Downregulation of MUC1 Inhibits Proliferation and Promotes Apoptosis by Inactivating NF-κB Signaling Pathway in Human Nasopharyngeal Carcinoma

Objective To investigate the effect of mucin 1(MUC1)on the proliferation and apoptosis of nasopharyngeal carcinoma(NPC)and its regulatory mechanism.Methods The 60 NPC and paired para-cancer normal tissues were collected from October 2020 to July 2021 in Quanzhou First Hospital.The expression of MUC1 was measured by real-time quantitative PCR(qPCR)in the patients with PNC.The 5-8F and HNE1 cells were transfected with siRNA control(si-control)or siRNA targeting MUC1(si-MUC1).Cell proliferation was analyzed by cell counting kit-8 and colony formation assay,and apoptosis was analyzed by flow cytometry analysis in the 5-8F and HNE1 cells.The qPCR and ELISA were executed to analyze the levels of TNF-αand IL-6.Western blot was performed to measure the expression of MUC1,NFкB and apoptosis-related proteins(Bax and Bcl-2).Results The expression of MUC1 was up-regulated in the NPC tissues,and NPC patients with the high MUC1 expression were inclined to EBV infection,growth and metastasis of NPC.Loss of MUC1 restrained malignant features,including the proliferation and apoptosis,downregulated the expression of p-IкB、p-P65 and Bcl-2 and upregulated the expression of Bax in the NPC cells.Conclusion Downregulation of MUC1 restrained biological characteristics of malignancy,including cell proliferation and apoptosis,by inactivating NF-κB signaling pathway in NPC.

Apatinib reduces liver cancer cell multidrug resistance by modulating NF-κB signaling pathway

Objectives:This investigation aimed to elucidate the inhibitory impact of apatinib on the multidrug resistance of liver cancer both in vivo and in vitro.Methods:To establish a Hep3B/5-Fu resistant cell line,5-Fu concentrations were gradually increased in the culture media.Hep3B/5-Fu cells drug resistance and its alleviation by apatinib were confirmed via flow cytometry and Cell Counting Kit 8(CCK8)test.Further,Nuclear factor kappa B(NF-κB)siRNA was transfected into Hep3B/5-Fu cells to assess alterations in the expression of multidrug resistance(MDR)-related genes and proteins.Nude mice were injected with Hep3B/5-Fu cells to establish subcutaneous xenograft tumors and then categorized into 8 treatment groups.The treatments included oxaliplatin,5-Fu,and apatinib.In the tumor tissues,the expression of MDRrelated genes was elucidated via qRT-PCR,immunohistochemistry,and Western blot analyses.Results:The apatinibtreated mice indicated slower tumor growth with smaller size compared to the control group.Both the in vivo and in vitro investigations revealed that the apatinib-treated groups had reduced expression of MDR genes GST-pi,LRP,MDR1,and p-p65.Conclusions:Apatinib effectively suppresses MDR in human hepatic cancer cells by modulating the expression of genes related to MDR,potentially by suppressing the NF-κB signaling pathway.

Kuicolong-yu enema decoction retains traditional Chinese medicine enema attenuates inflammatory response ulcerative colitis through TLR4/NF-κB signaling pathway

