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.

Quercetin regulates depression-like behavior in CUMS rat models via TLR4/NF-κB signaling

Background:Depression is becoming increasingly prevalent around the world,imposing a substantial burden on individuals,families,as well as society.Quercetin is known to be highly effective in treating depression.However,additional research is needed to dissect the mechanisms of its anti-depressive effects.Methods:For this study,Sprague-Dawley(SD)rats were randomized into the control,model,quercetin,or fluoxetine group.The latter three groups were exposed to chronic unpredictable mild stress(CUMS)for 42 d.The first two groups received saline solution daily via oral gavage.Meanwhile,the quercetin group was orally administered a quercetin suspension(52.08 mg/kg)every day,while the fluoxetine group was orally administered a fluoxetine solution(2.08 mg/kg).Here,fluoxetine served as the positive control drug to compare the therapeutic effects of quercetin.The experimental period was 6 weeks.Depressive behaviors in rats were assessed through various physiological and behavioral measures.Additionally,pathological changes in hippocampal tissues were examined using Nissl staining.Serum cytokines were detected using an enzymelinked immunosorbent assay(ELISA),and immunohistochemistry was employed to quantify the levels and integral optical density(IOD)values of ionized calcium binding adaptor molecule-1(Iba-1)expression in the brain.Real-time fluorescence quantitative PCR(RT-qPCR)was utilized to evaluate the mRNA levels of inflammatory indicators as well as toll-like receptor 4(TLR4),and nuclear factor-κappa B P65(NF-κB P65)in hippocampus.Western blot(WB)technique was employed to observe the protein levels of TLR4,NF-κB P65,and phospho-NF-κB P65(p-NF-κB P65).Results:After 42 d of exposure to CUMS,rats exhibited a slow increase in body weight,a reduction in food intake,an abnormal preference for sugar water,and aberrant open-field behaviors.Pathological analysis revealed the disintegration,rupture,interruption,and disorganization of hippocampal neuronal cells after CUMS exposure,along with a decrease in Nissl bodies in the CA1 region.This was accompanied by the elevated expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in the serum and the upregulation of IL-1β,IL-6,and TNF-αmRNA expression in the hippocampus.Increases in Iba-1-positive cells and the IOD values of Iba-1 were detected in hippocampal microglia.Furthermore,TLR4 and NF-κB P65 mRNA and protein levels were upregulated in hippocampal tissues.Quercetin,an antidepressant,could alleviate depression-like symptoms in rats and downregulate inflammatory factors associated with the TLR4/NF-κB signaling pathway in hippocampal microglia,and its therapeutic effect was comparable to fluoxetine.Conclusion:In rat models of CUMS,quercetin may act as an antidepressant by inhibiting inflammation in hippocampal microglia via TLR4/NF-κB signaling pathway.These results offer experimental and theoretical support for applying quercetin in the clinical management of depression.

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.

Anti-inflammation Effects of Sinomenine on Macrophages through Suppressing Activated TLR4/NF-kB Signaling Pathway

Sinomenine(SN)has been used in the clinical treatment of systemic lupus erythematosus and rheumatoid arthritis for many years.Studies showed that SN held protective effects such as anti-inflammation,scavenging free radicals and suppressing immune response in many autoimmune diseases.The purpose of the present study is to explore the mechanism of anti-inflammation of SN on lipopolysaccharide(LPS)-induced macrophages activation and investigate whether the TLR4/NF-κB signaling pathway participated in.Macrophages isolated from mouse peritoneal cavity were stimulated by 1 pg/mL LPS for 24 h.And then the cells were treated with various concentrations of SN,TLR4 inhibitor respectively for additional 48 h.Drug toxicity was detected by MTT assay and Transwell experiment was used to assess chemotaxis.Furthermore,TLR4 and MyD88 mRNA levels were detected by real-time PCR.Western blotting was used to examine TLR4,MyD88 and phosphorylated IκB protein expression in macrophages.Immunofluorescence assay was applied to observe p65 NF-κB protein expression in macrophage nucleus.We extracted macrophages with high purity and activity from the abdominal cavity of mice.SN remarkably inhibited the chemotaxis and secretion function of LPS-stimulated macrophages.It also down-regulated both the protein levels of inflammatory cytokines(TNF-α,IL-β and IL-6)and the RNA and protein levels of the key factors(TLR4,MyD88,p-IkB)in TLR4 pathway.The expression of p65 NF-κB protein in nuclei was down-regulated,which was correlated with a similar decrease in p-IκB protein level.In conclusion,SN can inhibit the LPS induced immune responses in macrophages by blocking the activated TLR4/NF-κB signaling pathway.These results may provide a therapeutic approach to regulate inflammatory responses.

