Non-SMC condensin Ⅰ complex subunit D2 and non-SMC condensin Ⅱ complex subunit D3 induces inflammation via the IKK/NF-κB pathway in ulcerative colitis

BACKGROUND Ulcerative colitis(UC)is a chronic,nonspecific intestinal inflammatory disease with undefined pathogenesis.Non-SMC condensin I complex subunit D2(NCAPD2)and non-SMC condensin II complex subunit D3(NCAPD3)play pivotal roles in chromosome assembly and segregation during both mitosis and meiosis.To date,there has been no relevant report about the functional role of NCAPD2 and NCAPD3 in UC.AIM To determine the level of NCAPD2/3 in intestinal mucosa and explore the mechanisms of NCAPD2/3 in UC.METHODS Levels of NCAPD2/3 in intestinal tissue were detected in 30 UC patients and 30 healthy individuals with in situ hybridization(ISH).In vitro,NCM60 cells were divided into the NC group,model group,si-NCAPD2 group,si-NCAPD3 group and si-NCAPD2+si-NCAPD3 group.Inflammatory cytokines were measured by ELISA,IKK and NF-κB were evaluated by western blot,and IKK nucleation and NF-κB volume were analyzed by immunofluorescence assay.RESULTS Compared with expression in healthy individuals,NCAPD2 and NCAPD3 expression in intestinal tissue was significantly upregulated(P<0.001)in UC patients.Compared with levels in the model group,IL-1β,IL-6 and TNF-αin the si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups were significantly downregulated(P<0.01).IKK and NF-κB protein expression in the si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups was significantly decreased(P<0.01).Moreover,IKK nucleation and NF-κB volume were suppressed upon si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 transfection.CONCLUSION NCAPD2/3 is highly expressed in the intestinal mucosa of patients with active UC.Overexpression of NCAPD2/3 promotes the release of pro-inflammatory cytokines by modulating the IKK/NF-κB signaling pathway.

Neuroprotective effect of Angiopep-2 peptide modified scutellarin-loaded PEGylated PAMAM dendrimer nanoparticles on ischemic stroke by modulating the Toll-like receptors-dependent MyD88/IKK/NF-κB signaling pathway

OBJECTIVE The greatest challenge in chemotherapy of ischemic stroke is the construction a suitable delivery system to overcome the poor physicochemical properties of drug and its low permeability across the blood brain barrier(BBB).METHODS In the present study,dendrimer,polyamidoamine(PAMAM),was synthesized as the nano-drug carriers.Angiopep-2,which has been proved excellent ability to cross the BBB,was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethylene glycol(PEG).Then scutellarin(STA)was encapsulated into the functionalized nanoparticles(NPs)to formulate Angiopep-2 modified STA-loaded PEG-PAMAM NPs.Ischemic stroke model was established to evaluate the treatment efficacy and protective mechanism of Angiopep-2-STA-PEG-PAMAM NPs.RESULTS The pharmacokinetics and biodistribu-tion demonstrated that Angiopep-2-STA-PEG-PAMAM NPs exhibited significantly higher plasma concentration from 1 h to 10 h after intravenous administration and improve accumulation in brain(4.7-fold)compared with STA solution.Moreover,prolonged elimination half-life(4.8-fold)and lower clearance(3.4-fold)were observed.The brain uptake study of 6-coumarin confirmed that Angiopep-2-PEG-PAMAM NPs possessed better brain targeting efficacy(3.2-fold)than PEG-PAMAM NPs.Angiopep-2-STA-PEG-PAMAM NPs obviously ameliorated infarct volume,neurological deficit,histopathological severity and neuronal apoptosis.In addition,Angiopep-2-STA-PEG-PAMAM NPs markedly inhibited the calcium content and the levels of IL-12p40,IL-13,IL-17 and IL-23.Furthermore,Angiopep-2-STA-PEG-PAMAM NPs significantly decreased the m RNA and protein expressions of HMGB1,TLR2,TLR4,TLR5,My D88,TRIF,TRAM,IRAK-4,TRAF6,IкBα,IKKβand NF-кBp65.CONCLUSION The results suggested that Angiopep-2modified scutellarin-loaded PEG-PAMAM nanocarriers possessed remarkable neuroprotective effects on ischemic stroke through modulation of inflammatory cascades and HMGB1/TLRs/MyD 88-induced NF-κB activation pathways.