BACKGROUND Ulcer colitis(UC)is a chronic,nonspecific,and noninfectious inflammatory bowel disease.Recently,Toll-like receptors(TLRs)have been found to be closely associated with clinical inflammatory diseases.Achieving complete remission in patients with intermittent periods of activity followed by dormancy is challenging.Moreover,no study has explored the mechanism by which Kuicolong-yu enema decoction retains traditional Chinese medicine enemas to attenuate the inflammatory response in UC.AIM To explore the mechanism by which Kuicolong-yu enema decoction retains traditional Chinese medicine enemas to attenuate the inflammatory response in UC.METHODS This prospective clinical study included patients who met the exclusion criteria in 2020 and 2021.The patients with UC were divided into two groups(control and experimental).The peripheral blood of the experimental and control groups were collected under aseptic conditions.The expression of TLR4 protein,NF-κB,IL-6,and IL-17 was detected in the peripheral blood of patients in the experimental group and control group before and 1 month after taking the drug.Linear co rrelation analysis was used to analyze the relationship between the expression level of TLR4 protein and the expression levels of downstream signal NF-κB and inflammatory factors IL-6 and IL-17,and P<0.05 was considered statistically significant.RESULTS There were no significant differences in the patient characteristics between the control and experimental groups.The results showed that the expression levels of TLR4 and NF-κB in the experimental group were significantly lower than those in the control group(P<0.05).The levels of IL-6 and IL-17 in the experimental group were significantly lower than those in the control group(P<0.05).The TLR4 protein expression in the experimental group was positively correlated with the expression level of downstream signal NF-κB and was positively correlated with the levels of downstream inflammatory cytokines IL-6 and IL-17(r=0.823,P<0.05).CONCLUSION Kuicolong-yu enema decoction retains traditional Chinese medicine enema attenuates the inflammatory response of UC through the TLR4/NF-κB signaling pathway.

Aszonapyrone A Isolated from Neosartorya spinosa IFM 47025 Inhibits the NF-κB Signaling Pathway Activated by Expression of the Ependymoma-Causing Fusion Protein ZFTA-RELA

Ependymoma is a rare and chemotherapy-resistant brain tumor, which has resulted in a delay in the development of drugs to treat it. A subclass of supratentorial ependymomas (ST-EPN), designated ST-EPN-zinc finger-translocation-associated (ZFTA, ST-EPN-ZFTA), exhibits the expression of a fusion protein comprising ZFTA and v-rel reticuloendotheliosis viral oncogene homolog A (RELA), an effector transcription factor of the nuclear factor-kappa B (NF-κB) pathway (ZFTA-RELA). The expression of ZFTA-RELA results in the hyperactivation of the oncogenic NF-κB signaling pathway, which ultimately leads to the development of ST-EPN-ZFTA. To identify inhibitors of the NF-κB signaling pathway activated by the expression of ZFTA-RELA, we used a doxycycline-inducible ZFTA-RELA-expressing NF-κB reporter cell line and found that extracts of the fungus Neosartorya spinosa IFM 47025 exhibited NF-κB inhibitory activity. We identified eight compounds [aszonapyrone A (2), sartorypyrone A (3), epiheveadride (4), acetylaszonalenin (5), (R)-benzodiazepinedione (6), aszonalenin (7), sartorypyrone E (8) and (Z, Z)-N,N’-(1,2-bis[(4-methoxyphenyl)methylene]-1,2-ethanediyl)bis-formamide (9)] from N. spinosa IFM 47025 culture extract using a variety of chromatographic techniques. The structures of these compounds were identified through the analysis of various instrumental data (1D, 2D-NMR, MS, and optical rotation). The NF-κB responsive reporter assay indicated that compounds 2, 3, 5, 7, and 9 exhibited inhibitory activity. We further evaluated the inhibitory activity of these compounds against the expression of endogenous NF-κB responsive genes (CCND1, L1CAM, ICAM1, and TNF) and found that compound 2 showed significant inhibitory activity. Further studies are required to elucidate the mechanism of action of compound 2, which may serve as a lead compound for the development of a novel therapy for ST-EPN-ZFTA.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

Acupuncture at Back-Shu point improves insomnia by reducing inflammation and inhibiting the ERK/NF-κB signaling pathway

