Silencing of Jumonji domain-containing 1C inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells via nuclear factor-κB signaling

BACKGROUND Osteoporosis is a common metabolic bone disorder induced by an imbalance between osteoclastic activity and osteogenic activity.During osteoporosis,bone mesenchymal stem cells(BMSCs)exhibit an increased ability to differentiate into adipocytes and a decreased ability to differentiate into osteoblasts,resulting in bone loss.Jumonji domain-containing 1C(JMJD1C)has been demonstrated to suppress osteoclastogenesis.AIM To examine the effect of JMJD1C on the osteogenesis of BMSCs and the potential underlying mechanism.METHODS BMSCs were isolated from mouse bone marrow tissues.Oil Red O staining,Alizarin red staining,alkaline phosphatase staining and the expression of adipo-genic and osteogenic-associated genes were assessed to determine the differen-tiation of BMSCs.Bone marrow-derived macrophages(BMMs)were incubated with receptor activator of nuclear factor-kappaΒligand to induce osteoclast differentiation,and osteoclast differen-tiation was confirmed by tartrate-resistant acid phosphatase staining.Other related genes were measured via reverse transcription coupled to the quantitative polymerase chain reaction and western blotting.Enzyme-linked immunosorbent assays were used to measure the levels of inflammatory cytokines,including tumor necrosis factor alpha,interleukin-6 and interleukin-1 beta.RESULTS The osteogenic and adipogenic differentiation potential of BMSCs isolated from mouse bone marrow samples was evaluated.JMJD1C mRNA and protein expression was upregulated in BMSCs after osteoblast induction,while p-nuclear factor-κB(NF-κB)and inflammatory cytokines were not significantly altered.Knockdown of JMJD1C repressed osteogenic differentiation and enhanced NF-κB activation and inflammatory cytokine release in BMSCs.Moreover,JMJD1C expression decreased during BMM osteoclast differentiation.CONCLUSION The JMJD1C/NF-κB signaling pathway is potentially involved in BMSC osteogenic differentiation and may play vital roles in the pathogenesis of osteoporosis.

Total polysaccharides of the Sijunzi decoction attenuate tumor necrosis factor-α-induced damage to the barrier function of a Caco-2 cell monolayer via the nuclear factor-κB-myosin light chain kinase-myosin light chain pathway

AIM To explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction(TPSJ) on the epithelial barriers in vitro. METHODS Caco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance(TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respecti-vely used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. western blotting was also used to evaluate the cellular expression of myosin light chain(MLC), phosphorylated MLC(pM LC), MLC kinase(MLCK), and nuclear factor(NF)-κB p65. RESULTS TPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cyto-kines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor(TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65. CONCLUSION Our results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

Background:G-protein coupled receptors(GPCRs)are recognized as attractive targets for drug therapy.However,it remains poorly understood how GPCRs,except for a few chemokine receptors,regulate the progression of liver fibrosis.Here,we aimed to reveal the role of GPR65,a proton-sensing receptor,in liver fibrosis and to elucidate the underlying mechanism.Methods:The expression level of GPR65 was evaluated in both human and mouse fibrotic livers.Furthermore,Gpr65-deficient mice were treated with either bile duct ligation(BDL)for 21 d or carbon tetrachloride(CCl4)for 8 weeks to investigate the role of GPR65 in liver fibrosis.A combination of experimental approaches,including Western blotting,quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR),and enzyme-linked immunosorbent assay(ELISA),confocal microscopy and rescue studies,were used to explore the underlying mechanisms of GPR65’s action in liver fibrosis.Additionally,the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated.Results:We found that hepatic macrophage(HM)-enriched GPR65 was upregulated in both human and mouse fibrotic livers.Moreover,knockout of Gpr65 significantly alleviated BDL-and CCl4-induced liver inflammation,injury and fibrosis in vivo,and mouse bone marrow transplantation(BMT)experiments further demonstrated that the protective effect of Gpr65knockout is primarily mediated by bone marrow-derived macrophages(BMMs).Additionally,in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited,while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and transforming growth factor-β(TGF-β),all of which subsequently promoted the activation of hepatic stellate cells(HSCs)and the damage of hepatocytes(HCs).Mechanistically,GPR65 overexpression,the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-αand IL-6 via the Gαq-Ca^(2+)-JNK/NF-κB pathways,while promoted the expression of TGF-βthrough the Gαq-Ca^(2+)-MLK3-MKK7-JNK pathway.Notably,pharmacological GPR65 inhibition retarded the development of inflammation,HCs injury and fibrosis invivo.Conclusions:GPR65 is a major regulator that modulates the progression of liver fibrosis.Thus,targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats

