Comparative study of anti-inflammatory effects of different processed products through the COX-2/PGE2 signaling pathway: based on network pharmacology and molecular docking

Background:Radix Aconiti Lateralis Preparata(Fu-zi)is a traditional Chinese medicinal herb,which has been widely used in the clinic and has potent anti-inflammatory activities.we aimed to explore the mechanisms of extract containing alkaloids from different Fu-zi Processed Products(FPP)in treating inflammation,especially rheumatoid arthritis(RA).Methods:Firstly,using network pharmacology technology,the ingredients,and targets of Fu-zi were obtained by searching and screening,the targets involving RA were acquired,the intersection targets were constructed a"component-target-pathway"network.A comprehensive investigation was conducted on the anti-rheumatoid arthritis mechanisms of 5 FPPs in lipopolysaccharide(LPS)induced RAW264.7 cells,which serve as a model for RA.The production of NO and inflammatory cytokines were measured by ELISA kit.Quantitative Real-time PCR(qRT-PCR)was utilized to measure the mRNA levels.COX-2/PGE2 signaling pathway-associated proteins were determined by western blot.Results:According to a network pharmacological study,16 chemical components and 43 common targets were found in Fu-zi and 6 key targets including PTGS2 were closely related to the mechanism of Fu-zi in treating RA.The in vitro study revealed that the levels of NO,TNF-α,and IL-1βwere substantially decreased by the 5 FPPs.The 5 FPPs significantly suppressed the expression of proteins COX-2,iNOS,and NF-κB,with particularly notable effects observed for PFZ and XFZ.Conclusion:Altogether,these results demonstrated that the 5 PPS containing alkaloids have a good anti-RA-related inflammatory effect,and the mechanism may be related to COX-2/PGE2 signaling pathway,particularly,Fu-zi prepared utilizing a traditional Chinese technique.

β-arrestin 2 attenuates lipopolysaccharide-induced liver injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation in mice

AIM To study the role and the possible mechanism of β-arrestin 2 in lipopolysaccharide(LPS)-induced liver injury in vivo and in vitro.METHODS Male β-arrestin 2^(+/+) and β-arrestin 2^(-/-)C57 BL/6 J mice were used for in vivo experiments, and the mouse macrophage cell line RAW264.7 was used for in vitro experiments. The animal model was established via intraperitoneal injection of LPS or physiological sodium chloride solution. Blood samples and liver tissues were collected to analyze liver injury and levels of pro-inflammatory cytokines. Cultured cell extracts were collected to analyze the production of pro-inflammatory cytokines and expression of key molecules involved in the TLR4/NF-κB signaling pathway.RESULTS Compared with wild-type mice, the β-arrestin 2 knockout mice displayed more severe LPS-induced liver injury and significantly higher levels of proinflammatory cytokines, including interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, and IL-10. Compared with the control group, pro-inflammatory cytokines(including IL-1β, IL-6, TNF-α, and IL-10) produced by RAW264.7 cells in the β-arrestin 2 si RNA group were significantly increased at 6 h after treatment with LPS. Further, key molecules involved in the TLR4/NF-κB signaling pathway, including phosphoIκBα and phosho-p65, were upregulated.CONCLUSION β-arrestin 2 can protect liver tissue from LPS-induced injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation.

Study on the Hepatoprotective Effect of Oxalis coriniculata L. and Related Mechanism by Regulating Oxidative Stress and TLR-2/NF-κB Signaling Pathway