BACKGROUND Insomnia is a disease where individuals cannot maintain a steady and stable sleep state or fail to fall asleep.Western medicine mainly uses sedatives and hypnotic drugs to treat insomnia,and long-term use is prone to drug resistance and other adverse reactions.Acupuncture has a good curative effect and unique advantages in the treatment of insomnia.AIM To explore the molecular mechanism of acupuncture at Back-Shu point for the treatment of insomnia.METHODS We first prepared a rat model of insomnia,and then carried out acupuncture for 7 consecutive days.After treatment,the sleep time and general behavior of the rats were determined.The Morris water maze test was used to assess the learning ability and spatial memory ability of the rats.The expression levels of inflammatory cytokines in serum and the hippocampus were detected by ELISA.qRTPCR was used to detect the mRNA expression changes in the ERK/NF-κB signaling pathway.Western blot and immunohistochemistry were carried out to evaluate the protein expression levels of RAF-1,MEK-2,ERK1/2 and NF-κB.RESULTS Acupuncture can prolong sleep duration,and improve mental state,activity,diet volume,learning ability and spatial memory.In addition,acupuncture increased the release of 1L-1β,1L-6 and TNF-αin serum and the hippocampus and inhibited the mRNA and protein expression of the ERK/NF-κB signaling pathway.CONCLUSION These findings suggest that acupuncture at Back-Shu point can inhibit the ERK/NF-κB signaling pathway and treat insomnia by increasing the release of inflammatory cytokines in the hippocampus.

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.

Low-temperature Plasma Promotes Fibroblast Proliferation in Wound Healing by ROS-activated NF-κB Signaling Pathway

Low-temperature plasma(LTP)has shown great promise in wound healing,although the underlying mechanism remains poorly understood.In the present study,an argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro and skin wounds in BALB/c mice.The in vitro analysis revealed that treatment of fibroblasts with LTP for 15 s resulted in a significant increase in cell proliferation,secretion of epidermal growth factor(EGF)and transforming growth factor-β1(TGF-β1),production of intracellular reactive oxygen species(ROS),and the percentage of cells in S phase,protein expression of phosphorylated p65(P-p65)and cyclin D1,but a noted decrease in the protein expression of inhibitor kappa B(IκB).The in vivo experiments demonstrated that 30-s LTP treatment enhanced the number of fibroblasts and the ability of collagen synthesis,while 50-s treatment led to the opposite outcomes.These results suggested that LTP treatment promotes the fibroblast proliferation in wound healing by inducing the generation of ROS,upregulating the expression of P-p65,downregulating the expression of IκB,and activating the NF-κB signaling pathway and consequently altering cell cycle progression(increased DNA synthesis in S phage).

Immunoregulatory polysaccharides from Apocynum venetum L.flowers stimulate phagocytosis and cytokine expression via activating the NF-κB/MAPK signaling pathways in RAW264.7 cells

Two immunomodulatory polysaccharides(Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cells. Both the level of released nitric oxide(NO) and expression of inducible nitric oxide synthase(iNOS) m RNA were significantly enhanced in the RAW264.7 macrophages cells treated by Vp2a-Ⅱ and Vp3. Vp2a-Ⅱ(100–800 μg/m L) and Vp3(400 μg/mL) could significantly increase the phagocytic activity of RAW264.7 cells and the secretion and m RNA expression of TNF-α and IL-6 in a concentrationdependent manner through affecting mitogen-activated protein kinase(MAPK) activity and nuclear factor κB(NF-κB) nuclear translocation. Vp2a-Ⅱ might activate the MAPK signaling pathways and induce the nuclear translocation of NF-κB p65, whilst Vp3 likely activated the NF-κB and MAPK signaling pathways without influencing the p38 MAPK route.

Effect of dexmedetomidine on the prevention of PSH in patients with severe craniocerebral injury by regulating TLR4/My D88/NF-kappa B signaling pathway