AIM:To observe the effects of N-acetylserotonin(NAS)administration on retinal ischemia-reperfusion(RIR)injury in rats and explore the underlying mechanisms involving the high mobility group box 1(HMGB1)/receptor for advanced glycation end-products(RAGE)/nuclear factor-kappa B(NF-κB)signaling pathway.METHODS:A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye.Eighty male Sprague Dawley were randomly divided into five groups:sham group(n=8),RIR group(n=28),RIR+NAS group(n=28),RIR+FPS-ZM1 group(n=8)and RIR+NAS+FPS-ZM1 group(n=8).The therapeutic effects of NAS were examined by hematoxylin-eosin(H&E)staining,and retinal ganglion cells(RGCs)counting.The expression of interleukin 1 beta(IL-1β),HMGB1,RAGE,and nod-like receptor 3(NLRP3)proteins and the phosphorylation of nuclear factorkappa B(p-NF-κB)were analyzed by immunohistochemistry staining and Western blot analysis.The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats.With NAS therapy,the HMGB1 and RAGE expression decreased significantly,and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression.Additionally,NAS exhibited an anti-inflammatory effect by reducing IL-1βexpression.The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression,so as to the IL-1βexpression and retinal edema,accompanied by an increase of RGCs in RIR rats.CONCLUSION:NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway,which may be a useful therapeutic target for retinal disease.

Integrin-linked kinase overexpression promotes epithelial-mesenchymal transition via nuclear factor-κB signaling in colorectal cancer cells

AIM: To investigate the effect of integrin-linked kinase (ILK) on proliferation, metastasis, and invasion of the colorectal cancer cell line SW480.METHODS: In this study, the colorectal cancer cell line SW480 was stably transfected with ILK plasmids, and small interfering RNA (siRNA) was used to knockdown expression of nuclear factor (NF)-κB/p65. Methylthiazole tetrazolium (MTT) assay was performed to measure proliferation, and the wound healing migration assay and matrigel invasion assay were used to test the metastasis and invasion ability of SW480 cells. To explore the epithelial-mesenchymal transition (EMT) process, embryonic development, and the invasion and metastasis of tumors, the protein level of E-cadherin, vimentin, snail, and slug was detected by western blot. Immunofluorescence was also used to detect E-cadherin expression. Western blot was used to determine the level of phosphorylated-inhibitor of kappa B (IκB)a, inhibitor of gamma B (IγB)a, and nuclear factor kappa B (NF-κB) expressions and to explore the ILK signaling pathway.RESULTS: Western blot results revealed that ILK expression significantly increased when ILK was overexpressed in SW480 cells (P < 0.05). Proliferation, metastasis, and invasion ability were improved in the vector-ILK group compared to the vector group (P < 0.05). Immunofluorescence results revealed that E-cadherin fluorescence intensity decreased after ILK was overexpressed (P < 0.05). Western blot results revealed that the protein expression of E-cadherin was reduced, while vimentin, snail, and slug were upregulated when ILK was overexpressed in SW480 cells (P < 0.05). In order to determine the role of the NF-κB signaling pathway in ILK overexpression promoted EMT occurrence, we overexpressed ILK in SW480 cells and found that levels of NF-κB/p65 and cytoplasmic phosphorylated-IκBa were increased and that cytoplasmic IкBa levels were decreased compared to the control group (P < 0.05). Furthermore, NF-κB/p65 knockout revealed that E-cadherin was increased in the overexpressed ILK group.CONCLUSION: ILK overexpression improved the proliferation, metastasis, and invasion ability of SW480 cells, and this effect may be mediated by the NF-κB signaling pathway.