[Objectives]This study aimed to explore the protective effect of Oxalis coriniculata L.on rats with acute liver injury induced by carbon tetrachloride(CCl4)and related mechanism by regulating oxidative stress and the TLR-2 TLR-2/NF-κB signaling pathway.[Methods]A total of 48 female rats were randomly and evenly divided into normal group,model group,silymarin group(0.12 g/kg),and high(16 g/kg),middle(8 g/kg)and low-dose(4 g/kg)O.coriniculata L.groups.The rats in the groups were intragastrically administered with 5 mL/kg of corresponding drugs(equal-volume distilled water for normal group and control group),respectively.The administration was conducted twice a day,for 10 consecutive days.After 2 h of the last administration,the rats in all the groups except the normal group were intraperitoneally injected with 12%carbon tetrachloride(CCl4)olive oil solution(5 mL/kg),respectively to establish liver injury rat models.After 16 h,the eyeball blood of the rats was collected,and their liver tissues were collected for preparation of HE sections.The biochemical indicators detected included aspartate aminotransferase(AST),alanine aminotransferase(ALT),total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)activity and malondialdehyde(MDA)content in the serum.The contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in the serum were detected by ELISA.The expression of Toll-like receptor-2(TLR-2)and nuclear factor-κB(NF-κB)in liver tissue was detected using Western blotting.The pathological changes of liver were observed under light microscope.[Results]Compared with the normal group,the ALT,AST activity and MDA,IL-1β,IL-6,TNF-αlevels in rat serum significantly increased(P<0.01),the GSH-Px,T-SOD activity in rat serum significantly decreased(P<0.01),and the expression of TLR-2 and NF-κB in liver tissue was up-regulated(P<0.01)in the model group.Compared with the model group,the ALT,AST activity and MDA,IL-1β,IL-6 and TNF-αlevels in rat serum reduced(P<0.05,P<0.01),the GSH-Px and T-SOD activity in rat serum increased(P<0.05,P<0.01),and the expression of TLR-2 and NF-κB in liver tissue was down-regulated(P<0.05,P<0.01)in the O.coriniculata L.administration groups.Pathological sections show that O.coriniculata L.had an improving effect on rats with acute liver injury induced by CCl4.[Conclusions]O.coriniculata L.has a good protective effect on rats with acute liver injury induced by CCl4.Its mechanism may be related to inhibition of oxidative stress,inhibition of inflammatory response and regulation of the TLR-2/NF-κB signaling pathway.

Fucoxanthin suppresses OxLDL-induced inflammation via activation of Nrf2 and inhibition of NF-κB signaling

Objective:To explore the impact of fucoxanthin on oxidized low-density lipoprotein(OxLDL)-induced stress and inflammation in human endothelial cells and its underlying mechanisms.Methods:HUVECs were treated with OxLDL and/or fucoxanthin for a range of time points and concentrations.We evaluated the effects of fucoxanthin on OxLDL-induced HUVECs using the MTT assay,reactive oxygen species accumulation assay,ELISA,RT-PCR,immunofluorescence,and Western blotting.Results:Fucoxanthin enhanced the cell viability in a dose dependent manner after OxLDL exposure.Furthermore,fucoxanthin pretreatment significantly decreased OxLDL-induced reactive oxygen species production and prevented the activation of the nuclear factor kappa-B pathway,which led to substantial suppression of pro-inflammatory gene expressions.OxLDL-induced upregulation of interleukin-6,intercellular adhesion molecule-1,vascular cell adhesion molecule-1,interleukin-1β,monocyte chemotactic protein-1,cyclooxygenase-1,and tumor necrosis factor-αwas significantly reduced by fucoxanthin.Conclusions:Fucoxanthin can inhibit OxLDL-induced vascular inflammation and oxidative stress in HUVECs by targeting Nrf2 signaling pathways.

机动车尾气诱发大鼠慢性阻塞性肺疾病并上调气道上皮细胞COX-2/PGE2/EP受体信号通路

目的:探究机动车尾气(MVE)长期暴露引起大鼠慢性阻塞性肺疾病(COPD)发生时,气道上皮细胞中环加氧酶2(COX-2)/前列腺素E;(PGE;)/E-前列腺素类激素(EP)受体信号通路成员的表达变化。方法:(1)动物实验:健康雄性SD纯系大鼠(SPF级)16只,随机分为2组:MVE暴露组(n=8)和空白对照(CTL)组(n=8)。采用MVE暴露6个月的方法建立COPD大鼠模型。造模结束后,使用Buxco小动物有创肺功能仪检测大鼠肺功能;肺组织切片行HE染色并评估肺组织病理变化;ELISA法检测大鼠支气管肺泡灌洗液(BALF)中炎症因子白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和PGE;的水平,评估大鼠肺部炎症情况;采用免疫荧光及Western blot法检测肺组织COX-2及EP受体蛋白水平;提取肺组织核蛋白,Western blot检测MVE对肺组织NF-κB核转位的影响。(2)细胞实验:采用MVE细颗粒物(PM2.5)标准品刺激人正常支气管上皮细胞BEAS-2B。ELISA法检测细胞培养液中PGE;、IL-6和IL-8的水平;Western blot检测细胞中COX-2及EP受体蛋白水平;收集细胞核蛋白,检测PM2.5对NF-κB核转位的影响。结果:(1)MVE长期暴露致大鼠出现类似COPD的症状,包括气道炎症、肺气肿及肺功能下降(P<0.05);(2)MVE长期暴露显著上调大鼠肺组织及气道上皮细胞COX-2和EP1/EP4受体表达(P<0.05),而对EP2/EP3受体表达无显著影响(P>0.05);(3)MVE PM2.5标准品长时间暴露导致BEAS-2B细胞COX-2、PGE;和EP1/EP4表达水平显著升高(P<0.05),但对EP2/EP3表达无显著影响(P>0.05);(4)PM2.5暴露诱导大鼠肺组织及BEAS-2B细胞中NF-κB活化增加(P<0.05)。结论:MVE诱导大鼠COPD发病过程中,气道上皮细胞COX-2-PGE;-EP1/4受体信号通路成员表达增加并伴随NF-κB信号通路的活化。