Objective:To investigate the clinical efficacy of dexmedetomidine in the regulation of TLR4/My D88/NF-κB in the prevention of paroxysmal sympathetic over-excitation (PSH) in patients with severe head injury. Methods:One hundred patients with severe head injury who were admitted to our hospital from September 2016 to May 2019 were enrolled. The randomized digital table method was divided into 50 cases in the study group and the control group. Patients in the study group were given dexmedetomidine at a dose of 1.0 μg/kg before anesthesia induction, followed by infusion at 0.4 μg / (kg·h), and the control group was injected with the same amount of normal saline. The incidence of PSH, clinical symptoms, imaging findings, mechanical ventilation time, tracheal intubation/incision duration, ICU hospitalization time, total length of hospital stay, and GCS scores three months after discharge were compared between the two groups. At the same time, the fluorescence intensity, TLR4, NF-κB expression level and tumor necrosis factor-α (TNF-α) expression levels in peripheral blood CD14+ monocytes of the two groups were detected. Results:The incidence of PSH was significantly lower in the study group than in the control group at 7 and 3 months (P<0.05). The total length of hospital stay, duration of ICU hospitalization, intraoperative tracheotomy, and mechanical ventilation time were significantly lower in the study group than in the control group. And the GCS score was higher than the control group, and the difference was statistically significant (P<0.05). In addition, the imaging results showed that there were some differences in the location of imaging lesions between the two groups. The proportion of lesions in the ventricular system and surrounding areas was higher in the control group than in the study group (P<0.05). And the T14-T3 CD14+ PBMC MyD88 fluorescence intensity, TLR4 and NK-κB positive expression rate were significantly higher than those of T0 (P<0.05), but the MyD88 fluorescence intensity, TLR4 and NK-κB positive expression rate in the study group were significantly lower than those in the control group at T1~T3 (P<0.05). The levels of serum TNF-α in T1~T3 groups were significantly higher than those in T0 (P<0.05), but the levels of serum TNF-α in T1~T3 in the study group were significantly lower than those in the control group (P< 0.05). Conclusions:Dexmedetomidine can reduce the oxidative stress response in patients with severe head injury by inhibiting TLR4/My D88/NF-κB signaling pathway, thus effectively reducing the risk of PSH and improving the prognosis of patients.

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.

Mechanism and effect of curcumin on apoptosis of EAE mice by regulating TLRs/NF-κB signaling pathway

Objective:To investigate theanti apoptosis effect of curcumin(cur)in experimental autoimmune encephalomyelitis(EAE)mice by regulating TLRs/NF-κB signaling pathway and its mechanism.Methods:45 C57BL/6 mice were randomly divided into the control group,EAE group,curcumin group,15 mice in each group.Blank groups are not processed.The EAE model was established by classical modeling method in the EAE group and the curcumin group.From the day of modeling,the blank group and the EAE group were intraperitoneally injected with 1ml/kg/d of normal saline,Curcumin group was given 100 mg/kg/d continuous intraperitoneal injection of curcumin extract.With Benson EAE group and Curcumin group mice were killed at the peak of the disease.The blank group and the rest of the mice were killed after 4 weeks of feeding,and the spinal cord tissue was taken out to separate the lumbar enlargement segment.The effects of curcumin on the pathological changes of spinal cord tissue in EAE mice were observed by HE staining and TUNEL staining,and the expression of apoptosis positive cells was calculated.The distribution and co aggregation of apoptosis related proteins Bcl-2 and Bax with spinal cord tissue were observed by double immunofluorescence staining The protein levels of TLR4,NF-κBp65 and MyD88 were detected by Western blot.Results:compared with the blank group,TUNEL staining increased the number of apoptotic cells and the apoptotic rate in EAE group(P<0.05);the expression of apoptosis related protein Bcl-2 decreased and the expression of Bax increased in EAE group(P<0.05),The protein of TLR4,NF-κBp65 and MyD88 in spinal cord tissue of mice were increased by blot detection(P<0.05);compared with EAE group,the number of apoptotic cells in spinal cord tissue of curcumin group was decreased by TUNEL staining,and the apoptosis rate was decreased(P<0.05);the expression of apoptosis related protein Bcl-2 was increased,the expression of Bax protein was decreased,and Western blot was used to detect the expression of apoptosis related protein The protein of TLR4,NF-κBp65 and MyD88 in spinal cord tissue of mice were decreased by blot(P<0.05).Conclusion:Curcumin has anti apoptotic effect on EAE mice,and its mechanism may be related to the inhibition of TLRs/NF-κB signaling pathway and the reduction of apoptotic protein production.