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.

基于TLR4/MAPK/NF-κB信号通路探讨萝卜硫素抗甲状腺乳头状癌的作用

目的探讨萝卜硫素对裸鼠人乳头瘤状甲状腺癌移植瘤的作用及机制。方法采用皮下注射TPC-1细胞的方法构建裸鼠人乳头瘤状甲状腺癌移植瘤模型,随后将模型小鼠随机分为模型组、萝卜硫素低剂量组和萝卜硫素高剂量组,每组10只。萝卜硫素低、高剂量组分别灌胃给予5 mg/kg和20 mg/kg萝卜硫素,模型组灌胃给予等量生理盐水,1次/d,连续25 d。每5天测量肿瘤生长情况;取材后称量肿瘤质量,计算肿瘤抑制率;TUNNEL法观察肿瘤组织细胞凋亡情况;ELISA法检测血清炎症因子和氧化应激指标水平;流式细胞仪检测血液CD4^(+)/CD8^(+)淋巴细胞亚群比值;Western blot法检测肿瘤组织TLR4、P38和p-NF-κB蛋白的表达水平。结果与模型组比较,萝卜硫素给药组的肿瘤生长速度均明显被抑制(P<0.01),肿瘤质量明显减轻(P<0.01),肿瘤抑制率明显升高(P<0.01),肿瘤细胞凋亡阳性率明显升高(P<0.01),血清TNF-α、IL-1β和IL-6水平均明显降低(P<0.01),血清GSH-Px和SOD水平均明显升高(P<0.01),而MDA水平明显降低(P<0.01),血液CD4^(+)/CD8^(+)淋巴细胞亚群比值明显升高(P<0.01),肿瘤组织TLR4、P38、p-NF-κB蛋白相对表达量明显降低(P<0.01),且呈剂量依赖性。结论萝卜硫素具有抗甲状腺乳头状癌的作用,其机制主要与调控TLR4/MAPK/NF-κB信号通路、抗炎和氧化应激以及免疫调节有关。

Effects of Polysaccharides from Dicliptera chinensis(L.)Nees.on TLR4/NF-κB Signaling Pathway in Cell Model of Non-alcoholic Fatty Liver

[Objectives]To observe the effects of polysaccharides from Dicliptera chinensis(L.)Nees.on the expression of TLR/NF-κB pathway related proteins in HepG2 cells induced by oleic acid,and to explore the possible mechanism of polysaccharides from D.chinensis(L.)Nees.in the treatment of non-alcoholic fatty liver disease(NAFLD).[Methods]HepG2 cells were induced with oleic acid to establish a non-alcoholic fatty liver cell model.After intervention with 0.25 and 0.5 mg/mL of D.chinensis(L.)Nees.polysaccharides,the ALT and AST activity and TG and TC contents were detected with kits,and the changes in the expression of CDK5,TLR4,p-NF-κB and NF-κB were analyzed using Western-blotting.[Results]In the HepG2 cells induced with oleic acid,the ALT and AST activity increased significantly,the TG and TC contents increased significantly,and the expression levels of CDK5,TLR4 and p-NF-κB proteins up-regulated significantly.In the HepG2 cells intervened with D.chinensis(L.)Nees.polysaccharides,the activity of ALT and AST,the contents of TG and TC,and the expression levels of CDK5,TLR4 and p-NF-κB proteins all reduced significantly.[Conclusions]Polysaccharides from D.chinensis(L.)Nees.may interfere with NAFLD by inhibiting the TLR4/NF-κB pathway.

Exopolysaccharides of Lactobacillus rhamnosus GG ameliorate Salmonella typhimurium-induced intestinal inflammation via the TLR4/NF-κB/MAPK pathway

