes, Akirin1 and Akirin2, were identified from Megalobrama amblycephala. The open reading frames (ORFs) of M. amblycephala Akirin1 and Akirin2 were 570 bp and 558 bp in length, encoding 189 and 185 amino acids, respectively. Quantitative real-time PCR analysis indicated that both Akirin1 and Akirin2 mRNA were ubiquitously expressed in all 10 examined tissues with different expression patterns. After Aeromonas hydrophila infection, the mRNA levels of Akirin1, Akirin2, nuclear factor κB subunit p50 (NF-κB p50) and nuclear factor κB subunit p65 (NF-κB p65) were significantly induced in the liver and spleen. Knockdown and overexpression of Akirins in L8824 cells showed that Akirin2 positively regulated the expression of NF-κB p50, NF-κB p65, liver-expressed antimicrobial peptide 1 (LEAP-1) and liver-expressed antimicrobial peptide 1 (LEAP-2), while Akirin1 had no significant effect on the expression of these genes above. In addition, the recombinant M. amblycephala Akirin1 (rAkirin1) and Akirin2 (rAkirin2) proteins were successfully obtained and intraperitoneal injection of them could help protect M. amblycephala from A. hydrophila infection, with rAkirin2 having a better protective effect than rAkirin1. Furthermore, in vitro glutathione S-Transferase (GST) pull-down assays showed that M. amblycephala Akirin2 could bind to the IPT domain of NF-κB p50 and luciferase assays showed that NF-κB p65 could promote the activities of LEAP-1 and LEAP-2 promoters, which suggested that Akirin2, rather than Akirin1, could interact with the IPT domain of NF-κB p50 and induce the transcription of downstream antimicrobial peptides LEAP-1 and LEAP-2 though NF-κB p65 to enhance the immunity of M. amblycephala to A. hydrophila infection. All the above results indicated that M. amblycephala Akirin2 plays an essential role in the defense against A. hydrophila via the NF–κB signaling pathway, providing a theoretical basis for in-depth study of the innate immune mechanism of fish and promotion of healthy aquaculture.

A Water-Soluble Polysaccharide from the Fibrous Root of Anemarrhena asphodeloides Bge. and Its Immune Enhancement Effect in Vivo and in Vitro.

Background The fibrous roots of Anemarrhena asphodeloides Bge. (FRAAB) are byproducts of the rhizome of Anemarrhena asphodeloides. Some studies have revealed secondary metabolic small molecules in FRAAB, but there are few reports on the polysaccharides of FRAAB (PFRAAB). Aim of the Study. The present study aimed to investigate the preliminary characterization and underlying mechanism of immune stimulation of PFRAAB. Materials and Methods The crude polysaccharide of FRAAB was obtained by hot water extraction and alcohol precipitation, and PFRAAB was purified by a diethylaminoethyl-52 (DEAE-52) cellulose chromatographic column and graphene dialysis membrane. The preliminary characterization of PFRAAB was studied by ultraviolet (UV) scanning and Fourier Transform Infrared Reflection (FTIR). The molecular weight and composition of PFRAAB were analysed by high-performance gel permeation chromatography (HPGPC) and high-performance liquid chromatography (HPLC), respectively. The immune stimulation of PFRAAB was investigated by using cyclophosphamide- (CCP-) treated mice and RAW264.7 cells. Results A water-soluble PFRAAB was obtained with a molecular weight of 115 kDa and was mainly composed of arabinose (ara), galactose (gal), glucose (glc), and mannose (man). Compared with CCP-induced mice, PFRAAB significantly (p < 0.05 or p < 0.01) increased the spleen and thymus index, ameliorated injury to the spleen and thymus, and evaluated immunoglobulin levels. In addition, PFRAAB also increased the secretion of nitric oxide (NO), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), and IL-6 in RAW264.7 cells and upregulated the expression of toll-like receptor 4 (TLR4), Myd88, nuclear factor kappa-B (NF-κB) P65, p–NF–κB P65, IKB-α, and p-IKB-α. Conclusion PFRAAB possesses immune stimulation activity and can be used as a potential resource for immune-enhancing drugs. Our present study provides a scientific basis for the comprehensive development of Anemarrhena asphodeloides medicinal plant resources.