miR-146a-5p affects inflammation response of trophoblast by inhibiting TRAF6/NF-кB signaling pathway

Objective:To investigate the association of Micro-rna(miR)-146a-5p expression with preeclampsia,and further explore the potential mechanism involved.Methods:Compared with the blank control group,the expressions of miR-146a-5p and TRAF6 were detected in lipopolysaccharide(LPS)-induced JEG-3 cells.Chorionic carcinoma cell JEG-3 in vitro culture are divided into control,miR-146a-5p mimic+lipopolysaccharide(lps),miR-146a-5p mimic and miR-146a-5p inhibitor groups.qRT-PCR analysis were used to detect the mRNA of miR-146a-5p,IL-1β,IL-6,IL-8 and TNF-α.Western blot assays were carried out to determine the protein expression of TRAF6/NF-кB pathway related proteins.Results:1.miR-146a expression in miR-146a mimic group were significantly higher than the other three groups(P<0.05).2.Compared with the control group,the expression level of miR-146a-5p in JEG-3 cells induced by LPS was significantly increased,and the expression level of TRAF6 was significantly reduced(P<0.05).3.Compared with the control group,the mRNA expression levels of IL-1β,IL-6,IL-8,and TNF-αdecreased significantly after using miR-146a mimic(P<0.05).After adding miR-146a inhibitor,the mRNA expression levels of IL-1β,IL-6,IL-8,and TNF-αwere significantly increased(P<0.05).However,compared with the mimic+LPS group,the difference was not statistically significant(all P>0.05).The results of Western Blot showed that the expression of TRAF6 and NF-κB protein in JEG-3 cells decreased significantly after adding miR-146a mimic and increased after adding miR-146a inhibitor.Conclusion:MiR-146-5p can affect the inflammation response of Maternal-fetal interface by inhibiting TRAF6/NF-кB signaling pathway in preeclampsia.

Research progreess on relevant diseases of RhoA/ROCK signaling pathway

RhoA (Ras homolog gene family member A) belongs to the Rho subfamily of GTPases. ROCK (Rho—associated coiled—coil forming protein kinase) is downstream of the active RhoA and affects the generation and secretion of cellular element, which will result in relevant biologic effects. The RhoA/ROCK signaling pathway consists of these serious reactions. Therefore, the activation and inhibition of this pathway are closely related to the occurrence and development of many diseases. The research on the molecular mechanism of these diseases may be instructive and helpful to the clinical treatmen and prognosis of diseases. Recent studies of these typical diseases related to RhoA/ROCK signaling pathway are viewed in this article.

Luteolin affects the EMT transformation and cell biological behavior of osteosarcoma U2OS cells via the PI3K/AKT/NF-κB signaling pathway

Objective:To investigate the effect of luteolin on the Epithelial-mesenchymal transition(EMT)of osteosarcoma cell line U2OS and its molecular mechanism by regulating phosphoinositide 3-kinase/protein kinase B/nuclear factor-kappa B(PI3K/AKT/NF-κB)signaling pathway.Methods:U2OS cells were treated in different concentrations(10,20 and 40μmol/L)of luteolin.MTT was used to detect the effect of luteolin on the proliferation of osteosarcoma U2OS cells.Transwell chamber assay was used to detect the influence of luteolin on migration of U2OS cells.qPCR was used to detect the influence of luteolin on the mRNA expression of Bax,Bcl-2 and Caspase-2 in U2OS cells.Western blot was used to observe the change of p-PI3K,p-AKT,p-IKK and NF-κB proteins.Immunofluorescence was used to detect the influence of luteolin on the protein expression of E-cadherin and vimentim.Results:Luteolin inhibited significantly the proliferation of U2OS cells(P<0.05)in a time-concentration-dependent manner.Luteolin inhibited significantly the migration of U2OS cells(P<0.05).After treatment with luteolin,the mRNA expression of Bax and Caspase-2 was increased significantly(P<0.05),but Bcl-2 was reduced significantly(P<0.05)in U2OS cells.The protein expression of p-PI3K,p-AKT,p-IKK,NF-κB,E-cadherin and vimentin was decreased significantly(P<0.05).Conclusions:Luteolin inhibits the proliferation,migration and EMT transformation of osteosarcoma U2OS cells,which may be related to the inhibition of PI3K/AKT/NF-κB signaling pathway.