Phosphoinositide 3-Kinase/Akt and Nuclear Factor-κB Are Involved in Staphylococcus Aureus-induced Apoptosis in U937 Cells

Objective To explore the mechanisms involved in Staphylococcus aureus （S. aureus） invading human monocytic U937 cells. Methods S. aureus were added to U937 cells at multiplicity of infections （MOI） of 20：1 for 0, 15, 30, 60, and 90 minutes, respectively. Cell apoptosis was analyzed with Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI） flow cytometry analysis. Akt and nuclear factor-κB （NF-κB） activities were detected by Western blotting. Results Infection of U937 cells with S. aureus induced rapid cell death in a time-dependent manner, and the cells displayed characteristic features of apoptosis. S. aureus-induced apoptosis was associated with a prominent downregulation of activated （phosphorylated） Akt and NF-κB. The inhibition of phosphorylated Akt by LY294002 led to the inhibition of NF-κB in a dose-dependent manner. Inhibition of Akt with LY294002 caused further increase in apoptosis of U937 cells. Conclusions S. aureus can stimulate the apoptosis of U937 ceils. S. aureus induces apoptosis of U937 cells by inhibiting Akt-regulated NF-κB.

An Experimental Study on the Role of Nuclear Factor-κB in the Signal Conduction of Protein Kinase C Regulating the Proliferation and Apoptosis of T Lymphocytes in Asthma

To explore the role of nuclear factor-κB(NF-κB) in the signal pathway of protein kinase C (PKC) regulating the proliferation and apoptosis of T lymphocytes in asthma. T lymphocytes were isolated from the asthmatic model of guinea pigs and the asthmatic patients. Either the T cells stimulated with PMA alone or those stimulated with PMA together with pyrrolidine dithiocarbamate (PDTC) were incubated for 1 and 24?h. The proliferation of and the presence of NF-κB in the cells incubated for 1?h were observed by MTT and immunohistochemical staining, respectively. And the cells incubated for 24?h were observed for the apoptosis by TUNEL. All the assays were paralleled with controls, and all the data were analyzedstatistically with the software SAS. The percentage of cells of nuclear positive staining of NF-κB and the proliferation of T lymphocytes from asthmatic guinea pigs and asthmatic patients stimulated with PMA were significantly higher than those of T lymphocytes from asthmatic guinea pigs and asthmatic patients stimulated without PMA respectively (P<0.01) and those of T lymphocytes from normal control guinea pigs and normal control persons stimulated with PMA respectively (P<0.01), and were significantly reduced by PDTC (P<0.01). The apoptosis index of T lymphocytes from asthmatic guinea pigs and asthmatic patients stimulated with PMA were significantly lower than those of T lymphocytes from asthmatic guinea pigs and asthmatic patients stimulated without PMA respectively (P<0.01) and those of T lymphocytes from normal control guinea pigs and normal control persons stimulated with PMA respectively (P<0.01), and were significantly induced by PDTC (P<0.01). There were good positive correlation between the percentage of cells of nuclear staining of NF-κB of T lymphocytes and the proliferation of T lymphocytes (r=0.51-0.72, P<0.001), and also good negative correlation between the percentage of cells of nuclear staining of NF-κB and the apoptosis index of T lymphocytes (r=-0.55-0.71, P<0.001, respectively). It concludes that the active PKC of asthmatic T lymphocytes promoting the proliferation and inhibiting the apoptosis of T lymphocytes may be mediated by activating NF-κB, the activation of PKC-NF-κB signal pathway of T lymphocytes NF-κB may play an important role in the pathogenesis of asthma.