Xianglian Pill attenuates ulcerative colitis through TLR4/MyD88/NF-κB signaling pathway

Ethnopharmacological relevanceXianglian Pill (XLP) is a classical Chinese medicine prescription applied for controlling ulcerative colitis (UC). Whereas, the underlying mechanism remains unclear. Aim of the studyThe present work was aimed to investigate the mechanism of XLP in dextran sulfate sodium (DSS)-induced UC via the Toll Like Receptor 4 (TLR4)/Myeloid Differentiation factor 88 (MyD88)/Nuclear Factor kappa-B (NF-κB) signaling in mice. Materials and methodsThe major components of XLP were detected by high-performance liquid chromatography-diode array detection (HPLC-DAD). The ulcerative colitis model was induced by DSS in mice. 5-Amino Salicylic Acid (5-ASA) group and XLP group were intragastrically treated. Disease activity index (DAI) and colon length were monitored and hematoxylin-eosin (HE) staining was conducted. Gasdermin D (GSDMD)-N and TLR4 expressions in colon tissues were visualized by immunofluorescence. TLR4 mRNA was measured by Real Time Quantitative PCR (RT-qPCR). The expressions of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), active-caspase-1, GSDMD-N, TLR4, MYD88, NF-κB, p–NF–κB, and the ubiquitination of TLR4 in colon tissues were detected by Western blot. Myeloperoxidase (MPO) enzyme activity was examined and serum inflammatory factors Interleukin (IL)-1β, IL-6, Tumor Necrosis Factor-α (TNF-α), and IL-18 were determined by Enzyme-linked Immunosorbent Assay (ELISA). TLR4-/- mice were applied for verifying the mechanism of XLP attenuated DSS symptoms. ResultsThe XLP treatment extended colon length, reduced DAI, and attenuated histopathological alteration in DSS-induced mice. XLP administration suppressed MPO activity and reduced the content of IL-1β, IL-6, TNF-α and IL-18 in serum. XLP also inhibited the expression levels of GSDMD-N, TLR4, NLRP3, active-caspase-1, MyD88, p–NF–κB/NF-κB in colon tissues of DSS-induced mice. TLR4-/- mice proved that TLR4 was involved in XLP-mediated beneficial effect on DSS-induced ulcerative colitis. ConclusionsXLP might treat ulcerative colitis by regulating the TLR4/MyD88/NF-κB signaling pathway.

Shufeng Jiedu capsule inhibits inflammation and apoptosis by activating A2AAR and inhibiting NF-κB to alleviate LPS-induced ALI

Ethnopharmacological relevanceShufeng Jiedu capsule (SFJDC) is a pure form of traditional Chinese medicine (TCM) that contains eight medicinal plants. Known for its anti-inflammatory and antipyretic effects, it is mostly used to treat upper respiratory tract infections and other infectious diseases, such as colds, pharyngitis, laryngitis, and tonsillitis. Both acute lung injury (ALI) and COVID-19 are closely related to lung damage, primarily manifesting as lung inflammation and epithelial cell damage. However, whether SFJDC can improve ALI and by what mechanism remain unclear. The purpose of this study was to explore whether SFJDC could be used as a prophylactic treatment for COVID-19 by improving acute lung injury. Aim of the studyThe purpose of this study was to determine whether SFJDC could protect against ALI caused by lipopolysaccharide (LPS), and we wanted to determine how SFJDC reduces inflammation and apoptosis pharmacologically and molecularly. Materials and methodsPreadministering SFJDC at 0.1 g/kg, 0.3 g/kg, or 0.5 g/kg for one week was followed by 5 mg/kg LPS to induce ALI in mice. Observations included the study of lung histomorphology, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) secretion, as well as the ratio of lung wet/dry weights. In addition, RAW264.7 cells were treated for 24 h with 1 μg/mL LPS after being pretreated for 1 h with 0.5 mg/mL SFJDC. In the samples, we detected TNF-α, IL-1β, and IL-6. Cell apoptosis was detected by stimulating A549 cells for 24 h with RAW264.7 supernatant. Both in vitro and in vivo, the levels of A2A adenosine receptor (A2AAR), PKA, IκB, p-IκB, NF-κB P65 (P65), p–NF–κB P65 (p-P65), cleaved caspases-3 (Cc3), Bcl-2 associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2) proteins were determined using Western blot analysis. ResultsLung tissue morphology was improved as SFJDC decreased cytokine secretion, the ratio of lung wet/dry weights, and lung tissue secretion of proinflammatory cytokines. The expression of A2AAR was increased by SFJDC, and the phosphorylation of NF-κB was inhibited. TUNEL staining and flow cytometry showed that SFJDC inhibited apoptosis by reducing the expression of Cc3 and the ratio of Bax/Bcl-2. ConclusionsAccording to the results of this study, SFJDC can reduce inflammation and inhibit apoptosis. A2AAR activation and regulation of NF-κB expression are thought to make SFJDC anti-inflammatory and anti-apoptotic. A wide range of active ingredients may result in an anti-inflammatory and antipyretic effect with SFJDC.