Attenuation of lipopolysaccharide-induced neuroinflammatory events in BV-2 microglial cells by Moringa oleifera leaf extract

Objective: To determine the anti-neuroinflammatory activity of Moringa oleifera leaf extract(MLE) under lipopolysaccharide stimulation of mouse murine microglia BV2 cells in vitro. Methods: The cytotoxicity effect of MLE was investigated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide assay. The inflammatory response of BV-2 cells were induced with lipopolysaccharide. The generation of nitric oxide levels was determined by using Griess assay and the level of pro-inflammatory cytokines(IL-1β, IL-6 and TNF-α) was evaluated by ELISA kit. The expression of iNOS, COX-2 as well as IκB-ααwas carried out by immunoblot analysis. Results: MLE reduced the nitric oxide production in concentration-dependent manner, and maintained the viability of BV-2 microglial cells which indicated absence of toxicity. In addition, MLE repressed the activation of nuclear factor kappa B by arresting the deterioration of IκB-α, consequently resulted in suppression of cytokines expression such as COX-2 and iNOS. Conclusions: MLE inhibitory activities are associated with the inhibition of nuclear factor kappa B transcriptional activity in BV2 microglial cells. Thus MLE may offer a substantial treatment for neuroinflammatory diseases.

Photothermal therapy with regulated Nrf2/NF-κB signaling pathway for treating bacteria-induced periodontitis

Periodontitis is an inflammatory disease initiated by bacterial infection,developed by excessive immune response,and aggravated by high level of reactive oxygen species(ROS).Hence,herein,a versatile metal-organic framework(MOF)-based nanoplatform is prepared using mesoporous Prussian blue(MPB)nanoparticles to load BA,denoted as MPB-BA.The established MPB-BA nanoplatform serves as a shelter and reservoir for vulnerable immunomodulatory drug BA,which possesses antioxidant,anti-inflammatory and anti-bacterial effects.Thus,MPB-BA can exert its antioxidant,anti-inflammatory functions through scavenging intracellular ROS to switch macrophages from M1 to M2 phenotype so as to relieve inflammation.The underlying molecular mechanism lies in the upregulation of phosphorylated nuclear factor erythroid 2-related factor 2(Nrf2)to scavenge ROS and subsequently inhibit the nuclear factor kappa-B(NF-κB)signal pathway.Moreover,MPB-BA also exhibited efficient photothermal antibacterial activity against periodontal pathogens under near-infrared(NIR)light irradiation.In vivo RNA sequencing results revealed the high involvement of both antioxidant and anti-inflammatory pathways after MPB-BA application.Meanwhile,micro-CT and immunohistochemical staining of p-Nrf2 and p-P65 further confirmed the superior therapeutic effects of MPB-BA than minocycline hydrochloride.This work may provide an insight into the treatment of periodontitis by regulating Nrf2/NF-κB signaling pathway through photothermal bioplatform-assisted immunotherapy.