黄芪阳和汤调控PI3K/AKT/NF-κB信号通路促进糖尿病足溃疡大鼠创面愈合

目的基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子-κB(NF-κB)信号通路探究黄芪阳和汤对糖尿病足溃疡(DFU)大鼠创面愈合的影响。方法构建DFU大鼠模型,将建模成功的48只大鼠随机分为模型组,黄芪阳和汤低(8.5 g/kg)、高(17 g/kg)剂量组,黄芪阳和汤高剂量(17 g/kg)+LY294002(PI3K/AKT通路抑制剂,0.3 mg/kg)组;每组12只;另取12只大鼠为对照组。各组大鼠给予对应药物干预,连续4周。第14、28天给药后,观察大鼠一般状态及创面变化,计算创面愈合率,检测大鼠空腹血糖(FBG)水平和大鼠创面周围组织经皮氧分压(TcpO2);酶联免疫吸附试验检测大鼠血清血管内皮生长因子(VEGF)、缺氧诱导因子-1α(HIF-1α)、C反应蛋白(CRP)、白细胞介素(IL)-6水平;苏木素-伊红染色观察大鼠创面组织病理学变化;免疫组织化学染色测定大鼠创面组织微血管密度;蛋白免疫印迹法检测大鼠创面组织中PI3K、磷酸化PI3K(p-PI3K)、AKT、磷酸化AKT(p-AKT)、NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)、NF-κB抑制蛋白α(IκB-α)蛋白表达。结果对照组大鼠毛色光滑,饮食、饮水、排泄均正常,较活跃,创面愈合快,创面组织炎症反应较轻,新生血管较多,肉芽组织中成纤维细胞及胶原基质丰富;模型组大鼠毛色暗淡无光泽,活动减少,且出现多饮、多食、多尿症状,创面颜色较深,且周围组织出现水肿、溃疡,创面组织可见大量炎性细胞浸润,伴组织坏死、渗出,新生血管及成纤维细胞较少,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平降低,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达升高(P<0.05);与模型组相比,黄芪阳和汤低、高剂量组大鼠状态逐渐改善,创面组织病变程度依次减轻,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平依次升高,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达依次降低(P<0.05);LY294002能部分逆转高剂量黄芪阳和汤对DFU大鼠的治疗作用(P<0.05)。结论黄芪阳和汤能调控PI3K/AKT/NF-κB信号通路,抑制DFU大鼠炎症反应,促进血管新生,从而促进创面愈合。

Inhibition of p38 mitogen-activated protein kinase attenuates experimental autoimmune hepatitis: Involvement of nuclear factor kappa B

To investigate the role of p38 mitogen-activated protein kinase （p38MAPK） in murine experimental autoimmune hepatitis （EAH）.METHODS： To induce EAH, the syngeneic S-100 antigen emulsified in complete Freud＇s adjuvant was injected intraperitoneally into adult male C57BI/6 mice. Liver injury was assessed by serum ALT and liver histology. The expression and activity of p38 MAPK were measured by Western blot and kinase activity assays. In addition, DNA binding activities of nuclear factor kappa B （NF-KB） were analyzed by electrophoretic mobility shift assay. The effects of SB203580, a specific p38 MAPK inhibitor, on liver injuries and expression of proinflammatory cytokines （interferon-y, IL-12, IL-1β and TNF-α） were observed.RESULTS： The activity of p38 MAPK and NF-~：B was increased and reached its peak 14 or 21 d after the first syngeneic S-100 administration. Inhibition of p38 MAPK activation by SB203580 decreased the activation of NF-~：B and the expression of proinflammatory cytokines. Moreover, hepatic injuries were improved significantly after SB203580 administration.

Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition

BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIM To explore the protective effects of panax notoginseng saponin(PNS) against dextran sulfate sodium(DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B(PI3K/AKT) signaling pathway inhibition in rats.METHODS Colitis rat models were generated via DSS induction, and rats were divided into control(no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3 K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTS Compared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers,and showed significantly increased M1 macrophages in spleen and colon tissues.They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway(all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency(all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group(P < 0.05).CONCLUSION PNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.