基于RhoA/ROCK/NF-κB信号通路探究益肾活血化痰方对动脉粥样硬化小鼠炎症反应的影响

目的探讨益肾活血化痰方对动脉粥样硬化(AS)小鼠炎症反应的影响及机制。方法采用高脂饲养法制备AS模型。将小鼠分为对照组、模型组、药物组(303 mg/kg益肾活血化痰方灌胃给药)、抑制剂组(仅给予5 mg/kg的ROCK抑制剂Y-27632腹腔注射)、阳性药物组(3.03 mg/kg的阿托伐他汀灌胃给药),每组10只。油红O染色观察小鼠主动脉组织斑块形成。ELISA法检测血清炎症因子(TNF-α、IL-1β、IL-6)水平,自动生化分析仪检测血脂(TG、TC、LDL-C、HDL-C)水平,免疫组化法检测主动脉组织IL-1β表达。Western blot法检测主动脉组织RhoA、ROCK蛋白表达及NF-κB p65磷酸化水平。结果与对照组比较,模型组小鼠血清TG、TC、LDL-C、TNF-α、IL-1β、IL-6水平显著升高(P<0.05),HDL-C水平显著降低(P<0.05),主动脉斑块面积增大(P<0.05),主动脉组织IL-1β、RhoA、ROCK蛋白表达及p-NF-κB p65/NF-κB p65显著升高(P<0.05)。与模型组比较,药物组、抑制剂组、阳性药物组小鼠血清TG、TC、LDL-C、TNF-α、IL-1β、IL-6水平显著降低(P<0.05),HDL-C水平显著升高(P<0.05),主动脉斑块面积减小(P<0.05),主动脉组织IL-1β、RhoA、ROCK蛋白表达及p-NF-κB p65/NF-κB p65显著降低(P<0.05)。药物组与阳性药物组、抑制剂组各指标比较差异无统计学意义(P>0.05)。结论益肾活血化痰方可能通过抑制RhoA/ROCK/NF-κB信号通路激活,改善AS小鼠血脂代谢,减轻AS小鼠炎症反应。

LncRNA Airsci increases the inflammatory response after spinal cord injury in rats through the nuclear factor kappa B signaling pathway

Spinal cord injury(SCI)is a serious traumatic event to the central nervous system.Studies show that long non-coding RNAs(lncRNAs)play an important role in regulating the inflammatory response in the acute stage of SCI.Here,we investigated a new lncRNA related to spinal cord injury and acute inflammation.We analyzed the expression profile of lncRNAs after SCI,and explored the role of lncRNA Airsci(acute inflammatory response in SCI)on recovery following acute SCI.The rats were divided into the control group,SCI group,and SCI+lncRNA Airsci-siRNA group.The expression of inflammatory factors,including nuclear factor kappa B[NF-κB(p65)],NF-κB inhibitor IκBαand phosphorylated IκBα(p-IκBα),and the p-IκBα/IκBαratio were examined 1–28 days after SCI in rats by western blot assay.The differential lncRNA expression profile after SCI was assessed by RNA sequencing.The differentially expressed lncRNAs were analyzed by bioinformatics technology.The differentially expressed lncRNA Airsci,which is involved in NF-κB signaling and associated with the acute inflammatory response,was verified by quantitative real-time PCR.Interleukin(IL-1β),IL-6 and tumor necrosis factor(TNF-α)at 3 days after SCI were measured by western blot assay and quantitative real-time PCR.The histopathology of the spinal cord was evaluated by hematoxylin-eosin and Nissl staining.Motor function was assessed with the Basso,Beattie and Bresnahan Locomotor Rating Scale.Numerous differentially expressed lncRNAs were detected after SCI,including 151 that were upregulated and 186 that were downregulated in the SCI 3 d group compared with the control group.LncRNA Airsci was the most significantly expressed among the five lncRNAs involved in the NF-κB signaling pathway.LncRNA Airsci-siRNA reduced the inflammatory response by inhibiting the NF-κB signaling pathway,alleviated spinal cord tissue injury,and promoted the recovery of motor function in SCI rats.These findings show that numerous lncRNAs are differentially expressed following SCI,and that inhibiting lncRNA Airsci reduces the inflammatory response through the NF-κB signaling pathway,thereby promoting functional recovery.All experimental procedures and protocols were approved by the approved by the Animal Ethics Committee of Jining Medical University(approval No.JNMC-2020-DW-RM-003)on January 18,2020.