Background Salmonella typhimurium(S.T),as an important foodborne bacterial pathogen,can cause diarrhea and gastroenteritis in humans and animals.Numerous studies have confirmed that exopolysaccharides(EPSs)have various biological functions,but the mechanism through which EPSs improve the immunity of animals against the invasion of pathogenic bacteria is unclear.Here,we explored the protective effect of EPSs of Lactobacillus rhamnosus GG(LGG)on the S.T-infected intestine.Methods Mice received adequate food and drinking water for one week before the start of the experiment.After 7 d of prefeeding,2×108 CFU/mL S.T solution and an equivalent volume of saline(control group)were given orally for 1 d.On the fourth day,the mice were treated with 0.5 mg/mL EPSs,1.0 mg/mL EPSs,2.0 mg/mL EPSs,or 2.0 mg/mL penicillin for 7 d.Finally,the body and relative organ weight,histological staining,and the levels of antioxidant enzyme activity and inflammatory cytokines were determined.Results The S.T-infected mice exhibited symptoms of decreased appetite,somnolence,diarrhea and flagging spirit.Treatment with EPSs and penicillin improved the weight loss of the mice,and the high dose of EPSs showed the best therapeutic effect.EPSs significantly ameliorated S.T-induced ileal injury in mice.High-dose EPSs were more effective than penicillin for alleviating ileal oxidative damage induced by S.T.The mRNA levels of inflammatory cytokines in the ileum of mice showed that the regulatory effects of EPSs on inflammatory cytokines were better than those of penicillin.EPSs could inhibit the expression and activation of key proteins of the TLR4/NF-κB/MAPK pathway and thereby suppress the level of S.T-induced ileal inflammation.Conclusions EPSs attenuate S.T-induced immune responses by inhibiting the expression of key proteins in the TLR4/NF-κB/MAPK signaling pathway.Moreover,EPSs could promote bacterial aggregation into clusters,which may be a potential strategy for reducing the bacterial invasion of intestinal epithelial cells.

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.

Inhibitory Effect of Bergenin on TLR-4/NF-κB Signal Pathway in Reducing Allergic Rhinitis in Mice

[Objectives]To explore the effect and possible mechanism of bergenin in relieving allergic rhinitis(AR)in mice.[Methods]50 C57/BL6 mice were randomly divided into blank group(n=10),model group(n=10)and high(100 mg/kg),medium(50 mg/kg)and low(25 mg/kg)dose bergenin groups with 10 mice in each group.Except for the blank group,the other mice were sensitized by basic ways combined with attack to replicate the AR model.From the 15th d of modeling(from the second d after the end of the basic modeling),the drug group was given bergenin orally for 15 d,and the blank group and model group were given the same volume of normal saline once a day.24 h after the last establishment of the model,the content of interleukin 4(IL-4),IL-6,TNF-αand IL-1βin nasal lavage fluid and serum of mice in each group was detected by ELISA.The expression of TLR-4,NF-κB and p-NF-κB in nasal mucosa of mice was detected by Western blot.[Results]Compared with the blank group,the content of inflammatory factors IL-4,IL-6,TNF-αand IL-1βin nasal lavage fluid and serum of model group was significantly increased,and the protein expression of TLR-4 and p-NF-κB was significantly increased.After the intervention of bergenin,the content of IL-4,IL-6,TNF-αand IL-1βin nasal lavage fluid and serum and TLR-4 and p-NF-κB protein in tissue was significantly inhibited in bergenin group.[Conclusions]Bergenin can effectively reduce allergic inflammation in AR model mice,and its mechanism may be related to inhibition of inflammation and down-regulation of TLR-4/NF-κB signal pathway.

水飞蓟素通过共同抑制TLR4/NF-κB和TNF-α/ROS/P38MAPK通路减轻糖尿病肾病大鼠肾脏损伤的研究

目的:探讨水飞蓟素通过共同抑制Toll样受体4/核因子-κB(TLR4/NF-κB)和肿瘤坏死因子α/活性氧簇/P38丝裂原活化蛋白激酶(TNF-α/ROS/P38MAPK)通路减轻糖尿病肾病(DN)大鼠肾脏损伤的机制。方法:选取健康SD大鼠50只,按随机数字表法分为对照组、模型组、低剂量水飞蓟素组、中剂量水飞蓟素组、高剂量水飞蓟素组,每组10只。建模成功并治疗8周后,生化分析仪测定各组大鼠空腹血糖(FBG)、血肌酐(Scr)、尿素氮(BUN)、胱抑素C(Cys-C)、β_(2)微球蛋白(β_(2)-MG)、24 h尿蛋白(UTP)水平;计算肾脏指数;试剂盒检测各组大鼠肾组织丙二醛(MDA)、总抗氧化能力(T-AOC)水平;RT-PCR检测各组大鼠TLR4,NF-κB,TNF-α和P38MAPK等mRNA的表达水平。结果:与对照组相比,模型组体质量、T-AOC均降低(P<0.05),肾脏指数和FBG,Scr,BUN,Hcy,Cys-C,β_(2)-MG,24 h UTP,MDA,TLR4 mRNA,NF-κB p65 mRNA,TNF-αmRNA,P38MAPK mRNA均升高(P<0.05)。低、中、高剂量水飞蓟素组体质量、T-AOC水平均低于对照组,且均高于模型组(P<0.05);低、中、高剂量水飞蓟素组肾脏指数和FBG,Hcy,Cys-C,β_(2)-MG,24 h UTP,MDA,TLR4 mRNA,NF-κB p65 mRNA,TNF-αmRNA,P38MAPK mRNA均高于对照组,且均低于模型组(P<0.05);低、中剂量水飞蓟素组Scr,BUN水平均高于对照组,且低于模型组(P<0.05);高剂量水飞蓟素组Scr,BUN水平均低于模型组(P<0.05),高于对照组,但差异无统计学意义(P>0.05)。结论:水飞蓟素可调控TLR4/NF-κB和TNF-α/ROS/P38MAPK通路,可通过抑制其激活减轻氧化应激、减轻DN大鼠的肾脏损伤。