Antidepressants can treat inflammatory bowel disease through regulation of the nuclear factor-κB/nitric oxide pathway and inhibition of cytokine production:A hypothesis

Inflammatory bowel disease (IBD) is a group of inflammatory disorders mainly affecting the colon and small intestine. The main types of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). UC is restricted to the large intestine whereas CD can affect any part of the gastrointestinal tract. Treating this disorder depends on the form and level of severity. Common treatment involves an anti-inflammatory drug, such as mesalazine, and an immunosuppressant, such as prednisone. Several signaling pathways, including nuclear factor (NF)-κB and nitric oxide (NO), and genetic and environmental factors are believed to play an important role in IBD. Amitriptyline is a commonly used antidepressant with known anti-inflammatory activities. Amitriptyline also acts on the NF-κB/NO pathway or cytokine production. Therefore, we hypothesize that antidepressants like amitriptyline can be pioneered and considered effective as an innovative and effective therapeutic in the treatment and attenuation of development of IBD in adjusted doses.

Iguratimod promotes transformation of mononuclear macrophages in elderly patients with rheumatoid arthritis by nuclear factor-κB pathway

BACKGROUND The role of macrophages in rheumatoid arthritis(RA)and its mechanism have attracted much attention in RA pathogenesis.Macrophages accumulate in the synoviums of RA,and the proportion of M1 type pro-inflammatory macrophages is higher than that of M2 type anti-inflammatory macrophages,leading to the secretion of inflammatory molecules and the aggravation of inflammatory reaction,which has made macrophages a potential target of RA drugs.Iguratimod is a kind of cyclo-oxygenase-2 inhibitor that affects macrophage polarity.It is speculated that its anti-inflammatory and anti-rheumatic effects may be related to the regulation of macrophage M1/M2 ratio.AIM To investigate the effects of Iguratimod on the polarity of mononuclear macrophages in elderly patients with RA.METHODS Elderly patients with RA and joint effusion were selected,including 10 men and 25 women,with an average age of 66.37±4.42 years.Patients were treated with oral administration of 25 mg Iguratimod(Iremod,State Food and Drug Administration Approval No.H20110084)twice daily for 12 wk.Disease Activity Score 28 and Health Assessment Questionnaire score were collected according to the disease severity before and after treatment.Venous blood and joint effusion fluid were collected,mononuclear macrophages were extracted and expression of cell surface markers CD86,CD64,CD163,and CD206 was analyzed by flow cytometry.The concentration of inflammatory factors interleukin(IL)-6,IL-1β,transforming growth factor-β,and IL-4 in the joint effusion fluid was analyzed by enzyme-linked immunosorbent assay.Expression of mononuclear cells inhibitor of nuclear factor-κB(IκB)and phosphorylated IκB in peripheral blood was analyzed by western blotting.RESULTS Disease Activity Score 28 score and Health Assessment Questionnaire score of patients treated with Iguratimod decreased significantly.The percentage of cell surface markers CD86 and CD64 decreased significantly,and the percentage of CD163 and CD206 increased significantly(P<0.05).The inflammatory factors IL-6 and IL-1βdecreased significantly,and transforming growth factor-βand IL-4 increased significantly.Western blot analysis showed that mononuclear cell inhibitor of nuclear factor-κB in peripheral blood was significantly increased after treatment,and its phosphorylation level was significantly decreased(P<0.05).CONCLUSION Iguratimod can promote the transformation of mononuclear macrophages from M1 to M2 in elderly patients with RA by inhibiting the nuclear factor-κB pathway,thus improving symptoms of RA.

Testosterone alleviates tumor necrosis factor-alpha-mediated tissue factor pathway inhibitor downregulation via suppression of nuclear factor-kappa B in endothelial cells

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor(TFPI)gene expression through the androgen receptor in endothelial cells.This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha(TNF-α).Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-α.TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction.To study the cellular mechanism of testosterone’s action,nuclear factor-kappa B(NF-κB)translocation was confirmed by electrophoretic mobility shift assays.We found that after NF-κB was activated by TNF-α,TFPI protein levels declined significantly by 37.3%compared with controls(P<0.001),and the mRNA levels of TFPI also decreased greatly(P<0.001).A concentration of 30 nmol L-1 testosterone increased the secretion of TFPI compared with the TNF-α-treated group.NF-κB DNA-binding activity was significantly suppressed by testosterone(P<0.05).This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-κB activity.