PM2.5暴露后激活核转录因子/环氧化酶2/前列腺素E2信号通路引起氧化应激损伤雄性生殖功能的机制研究

目的分析大气细颗粒物(PM2.5)暴露后激活核转录因子κB(NF-κB)/环氧化酶2(COX-2)/前列腺素E2(PGE2)信号通路引起氧化应激损伤雄性生殖功能的机制。方法20只清洁级别雄性小鼠随机分为对照组、暴露组各10只,对照组仅饲养于SPF级动物中心,暴露组予以PM2.5暴露,连续12周后均麻醉处死,评估生殖功能,进行考马斯亮蓝染色法、免疫组化法观察,测定睾丸组织匀浆及血清中睾酮(T)、雄性激素结合蛋白(ABP)、肿瘤坏死因子-α(TNF-α)、白介素-1(IL-1)、白介素-6(IL-6),实时荧光定量PCR及Western blot法测定NF-κB、COX-2、PGE2。结果暴露组的睾丸系数、附睾系数、精子计数、精子活动率、精子成活率低于对照组,精子畸形率高于对照组(P<0.05);考马斯亮蓝染色中暴露组细胞质骨架逐渐松弛回缩甚至断裂、消失,分布紊乱,生精细胞脱落;免疫组化染色中暴露组睾丸间质细胞体积较对照组变小,染色加深,细胞核固缩,胞浆含量少;暴露组睾丸组织匀浆及血清中T、ABP表达水平低于对照组,TNF-α、IL-1、IL-6水平高于对照组(P<0.05);暴露组NF-κB、COX-2、PGE2的mRNA及蛋白表达水平均高于对照组(P<0.05)。结论PM2.5暴露可能增加睾丸Sertoli细胞形态结构及功能损伤,影响雄性大鼠生殖功能,机制可能为激活NF-κB/COX-2/PGE2信号通路引起氧化应激损伤。

基于NF-κB信号通路探讨通管助孕方治疗输卵管阻塞性不孕症大鼠的作用机制

[目的]基于核转录因子-κB(NF-κB)信号通路探讨通管助孕方治疗输卵管阻塞性不孕症的作用机制。[方法]将140只雌性SD大鼠随机分为正常组、模型组、中成药组、西药组及通管助孕方高、中、低剂量组,每组20只。除正常组外,其余各组采用金黄色葡萄球菌建立输卵管阻塞性不孕症大鼠模型,造模后第7天,各组分别给予生理盐水、丹黄祛瘀胶囊混悬液、人胎盘组织液、通管助孕方高、中、低剂量治疗21 d后,药效组大鼠取材处理,生殖组大鼠进行雌雄合笼实验。采用酶联免疫吸附(ELISA)法检测各组大鼠血清中NF-κB、核因子κB抑制蛋白(IκB)、环氧合酶2(COX-2)、前列腺素E2(PGE2)的水平,采用蛋白免疫印迹(Western Blot)法和实时定量荧光聚合酶链反应(RT-PCR)法分别检测输卵管组织中NF-κB、IκB、COX-2、PGE2的蛋白和基因表达情况。采用肉眼观察以及光镜下观察输卵管组织病理形态学变化。观察并记录生殖组大鼠受孕及产仔情况。[结果] 1)与正常组比较,模型组大鼠NF-κB、COX-2、PGE2蛋白和基因表达升高,IκB蛋白和基因表达降低(P<0.05);与模型组比较,通管助孕方高、中、低剂量组NF-κB、COX-2、PGE2蛋白和基因表达降低,IκB蛋白和基因表达升高(P<0.05);与中成药组及西药组比较,通管助孕方高剂量组中NF-κB、COX-2、PGE2蛋白和基因表达降低,IκB蛋白和基因表达升高(P<0.05)。2)通过肉眼观察及光镜观察后,与模型组比较,各给药组输卵管组织形态均有不同程度改善,尤以通管助孕方高剂量组改善最为明显,基本接近正常。3)对生殖功能的影响:模型组妊娠率为0%,正常组妊娠率为100%,两组比较差异有统计学意义(P<0.05),表明金黄色葡萄球菌接种法可以成功建立输卵管阻塞性不孕症大鼠模型。通管助孕方高、中、低剂量组妊娠率及平均产仔数与模型组比较,差异有统计学意义(P<0.05),说明通管助孕方对输卵管阻塞性不孕症有确切疗效。[结论]通管助孕方可以抑制输卵管阻塞性不孕症模型大鼠中促炎因子NF-κB、COX-2、PGE2的表达,促进抗炎因子IκB的表达,发挥抗炎抗粘连作用,其治疗作用机制可能与阻断NF-κB信号传导通路有关。