真武汤调控ROCK/IKK/NF-κB通路关键分子表达改善脾肾阳虚型DN小鼠肾脏炎症损伤的机制研究

基于Rho相关卷曲螺旋激酶(Rho-associated coiled helix kinase,ROCK)/核因子-κB上游激酶(IκB kinase,IKK)/核因子-κB(nuclear factor kappaB,NF-κB)通路探讨真武汤对脾肾阳虚型糖尿病肾病(diabetic nephropathy,DN)小鼠的干预作用及机制。95只7周龄db/db雄性小鼠和25只7周龄db/m雄性小鼠适应性喂养1周,采用灌服大黄水煎液联合氢化可的松法复制脾肾阳虚型DN小鼠模型,随后进行模型评价,造模成功后随机分为模型组,真武汤高、中、低剂量组(33.8、16.9、8.45 g·kg^(-1)·d^(-1)),厄贝沙坦组(25 mg·kg^(-1)·d^(-1)),每组至少15只,持续干预8周。干预结束后,检测体质量、饮食量;测定血清肌酐(serum creatinine,Scr)、尿素氮(blood urea nitrogen,BUN)、空腹血糖(fasting blood glucose,FBG)、尿微量白蛋白(urinary albumin,uALb)、尿肌酐(urine creatinine,Ucr)含量,计算尿微量白蛋白与尿肌酐比值(ACR)、24 h尿蛋白(urinary protein,UTP);HE染色、Masson染色评价肾脏病理形态;采用蛋白免疫印迹(Western blot)法检测ROCK/IKK/NF-κB通路关键分子蛋白水平;采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-8(interleukin-8,IL-8)、白细胞介素^(-1)0(interleukin-10,IL-10)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量。与空白组相比,模型组小鼠BUN、uALb、SCr含量上升,24 h UTP、ACR上升,Ucr含量下降(P<0.05);肾小球增大,基底膜变厚且系膜基质出现增生,炎性细胞浸润,胶原纤维沉积;肾脏中ROCK1、ROCK2、IKK、NF-κB以及磷酸化后的核因子-κB上游激酶(p-IκB kinase,p-IKK)、磷酸化后的核因子-κB(p-nuclear factor-kappaB,p-NF-κB)、磷酸化后的NF-κB抑制蛋白(p-inhibitor of NF-κB,p-IκB)蛋白表达上升(P<0.05),NF-κB抑制蛋白(inhibitor of NF-κB,IκB)蛋白表达下降(P<0.05);炎症因子IL-1β、IL-6、IL-8、TNF-α含量升高(P<0.05),IL-10含量降低(P<0.05)。与模型组相比,各组小鼠BUN、uALb、SCr含量下降,24 h UTP、ACR均下降,Ucr含量升高(P<0.05);肾脏病理状态与模型组相比均有不同程度改善;真武汤高、中剂量组及厄贝沙坦组小鼠肾脏中ROCK1、ROCK2、IKK、NF-κB、p-IKK、p-NF-κB、p-IκB蛋白表达下降(P<0.05),IκB蛋白表达上升(P<0.05),血清炎症因子IL-1β、IL-6、IL-8、TNF-α含量降低(P<0.05),IL-10含量升高(P<0.05)。真武汤能够显著改善脾肾阳虚型DN小鼠肾功能及肾脏病理损伤,其具体机制与真武汤下调肾脏中ROCK/IKK/NF-κB通路关键分子表达抑制炎症反应发生有关。

MicroRNA-23a reduces lipopolysaccharide-induced cellular apoptosis and inflammatory cytokine production through Rho-associated kinase 1/sirtuin-1/nuclear factor-kappa B crosstalk

Background:MicroRNAs are closely associated with the progression and outcomes of multiple human diseases,including sepsis.In this study,we examined the role of miR-23a in septic injury.Methods Lipopolysaccharide(LPS)was used to induce sepsis in a rat model and H9C2 and HK-2 cells.miR-23a expression was evaluated in rat myocardial and kidney tissues,as well as H9C2 and HK-2 cells.A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability,apoptosis,and the secretion of inflammatory cytokines.Furthermore,the effect of Rho-associated kinase 1(ROCK1),a miR-23a target,on cell damage was evaluated,and molecules involved in the underlying mechanism were identified.Results:In the rat model,miR-23a was poorly expressed in myocardial(sham vs.sepsis 1.00±0.06 vs.0.27±0.03,P<0.01)and kidney tissues(sham vs.sepsis 0.27±0.03 vs.1.00±0.06,P<0.01).Artificial overexpression of miR-23a resulted in increased proliferative activity(DNA replication rate:Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:34.13±3.12 vs.12.94±1.21 vs.13.31±1.43 vs.22.94±2.26,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),decreased cell apoptosis(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:11.39±1.04 vs.32.57±2.29 vs.33.08±3.12 vs.21.63±2.35,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),and decreased production of inflammatory cytokines,including interleukin-6(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:59.61±5.14 vs.113.54±12.30 vs.116.51±10.69 vs.87.69±2.97 ng/mL;P<0.05,F=12.67,HK-2 cells:68.12±6.44 vs.139.65±16.62 vs.143.51±13.64 vs.100.82±9.74 ng/mL,P<0.05,F=9.83)and tumor necrosis factor-α(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:103.20±10.31 vs.169.67±18.84 vs.173.61±15.91 vs.133.36±12.32 ng/mL,P<0.05,F=12.67,HK-2 cells:132.51±13.37 vs.187.47±16.74 vs.143.51±13.64 vs.155.79±15.31 ng/mL,P<0.05,F=9.83)in cells.However,ROCK1 was identified as a miR-23a target,and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells.Moreover,ROCK1 suppressed sirtuin-1(SIRT1)expression to promote the phosphorylation of nuclear factor-kappa B(NF-κB)p65,indicating the possible involvement of this signaling pathway in miR-23a-mediated events.Conclusion:Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1,mediated through the potential participation of the SIRT1/NF-κB signaling pathway.