Apigenin ameliorates imiquimod-induced psoriasis in C57BL/6J mice by inactivating STAT3 and NF-κB

Psoriasis is a chronic autoimmune disease featured by patches on the skin.It is caused by malfunction of immune cells and keratinocytes with inflammation as one of its key features.Apigenin(API)is a natural flavonoid with anti-inflammatory and immunoregulatory properties.Therefore,we speculated that API can ameliorate psoriasis,and determined its effect on the development of psoriasis by using imiquimod(IMQ)-induced psoriasis mouse model.Our results showed that API attenuated IMQ-induced phenotypic changes,such as erythema,scaling and epidermal thickening,and improved splenic hyperplasia.Abnormal differentiation of immune cells was restored in API-treated mice.Mechanistically,we revealed that API is a key regulator of signal transducer activator of transcription 3(STAT3).API regulated immune responses by reducing interleukin-23(IL-23)/STAT3/IL-17A axis.Moreover,it suppressed IMQ-caused cell hyperproliferation by inactivating STAT3 through regulation of extracellular signal-regulated kinase 1/2 and nuclear factor-κB(NF-κB)pathway.Furthermore,API reduced expression of inflammatory cytokines through inactivation of NF-κB.Taken together,our study demonstrates that API can ameliorate psoriasis and may be considered as a strategy for psoriasis treatment.

肠病药方调控PI3K/AKT/NF-κB信号通路改善溃疡性结肠炎的研究

目的探讨肠病药方调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子kappa B(NF-κB)信号通路对抗溃疡性结肠炎(UC)的作用机制。方法42只C57BL/6小鼠随机分为正常对照组(n=6,Control组,自由饮去离子水)、模型组(n=12,DSS组)、美沙拉嗪组(n=12,MES组)及肠病药方组(n=12,CBD组),后3组小鼠自由饮用3%葡聚糖硫酸钠(DSS)溶液7 d诱导UC模型,后两组同期灌胃美沙拉嗪或肠病药方,前两组灌胃等体积去离子水;观察实验期间小鼠体质量改变,评测小鼠疾病活动指数(DAI);造模结束后麻醉处死小鼠,测量结肠长度、观察结肠内容物及黏膜,采用苏木素-伊红(HE)染色观察各组小鼠结肠病理组织学变化,酶联免疫吸附试验(ELISA)检测各组小鼠结肠组织匀浆白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平,蛋白免疫印迹法(WB)检测各组小鼠结肠组织中磷酸化PI3K(p-PI3K)-p85、磷酸化AKT1(p-AKT1)、磷酸化NF-κB(p-NF-κB)-p65蛋白表达,实时荧光定量PCR技术(qRT-PCT)检测各组小鼠结肠组织PI3K、AKT、NF-κB信使RNA(mRNA)表达水平。结果与DSS组比较,CBD组治疗后,有效缓解UC小鼠症状,粪便性状及便血情况改善,结肠黏膜炎症浸润减少,体质量下降有所缓解,DAI评分降低及结肠长度接近正常,结肠组织匀浆中IL-6、TNF-α表达降低(P<0.05),p-PI3K-p85、p-AKT1、p-NF-κB-p65蛋白及mRNA表达明显减少(P<0.05)。结论肠病药方可缓解UC小鼠的症状、减轻结肠黏膜损伤,其机制可能与调解结肠组织中PI3K/AKT/NF-κB信号通路中相关蛋白质及mRNA表达、降低结肠组织炎症因子表达有关。