Tollip suppresses MyD88-mediated NF-κB activation by enhancing MyD88 ubiquitination levels in large yellow croaker (Larimichthys crocea)

Toll-interacting protein (Tollip) plays an important role in the innate immune response by negative regulation of the TLR-IL-1R signaling pathway. MyD88 serves as a universal adaptor in TLR-mediated NF-κB activation. However, the regulation mechanisms of Tollip in piscine MyD88-mediated NF-κB activation is largely unknown. In the present study, the cDNA sequence of LcTollip was identified from the large yellow croaker (Larimichthys crocea). The putative LcTollip protein encoded 275 amino acid residues, containing a N-terminal TBD domain, a central C2 domain, and a C-terminal CUE domain. Quantitative PCR showed that the most predominant constitutive expression of LcTollip was detected in spleen. In addition, LcTollip transcripts enhanced significantly after LPS and poly I:C challenge (P < 0.05). Cellular localization revealed that LcTollip existed in the cytoplasm and nucleus. Furthermore, the overexpression plasmids of wild type LcTollip as well as its six domain truncated mutants of LcTollip were constructed by overlap PCR. Dual luciferase analysis showed that NF-κB activation could not be induced by overexpression of LcTollip or its domain truncated mutants alone. However, the LcMyD88-induced–NF–κB activation was significantly suppressed by overexpression with LcTollip, and the truncated mutants LcTollip-ΔTBD, LcTollip-ΔC2, LcTollip-ΔCUE and LcTollip-ΔTBDΔCUE while not by LcTollip-ΔLR and LcTollip-ΔTBDΔC2. Moreover, co-immunoprecipitation (Co-IP) assay revealed that the interaction between LcTollip and LcMyD88 was through CUE domain. More interesting, IP and immunoblotting examination of HEK293T cells co-transfected with LcMyD88, LcTollip and HA-ubiquitin showed that LcMyD88 induced a dose-dependent de-ubiquitination of LcTollip while LcTollip enhanced a dose-dependent ubiquitination of LcMyD88. However, protein degradation investigation displayed that the proteolysis and ubiquitination of LcMyD88 were not connected. Our findings suggested that the LcTollip might involve in negative regulation TLR pathway by suppressing LcMyD88-mediated immune activation and improving the ubiquitination level of LcMyD88.

Hepatocyte Nuclear Factor 1α Proinflammatory Effect Linked to the Overexpression of Liver Nuclear Factor-κB in Experimental Model of Chronic Kidney Disease.

Chronic kidney disease (CKD) is associated with low-grade inflammation that activates nuclear factor–κB (NF–κB), which upregulates the expression of numerous NF–κB responsive genes, including the genes encoding IL-6, ICAM-1, VCAM-1, and MCP-1. Herein, we found the coordinated overexpression of genes encoding RelA/p65 (a subunit of NF–κB) and HNF1α in the livers of chronic renal failure (CRF) rats—an experimental model of CKD. The coordinated overexpression of RelA/p65 and HNF1α was associated with a significant increase in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. A positive correlation between liver RelA/p65 mRNA levels and a serum concentration of creatinine and BUN suggest that RelA/p65 gene transcription is tightly related to the progression of renal failure. The knockdown of HNF1α in the HepG2 cell line by siRNA led to a decrease in Rel A/p65 mRNA levels. This was associated with a decrease in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. The simultaneous repression of HNF-1α and RelA/p65 by clofibrate is tightly associated with the downregulation of IL-6, ICAM-1, VCAM-1, and MCP-1 gene expression. In conclusion, our findings suggest that NF–κB could be a downstream component of the HNF1α-initiated signaling pathway in the livers of CRF rats.

Improved biopharmaceutical performance of antipsychotic drug using lipid nanoparticles via intraperitoneal route

This study aims to develop, characterize and examine Olanzapine-loaded solid lipid nanocarriers (OLAN-SLNs) for effective brain delivery. OLAN has poor water solubility and low penetration through blood brain barrier (BBB). Herein, OLAN-SLNs were fabricated using high pressure homogenization (HPH) method followed by their investigation for particle properties. Moreover, in-vitro release and in-vivo pharmacokinetics profiles of OLAN-SLNs were compared with pure drug. Anti-psychotic activity was performed in LPS-induced psychosis mice model. Furthermore, expressions of the COX-2 and NF-κB were measured trailed by histopathological examination. The optimized formulation demonstrated nano particle size (149.1nm) with rounded morphology, negative zeta potential (-28.9mV), lower PDI (0.334) and excellent entrapment efficiency (95%). OLAN-SLNs significantly retarded the drug release and showed sustained release pattern as compared to OLAN suspension. Significantly enhanced bioavailability (9-fold) was demonstrated in OLAN-SLNs when compared with OLAN suspension. Behavioral tests showed significantly less immobility and more struggling time in OLAN-SLNs treated mice group. Additionally, reduced expression of COX-2 and -NF κB in brain were found. Altogether, it can be concluded that SLNs has the potential to deliver active pharmaceutical ingredients to brain, most importantly to enhance their bioavailability and antipsychotic effect, as indicated for OLAN in this study.