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.

Ragweed pollen induces allergic conjunctivitis immune tolerance in mice via regulation of the NF-κB signal pathway

AIM:To investigate the feasibility and mechanism of immune tolerance in allergic conjunctivitis.METHODS:The allergic conjunctivitis immune tolerance mice model was established by ragweed pollen(RW)and the related cytokines were detected.The mice were divided into 9 groups and the maslinic acid(MA)or PBS were given for different group after modeling.The expression levels of chemokine ligand 5(CCL5)and P-65 in the conjunctival tissue were analyzed by immunohistochemistry,quantitative reverse transcription polymerase chain reaction(q RT-PCR)and Western blot.The percentage of interleukin-17(IL-17)and CD4+CD25+in the splenocyte supernatant was analyzed by flow cytometry.Fur thermore,the serum and splenocyte supernatant concentration of total-IgE,interleukin-10(IL-10),and IL-17 was analyzed by enzyme linked immune response(ELISA).RESULTS:After the model was established,symptoms of conjunctivitis were alleviated,the level of P-65,CCL5,IL-17,and total-IgE was raised,while the expression of IL-10,CD4+CD25+was decreased.This result fully demonstrated that a typical IL-17/regulatory-T-cells(Treg cells)imbalance and NF-κB activation.When the NF-κB signal pathway was suppressed,it showed that there was a further relief of conjunctivitis in mice.At the same time,the expression of total-IgE,IL-17,and CCL5 was decreased and the expression of anti-inflammatory factor(IL-10,CD4+CD25+)was increased.CONCLUSION:In the state of immune tolerance,symptoms of conjunctivitis in mice are alleviated,the Th-17 cells of allergic conjunctivitis mice are inhibited,and Treg cells activity is enhanced.

Activation of CD40 by Soluble Recombinant Human CD40 Ligand Inhibits Human Glioma Cells Proliferation via Nuclear Factor-κB Signaling Pathway

As CD40 transduces activation signals involved in inflammatory and immune disorders,we explored the expression and response to CD40 engagement in human glioma cell lines in this study.The CD40 expression in BT-325 and U251 cells was flow cytometrically detected.The cells were incubated with srhCD40L for 72 h to assess its effects on cell growth in vitro.TNF-α expression was quantified by real-time PCR,and protein expression was analyzed by ELISA.The I-κb mRNA was detected by RT-PCR.I-κB expression decreased after stimulation with 1 μg/mL srhCD40L,but it was upregulated after the cells were pretreated with CD40 antibody.srhCD40L significantly inhibited the proliferation of the CD40+ human glioma cells.The stimulation of CD40+ glioma cells with soluble CD40L (CD154) up-regulated the expression of TNF-α at both mRNA and protein levels.We are led to conclude that CD40L/CD40 could inhibit human glioma cells through I-κb signaling pathway.Interferon-γ can augment CD40 expression and the inhibitory effect of CD40 ligand on cell growth in vitro.These results suggest that srhCD40L may benefit the therapy strategy of glioma.