大鼠局灶性脑缺血损伤后脑组织COX-2、PGE_2、15d-PGJ_2的表达及青心酮的干预作用

目的:观察大鼠局灶性脑缺血损伤后大脑组织COX-2、PGE2、PGJ2的表达及青心酮的干预作用,并对其机制进行初步探讨。方法:SD大鼠随机分为模型组和青心酮(4、12、36mg/kg)4组,改良LONGA法制备脑缺血-再灌注模型,取大脑组织分别采用realtime RT-PCR、ELISA和放射免疫法检测大脑组织中COX-2、NF-κB mRNA和蛋白,PGE2、PGJ2表达。结果:青心酮低剂量能明显降低大脑组织COX-2、NF-κB、PGE2的表达,明显提高PGJ2表达;中、高剂量能显著降低大脑组织COX-2、NF-κB、PGE2的表达,显著提高PGJ2表达。结论:青心酮能降低大鼠局灶性脑缺血损伤后大脑组织COX-2、PGE2的表达,提高PGJ2表达,对大鼠缺血-再灌注损伤有保护作用。其机制可能与抑制NF-κB表达有关。

Ellagic acid supplementation ameliorates cisplatin-induced liver injury in mice by inhibiting the NF-κB pathway and activating the Nrf2/HO-1 pathway

Cisplatin(cis-diaminodichloroplatinum II,CDDP),an essential chemotherapeutic agent,can cause potential hepa-totoxicity,but the underlying molecular mechanisms remain unclear.In this study,the protective effects of ellagic acid(EA)on CDDP exposure-induced hepatotoxicity and the underlying molecular mechanisms were investigated in a mouse model.Mice were randomly divided into control,CDDP model,EA100(i.e.,100 mg/kg/day),and CDDP plus 25,50,or 100 mg/kg/day EA groups.Mice in all the CDDP-treated groups were intraperitoneally injected with 20 mg/kg/day CDDP for two days.For all EA cotreatments,the mice were orally administered EA for seven days.Our results revealed that CDDP treatment resulted in liver dysfunction,oxidative stress,and caspase activation,which were effectively attenuated by EA cotreatment in a dose-dependent manner.Furthermore,EA supplementation sig-nificantly downregulated the CDDP exposure-induced protein and mRNA expression of NF-κB,IL-1β,TNF-α,and IL-6 but further upregulated the protein and mRNA expression of Nrf2 and HO-1.Molecular docking analysis revealed strong interactions between EA and the NF-κB or Keap1 proteins.In conclusion,our results revealed that EA sup-plementation could ameliorate CDDP-induced liver toxicity in mice by activating the Nrf2/HO-1 signaling pathway and inhibiting the NF-kB signaling pathway.

Discovery of novel aporphine alkaloid derivative as potent TLR2 antagonist reversing macrophage polarization and neutrophil infiltration against acute inflammation

Toll-like receptor 2(TLR2)mediated macrophages regulate the protective immune response to infectious microorganisms,but the aberrant activation of macrophages often leads to pathological inflammation,including tissue damage.In this study,we identified antagonists of TLR2 by screening2100 natural products and subsequently identified Taspine,an aporphine alkaloid,as an excellent candidate.Furthermore,analysis of the 10 steps chemical synthesis route and structural optimization yielded the Taspine derivative SMU-Y6,which has higher activity,better solubility,and improved drug-feasible property.Mechanistic studies and seq-RNA analysis revealed that SMU-Y6 inhibited TLR2 over other TLRs,hindered the formation of TLR2/MyD88 complex,and blocked the downstream NF-κB and MAPK signaling pathway,thus suppressing the release of inflammatory cytokines.SMU-Y6 could stabilize TLR2 and bind to TLR2 protein with a Kdof 0.18μmol/L.Additionally,SMU-Y6 could efficiently reverse the M1 phenotype macrophage polarization,reduce the production of cytokines as well as infiltration of neutrophiles and alleviate the local inflammation in mice with acute paw edema and colitis.Collectively,we reported the first aporphine alkaloid derivative that selectively inhibits TLR2 with high binding affinity and superior drug-feasible property,thus providing an urgently-needed molecular probe and potential drug candidate for inflammatory and autoimmune disease therapy.