L-theanine protects rat kidney from D-galactose-induced injury via inhibition of the AGEs/RAGE signaling pathway

As the irreversible products of the non-enzymatic reduction of sugars and the amino groups of proteins or peptides, advanced glycation end products (AGEs) are metabolized and excreted via the kidneys. However, if AGEs are not metabolized, they are deposited in the kidneys and bind to AGE receptors (RAGE), which can induce various pathological changes, including oxidative stress, apoptosis, and inflammation. This study used the D-galactose (DG)-induced rat model to explore the potential role and mechanism of L-theanine in inhibiting AGEs/RAGE-related signaling pathways in renal tissues. L-theanine increased the activities of glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) while downregulating the contents of malondialdehyde (MDA) and AGEs in renal tissues induced by DG (P < 0.05). By inhibiting the upregulation of RAGE protein expression attributed to AGEs accumulation (P < 0.05), L-theanine downregulated phosphorylated nuclear factor (p–NF–κB (p65)), Bax, and cleaved-caspase-3 expression and increased Bcl-2 protein expression (P < 0.05), thereby alleviating the oxidative stress damage and reducing the inflammation and cell injury induced by DG. In addition, the Congo red staining section of renal tissue also showed that the natural product L-theanine can protect against AGEs-induced renal damage in DG-induced rat model.

C49 SACUBITRIL–VALSARTAN IMPROVES LONGITUDINAL STRAIN AND EJECTION FRACTION IN MICE TREATED WITH DOXORUBICIN THROUGH NLRP3, MYD88 PATHWAYS RESULTING IN A REDUCTION OF MYOCARDIAL IL–1Β, IL–6, TNF–Α, G–CSF AND GM–CSF LEVELS

Abstract Introduction Doxorubicin–mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in breast cancer patients. Sacubitril–valsartan (LCZ 696) is a combination drug, made up of neprilysin inhibitor sacubitril and angiotensin II receptor blocker valsartan, used for the treatment of heart failure in patients with a reduced ejection fraction. Purpose We hypothesized that LCZ 696, administered during doxorubicin, could improve cardiac function and cardiac inflammation in preclinical models Methods Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin (200 nM) alone or in combination with LCZ–696 (100 mM) for 72 h. After the incubation period, we performed the following tests: cell viability, apoptosis and necrosis; expression of malondialdehyde and 4–hydroxynonenal and concentration of intracellular Ca2+. Moreover, pro–inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; mTORC1 Fox01/3a; NF–κB). C57Bl/6 mice were untreated (Sham, n = 6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n = 6), LCZ–696 at 60 mg/kg (LCZ, n = 6) or doxorubicin combined to LCZ–696 (DOXO–LCZ, n = 6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100). Cardiac tissue expression of NLRP3 inflammasome, Myd88, NF–kB and 13 chemokines (IL–1α, IL–1β, IL–2, IL–4, IL–6, IL–10, IL–12, IL17–α, IL–18, IFN–γ, TNF–α, G–CSF, and GM–CSF) were quantified. Results LCZ–696 exerts cardioprotective effects, enhancing cell viability of 48–54.6% compared to only doxorubicin–treated cells (p &amp;lt; 0,001 for all); LCZ 696 decreased NLRP3, MyD88 and NF–kB expression in cardiac cells. In preclinical study, LCZ 696 improved significantly the EF and prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin. A reduced expression of NLRP3, MyD88 and NF–kB in cardiac tissues was seen in DOXO–LCZ group compared to DOXO mice (p &amp;lt; 0.001). Cardiac expression of IL–1β, IL–6, TNF–α, G–CSF and GM–CSF were significantly reduced after treatment with LCZ–696 indicating anti–inflammatory properties. Conclusion LCZ–696 exerts direct beneficial effects in cardiomyocytes exposed to doxorubicin. In preclinical models, LCZ–696 reduced inflammation and cytokine expression involved in doxorubicin–mediated cardiotoxicity.