黄芩素调节PI3K/Akt/NF-κB信号通路及对细菌性脑膜炎大鼠血脑屏障的影响

目的:探讨黄芩素调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/核因子κB(NF-κB)信号通路对细菌性脑膜炎(BM)大鼠血脑屏障的影响。方法:制备大鼠BM模型,将大鼠分为对照组、模型组、黄芩素组(腹腔注射30 mg/kg黄芩素)、黄芩素+抑制剂组(腹腔注射30 mg/kg黄芩素+7.5 mg/kg LY294002),对大鼠进行Loeffler神经行为评分,H-E染色观察大鼠脑组织病理学变化,伊文思蓝染色检测血脑屏障通透性,分别检测脑脊液IL-1β、IL-6水平及白细胞计数(WBC)、脑组织含水量,水通道蛋白4(AQP4)、闭锁蛋白-5(claudin-5)、PI3K/Akt/NF-κB通路相关蛋白表达。结果:对照组大鼠脑组织结构正常,细胞排列整齐,模型组大鼠脑组织细胞排列紊乱,大量炎症细胞浸润。与对照组比较,模型组Loeffler评分及脑组织AQP4、claudin-5、p-PI3K、p-Akt表达水平显著降低,脑脊液IL-1β、IL-6水平及WBC、脑组织EB含量及含水量、p-NF-κB p65表达水平显著升高;黄芩素组较模型组病理损伤减轻,炎症细胞减少。与模型组比较,黄芩素组Loeffler评分及脑组织AQP4、claudin-5、p-PI3K、p-Akt表达水平显著升高,脑脊液IL-1β、IL-6水平及WBC、脑组织EB含量及含水量、p-NF-κB p65表达水平显著降低;LY294002可部分逆转黄芩素对BM大鼠血脑屏障通透性的改善作用。结论:黄芩素可改善BM大鼠脑水肿及血脑屏障通透性,可能与调控PI3K/Akt/NF-κB信号通路有关。

茯苓酸调节PI3K/AKT/NF-κB信号通路对大鼠幽门螺旋杆菌相关性胃炎的治疗作用

目的 探讨茯苓酸(PA)对大鼠幽门螺旋杆菌(Hp)相关性胃炎的治疗效果及作用机制。方法 建立Hp相关性胃炎大鼠模型;所有大鼠分为对照组(CT组)、模型组(M组)、PA低剂量组(PA L组)和PA高剂量组(PA H组)、PA H+磷脂酰肌醇3-激酶(PI3K)激活剂(740 Y-P)组;评估各组大鼠胃黏膜损伤指数(UI),透射电子显微镜观察胃黏膜细胞形态学,HE染色评价胃黏膜病理学特征,ELISA检测胃组织白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、IL-10、诱导型一氧化氮合酶(iNOS)、超氧化物歧化酶(SOD)的水平,Western blot法检测PI3K、磷酸化-PI3K(p-PI3K)、蛋白激酶B(AKT)、p-AKT、核因子(NF)-κB p65、p-NF-κB p65蛋白表达。结果 与CT组比较,M组大鼠胃黏膜糜烂,上皮水肿、充血、溃疡严重,上皮细胞固缩,炎性细胞浸润,UI、IL-6、TNF-α、iNOS以及p-PI3K/PI3K、p-AKT/AKT、p-NF-κB p65/NF-κB p65蛋白表达水平升高,IL-10和SOD水平降低(P<0.05);与M组比较,PA L组、PA H组大鼠胃黏膜损伤改善,炎性细胞浸润减少,UI、IL-6、TNF-α、iNOS以及p-PI3K/PI3K、p-AKT/AKT、p-NF-κB p65/NF-κB p65蛋白表达水平降低,IL-10和SOD水平升高(P<0.05);与PA H组比较,PA H+740 Y-P组大鼠胃黏膜病理损伤加重,上皮细胞固缩,UI、IL-6、TNF-α、iNOS以及p-PI3K/PI3K、p-AKT/AKT、p-NF-κB p65/NF-κB p65蛋白表达水平升高,IL-10和SOD水平降低(P<0.05)。结论 PA可能通过抑制PI3K/AKT/NF-κB信号通路发挥对大鼠Hp相关性胃炎的治疗作用。