Cyclosporin A up-regulated thromboxane A2 receptor through activation of MAPK and NF-κB pathways in rat mesenteric artery

Cyclosporin A (CsA) is an immunosuppressant used in transplantation patients and inflammatory diseases. CsA-induced local vasoconstriction can lead to serious side effects including nephrotoxicity and hypertension. However, the underlying mechanisms are not fully understood.Mesenteric artery rings of rats were cultured with CsA and specific inhibitors for mitogen-activating protein kinases (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. A sensitive myograph recorded thromboxane (TP) receptor-mediated vasoconstriction. Protein levels of key signaling molecules were assessed by Western blotting. The results show that CsA up-regulated the TP receptor expression with the enhanced vasoconstriction in a dose- and time-dependent manner. Furthermore, the blockage of MAPKs or NF-κB activation markedly attenuated CsA-enhanced vasoconstriction and the TP receptor protein expression.Rats subcutaneously injected with CsA for three weeks showed increased blood pressure in vivo and increased contractile responses to a TP agonist ex vivo. CsA also enhanced TP receptor, as well as p-ERK1/2, p-p38, p- IκBα, p–NF–κB P65 protein levels and decreased IκBα protein expression, demonstrating that CsA induced TP receptor enhanced-vasoconstriction via activation of MAPK and NF-κB pathways.In conclusion, CsA up-regulated the expression of TP receptors via activation of MAPK and NF-κB pathways. The results may provide novel options for prevention of CsA-associated hypertension.

Diversified Polyketides With Anti-inflammatory Activities From Mangrove Endophytic Fungus Daldinia eschscholtzii KBJYZ-1.

In total, five new polyketide derivatives: eschscholin B (2), dalditone A and B (3 and 4), (1R, 4R)-5-methoxy-1,2,3,4-tetrahydronaphthalene-1,4-dio (5), and daldilene A (6), together with 10 known as analogs (1, 7–15) were isolated from the mangrove endophytic fungus Daldinia eschscholtzii KBJYZ-1. Their structures and absolute configurations were established by extensive analysis of NMR and HRESIMS spectra data combined with ECD calculations and the reported literature. Compounds 2 and 6 showed significant cell-based anti-inflammatory activities with IC50 values of 19.3 and 12.9 μM, respectively. In addition, western blot results suggested that compound 2 effectively inhibits the expression of iNOS and COX-2 in LPS-induced RAW264.7 cells. Further molecular biology work revealed the potential mechanism of 2 exerts anti-inflammatory function by inactivating the MAPK and NF–κB signaling pathways.

Anti-inflammatory Effect of Predimenol, A Bioactive Extract from Phaleria macrocarpa, through the Suppression of NF-κB and COX-2.

Inflammation is the response to the reaction of any type of bodily injury by elevating cellular metabolism and releasing soluble mediators. It is also a contributing factor of pain. Predimenol, which has previously been known as DLBS1442, is a bioactive extract from Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae). It can be an alternative treatment for pain relief, especially for long-term use. The objective of this study is to evaluate the anti-inflammatory activities of predimenol through the evaluation of several parameters involved in the inflammatory pathway. Cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-  (TNF-), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), and nuclear factor B (NF-B) were observed after 24 h exposure of predimenol (0-180 µg/mL) to lipopolysaccharides (LPS)-activated RAW 264.7 cell. The inflammatory markers were measured using nitric oxide (NO) assay and enzyme-linked immunosorbent assay (ELISA) for COX-2 inhibitor assay. The gene expressions of TNF-α, IL-1β, IL-2 and IL-6 were quantified using the polymerase chain reaction (PCR) method. Western blotting was applied to detect phosphorylated IB kinase (IKK) protein to confirm the activation of NF-κB. Our study showed a similar mechanism with most non-steroidal anti-inflammatory drugs (NSAIDs). Predimenol consistently downregulated the expression of iNOS and inhibited COX-2 activity. Moreover, predimenol significantly inhibited the LPS-induced production of NO, TNF-α, IL-1β, IL-2 and IL-6. Down-regulation of these markers was suggested due to the reduction of NF-κB transcription level and activation by predimenol. Predimenol exhibits anti-inflammatory activities through NF-kB inactivation-mediated COX-2 suppression, which may suggest that predimenol is a potential analgesic and anti-inflammatory agent.

Astaxanthin ameliorates hyperglycemia induced inflammation via PI3K/Akt–NF–κB signaling in ARPE-19 cells and diabetic rat retina

Astaxanthin has been reported to possess anti-inflammatory effect but the exact mechanism in protecting the retinal pigment epithelial (RPE) cells is not clear. Hence, we hypothesized that astaxanthin could protect RPE by inhibiting ROS-mediated inflammation. The purpose of this study is to understand the retinal protective mechanism of astaxanthin in modulating hyperglycemia (HG) induced inflammation in ARPE-19 cell and diabetic rat retina. ARPE-19 cells were treated with 30 mM glucose to induce hyperglycemia whereas diabetes was induced in rats with streptozotocin followed by astaxanthin treatment. The level of oxidative stress markers, antioxidant enzyme activity, inflammatory markers (NF-κB, TNF-α, ICAM-1), signaling mediators (PI3K, p-Akt) and nuclear translocation of NF-κB were analyzed in ARPE-19 cells and rat retina. HG-mediated ROS generation and lipid peroxidation were declined upon astaxanthin treatment in ARPE-19 cells. Similarly, astaxanthin treatment found to reduce the elevated levels of nitric oxide, protein carbonyl, and lipid peroxides in diabetic group. Astaxanthin restored the activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione transferase in serum and retina of diabetic rats. NF-κB, TNF-α, and ICAM-1 levels were higher in HG-treated ARPE-19 cells and diabetic retina compared to control group, whereas astaxanthin treatment lowered their expression. PI3K and p-Akt were higher in high glucose treated ARPE-19 cells and diabetic retina. NAC, LY294002 and PDTC treatment resulted in reduced nuclear translocation of NF-κB and decreased expression of inflammatory markers in HG treated ARPE-19 cells. Thus, we conclude that astaxanthin protected the retinal cells from HG-induced inflammation by modulating NF-κB through ROS-PI3K/Akt signaling cascade.

Cathepsin K contributed to disturbed flow-induced atherosclerosis is dependent on integrin-actin cytoskeleton–NF–κB pathway

Atherosclerosis is a chronic inflammatory disease, occurring preferentially in bifurcation, branching, and bending of blood vessels exposed to disturbed flow. Disturbed flow in atheroprone areas activates elevated proteases, degrading elastin lamellae and collagenous matrix, resulting in endothelial dysfunction and vascular remodeling. As a mediator for extracellular matrix protein degradation, cathepsin K (CTSK) was directly regulated by hemodynamics and contributed to atherosclerosis. The mechanism of CTSK responding to disturbed flow and contributing to disturbed flow-induced atherosclerosis is unclear. In this study, the partial carotid ligation model of mice and in vitro disturbed shear stress model were constructed to explore the contribution and potential mechanism of CTSK in atherosclerosis. Our results indicated that CTSK elevated in the disturbed flow area in vivo and in vitro along with endothelial inflammation and atherogenesis. Additionally, the expression of integrin αvβ3 was upregulated in these atheroprone areas. We found that inhibition of the integrin αvβ3-cytoskeleton pathway could significantly block the activation of NF-κB and the expression of CTSK. Collectively, our findings unraveled that disturbed flow induces increased CTSK expression, and contributes to endothelial inflammation and vascular remodeling, leading to atherogenesis eventually. This study is helpful to provide new enlightenment for the therapy of atherosclerosis.

Total flavonoids of sea buckthorn (Hippophae rhamnoides L.) improve MC903-induced atopic dermatitis-like lesions

Ethnopharmacological relevanceSea buckthorn (Hippophae rhamnoides L.) is popularly used as a herbal medicine and food additive in the world. Total flavonoids of Hippophae rhamnoides (TFH) are reported to have anti-inflammatory and immunomodulatory activities. AimThe effects of TFH on atopic dermatitis (AD)-like lesions induced by MC903 in mice was elucidated in the study. MethodsTo induce AD-like lesions, MC903 was adopted to apply repeatedly on the left ear in C57BL/6 mice. After induction of AD-like lesions, 0.5% and 1% TFH cream was applied topically on ears of mice once a day for 8 days. The degree of skin lesions was evaluated by macroscopical and histological methods. Expressions of filaggrin (FLG) was evaluated by Western blotting. Real-time polymerase chain reaction (qPCR) was adopted to detect the mRNA expression of thymic stromal lymphopoietin (TSLP), interferon (IFN)-γ, interleukin (IL-4), tumor necrosis factor (TNF)-α in skin lesions. In vitro, Cytokine Antibody Arrays were performed to measure production of cytokines in IFN-γ/TNF-α-treated HaCaT cells, Western blotting was employed to detect the expressions of p–NF–κB, p-ERK and p-P38. ResultsTopical application of TFH significantly improved the severity of dermatitis by inhibiting the infiltration of mast cell, increasing expression of FLG, decreasing the expressions of TNF-α, IL-4, IFN-γ and TSLP in skin lesions. TFH decreased the levels of IL-1α, IL-1β, IL-6, monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage-derived chemokine (MDC), platelet-derived growth factor (PDGF)-BB, thymus and activation regulated chemokine (TARC) in the supernatants of the HaCaT cells treated by IFN-γ/TNF-α. Furthermore, expressions of p–NF–κB, p-ERK and p-P38 were also decreased by TFH administration with dose dependent manner in HaCaT cells treated by IFN-γ/TNF-α. ConclusionsTopical application of TFH improved AD-like lesions in mice induced by MC903. Which exerted the effects of anti-inflammation and repairing skin barrier by regulating Th1/Th2 balance. This finding indicates that TFH is a novel potential agent for the external treatment of AD.

In Vitro and In Vivo Dendritic Cell Immune Stimulation Effect of Low Molecular Weight Fucoidan from New Zealand Undaria pinnatifida.

Low molecular weight fucoidan (LMWF) has been reported to have immunomodulation effects through the increase of the activation and function of macrophages. In this study, the regulating effect of LMWF from Undaria pinnatifida grown in New Zealand on dendritic cells (DCs) was investigated. We discovered that LMWF could stimulate DCs’ maturation and migration, as well as CD4+ and CD8+ T cells’ proliferation in vitro. We proved that this immune promoting activity is activated through TLR4 and its downstream MAPK and NF–κB signaling pathways. Further in vivo (mouse model) investigation showed that LMWF has a strong immunological boosting effect, such as facilitating the proliferation of immune cells and increasing the index of immune organs. These findings suggest that LMWF has a positive immunomodulatory effect and is a promising candidate to supplement cancer immunotherapy.

CRB2 enhances malignancy of glioblastoma via activation of the NF-κB pathway

Glioblastoma (GBM) is one of the most lethal types of primary brain tumors in adults with a median survival of less than 15 months. Although comprehensive clinical treatment strategies including surgical resection followed by radiotherapy and chemotherapy are widely applied, the prognosis for GBM patients remains dismal. The Nuclear Factor-κB (NF-κB) signaling pathway is a complex network linking extracellular stimuli to cell survival and proliferation, and aberrant activation of NF-κB signaling has been implicated in the propagation of a wide range of cancers. However, the underlying mechanism of NF-κB activation still requires further investigation. Here, we report that crumbs homolog 2 (CRB2) is markedly up-regulated in human GBM relative to non-tumor tissues or normal astrocytes. Clinically, enriched CRB2 could be observed in high grade glioma with IDH IDH wild-type and 1p19q co-deletion and implied poor outcome in GBM. Consistent with this, malignant characteristics of GBM cells including proliferation, migration, invasion and tumorigenesis were significantly suppressed by lentivirus knock-down of CRB2. Furthermore, exogenous overexpression of CRB2 enhanced the malignant biological signatures of GBM cells as well as therapy resistance to temozolomide (TMZ). To further investigate the molecular mechanisms responsible, bioinformatics analysis was performed using 3 public databases, with the result that CRB2 was found to correlate closely with tumor necrosis factor α (TNFα)–NF–κB signaling. Mechanistically, elevated CRB2 increased the phosphorylation of IκB-kinase α (IKKα), thus activating NF-κB via reduction of Ikβ protein. Taken together, these data suggest that CRB2 might be a reliable prognostic biomarker and potential therapeutic target for GBM.

Dapagliflozin exerts anti-inflammatory effects via inhibition of LPS-induced TLR-4 overexpression and NF-κB activation in human endothelial cells and differentiated macrophages

Evidence has demonstrated that a new class of anti-diabetic drugs, sodium-glucose co-transporter 2 (SGLT2) inhibitors, could exert beneficial effects on atherosclerotic complications of diabetes. Atherosclerosis is widely accepted as an inflammatory disease. Therefore, we aimed to assess the direct anti-inflammatory effects of SGLT2 inhibitors dapagliflozin (DAPA) on two cell types involved in the process of atherogenesis. Human umbilical vein endothelial cells (HUVECs) and macrophages were exposed to DAPA and lipopolysaccharide (LPS 20 ng/mL) for 24 h under normal (5.5 mmol/L, NG) or high glucose (25 mmol/L, HG) conditions. Then, levels of TLR-4/p–NF–κB, inflammatory cytokines, inflammation-related miR-146a and miR-155 as well as alteration in the ratio of M1/M2 macrophage polarization was assessed. DAPA (0.5 μM) could significantly attenuate LPS-induced TLR-4 overexpression (23.9% and 33.1% under NG and HG conditions in HUVECs and 53.3% and 52.4% under NG and HG states in macrophages, respectively). NF-κB p65 phosphorylation was also significantly decreased to 30.1% under NG condition in HUVECs and 51.9% and 34.5% under NG and HG states in macrophages by 0.5 μM DAPA. Moreover, DAPA elevated expression levels of anti-inflammatory miR-146a, while values of miR-155 decreased in those cells. DAPA also caused a shift from inflammatory M1 macrophages toward M2-dominant macrophages. These data suggest that regardless of glucose concentrations, DAPA could exert direct anti-inflammatory effects, at least partly, by inhibiting the expression of TLR-4 and activation of NF-κB along with the secretion of pro-inflammatory mediators.

Gender-based differences in neuroprotective effects of hydrogen gas against intracerebral hemorrhage-induced depression

BackgroundPost-stroke depression (PSD) severely affects recovery in patients with intracerebral hemorrhage (ICH). Although hydrogen gas (H2) exerts excellent neuroprotective effects in patients with ICH, there are sex-based differences in H2 efficacy in several diseases. Herein, we determined whether estrogen increases susceptibility to the neuroprotective effects of H2 in males with ICH-induced depression. MethodsA rodent model of ICH in the basal ganglia was established using autologous blood injection (30 μL). Mice were treated with 2.9% H2 for 2 h daily for 3 days post-ICH. Estrogen (1 mg/kg) was administered by subcutaneous injection daily for 3 days to male mice post-ICH. Thirty days post-ICH, PSD was evaluated by sucrose preference, forced swimming, and 3-chamber social tests. Following the completion of behavioral tests, levels of superoxide dismutase (SOD) and reactive oxygen species (ROS), astrocytic activation, phosphorylated (p)–NF–κB-positive astrocytes, p–NF–κB, p-IKKβ, IL-1β, and IL-6 expression were determined. ResultsCompared with female mice, H2 administration post-ICH exhibited fewer neuroprotective effects, including decreased sucrose consumption and time spent sniffing a novel mouse, increased immobility time, downregulated total SOD content, upregulated ROS content and p–NF–κB levels, and elevated astrocyte branches, whereas estrogen enhanced the neuroprotective effects of H2 in male mice. A reduced number of p–NF–κB-positive astrocytes, downregulated expression of p–NF–κB, p-IKKβ, IL-1β, and IL-6 in the amygdala were demonstrated in ICH-males treated with estrogen plus H2. ConclusionsEstrogen was responsible for increased H2 sensitivity in male mice with ICH. The underlying mechanism may be associated with the suppression of NF-κB signaling in astrocytes.

Cangma Huadu granules, a new drug with great potential to treat coronavirus and influenza infections, exert its efficacy through anti-inflammatory and immune regulation

Ethnopharmacological relevanceCoronavirus and influenza virus infection seriously threaten human health. Cangma Huadu Granules (CMHD) is an in-hospital preparation composed of eight traditional Chinese medicines (TCM), which has been clinically used against COVID-19 in China and may be a promising candidate for the treatment of influenza. However, the role of its treatment urgently needs to be studied.Aim of the study: To evaluate the therapeutic effects of CMHD on pneumonia induced by coronavirus (HCoV-229E) and influenza A virus (H1N1/FM1) in mice and explore its mechanism of anti-infection. Materials and methodsMice were infected with HCoV-229E or H1N1/FM1 virus through the nasal cavity. CMHD (12.1, 6.05 and 3.03 g/kg/d) or the positive control drugs were administered intragastrically. The lung index and histopathological changes were used to evaluate the therapeutic effect of CMHD. The expression of TNF-α, IL-1β, IL-6 and IL-4 in Serum and the proportion of CD4+ and CD8+ T lymphocytes in peripheral blood were detected to evaluate the anti-inflammatory and immune regulation effects of CMHD, respectively. Furthermore, the levels of p–NF–κBp65/ NF-κB p65, which was the key targets of the NF-κB pathway was analyzed. ResultsIn HCoV-229E-induced pneumonia, the lung index was markedly reduced, and lung pathology was improved in mice that treated with CMHD (12.1, 6.05 g/kg/d). Meanwhile, the expression of TNF-α, IL-6 were obviously inhibited, but the expression of IL-4 was significantly increased in CMHD groups. Compared with the model group, CMHD could also markedly upregulate the level of CD4+ and CD8+. Furthermore, CMHD has a markedly effect on inhibit the expression of p–NF–κB p65/NF-κB p65 in the lung. In H1N1-induced pneumonia, the lung index of mice in the CMHD (12.1 g/kg/d) treatment group was lower than that in the model group, and less inflammatory infiltration could be seen in the lung pathological. Moreover, CMHD could also obviously decrease the expression of TNF-α, IL-1β, IL-6, but significantly increase the expression of IL-4. Except for that, CMHD could also markedly downregulate the level of CD4+ and upregulate the level of CD8+ compared with the model group. In addition, CMHD has a markedly effect on inhibit the expression of p–NF–κB p65/NF-κB p65 in the lung. ConclusionCMHD can significantly combats viral infections caused by HCoV-229E and H1N1, and the mechanism may be related to its multiple functions of anti-inflammatory, immunity regulating and inhibiting NF-κB signal transduction pathway.