猪链球菌2型表面蛋白分支酸合成酶通过p38MAPK和NF-κB通路促进TLR4依赖的炎性反应

在大肠杆菌中表达与纯化猪链球菌表面蛋白分支酸合成酶,研究其对小鼠巨噬细胞RAW264.7细胞分泌细胞因子的影响,并试图解释其分子机制。以SC19基因组为模板,PCR扩增aroC基因,构建表达质粒,转化到BL21(DE3)中并诱导表达,所得融合蛋白主要以包涵体的形式存在,以8 mol·L-1尿素(含有50 mmol·L-1Tris,pH8.0)为变性剂,对构建于pET-28a(+)的猪链球菌aroC基因在大肠杆菌BL21中表达的包涵体进行变性、复性、纯化、去除LPS以及除菌。以aroC蛋白体外刺激RAW264.7细胞,共同孵育4h后用real-time PCR的方法分析IL-1β、TNF-α的mRNA转录量。用ERK1/2、JNK、NF-κB和P38的抑制剂以及TLR2和TLR4的特异性抗体来解释其引起炎性反应的分子基础。结果显示成功对aroC蛋白进行了变性、复性及纯化,该蛋白具有刺激巨噬细胞表达细胞因子的能力,用NF-κB的抑制剂以及TLR4的特异性抗体预处理细胞后能明显降低aroC导致的IL-1β、TNF-αmRNA的转录,用P38的抑制剂预处理细胞后能明显降低aroC导致的TNF-αmRNA的转录量。结果证明aroC在RAW264.7中通过p38MAPK和NF-κB通路促进TLR4依赖的炎性反应。

基于TLR4/NF-κB/MAPK信号通路探讨大黄灵仙方对胆管细胞炎性反应的影响效果

目的通过观察大黄灵仙方调控TLR4/NF-κB/MAPK信号通路相关因子基因及蛋白的影响,探讨其对胆管细胞炎性反应的作用。方法采用大鼠胆总管注射脂多糖(lipopolysaccharide,LPS)构建原发性胆管结石胆管慢性炎症状态,取大鼠肝内胆管内皮细胞,通过qPCR技术同时结合使用信号通路阻断剂,观察胆管细胞炎性状态下TLR4/NF-κB/MAPK信号通路相关因子基因及蛋白影响变化及大黄灵仙方的干预作用。结果①Myd88、p-p38、TLR4、NF-κB mRNA变化情况:与空白组比较,L组、L+P组、L+S组、L+P+S组的各指标,均增加明显,剩余各组则降低明显,差异均具统计学意义(P<0.05)。与L组比较,L+P组、L+S组、L+P+S组的各指标有所降低,但差异无统计学意义(P>0.05),L+T组、L+P+T组、L+S+T组、L+P+S+T组则降低明显,差异均具统计学意义(P<0.05);与L+T组比较,L+P+T组、L+S+T组、L+P+S+T组的各指标有所降低,差异无统计学意义(P>0.05)。②Myd88、p-p38、TLR4、NF-κB蛋白变化情况:变化趋势与mRNA相同。结论大黄灵仙方可有效逆转胆管细胞炎性反应过程中存在的TLR4/NF-κB/MAPK信号通路相关因子及蛋白表达异常现象,其机制可能与NF-κB、MAPK信号阻断有关。

汉黄芩素调控TLR4/MAPK/NF-κB信号通路对糖尿病肾病大鼠肾纤维化的影响

目的研究汉黄芩素对糖尿病肾病(diabetic nephropathy,DN)大鼠肾纤维化的保护作用及其作用机制。方法构建DN大鼠模型。将大鼠随机分为对照组、模型组、汉黄芩素低剂量组、汉黄芩素中剂量组、汉黄芩素高剂量组、二甲双胍组,每组各10只。除对照组外,其余各组大鼠均采用腹腔注射链脲佐菌素65 mg·kg^(-1)联合高脂高糖喂养12周的方法构建DN大鼠模型。造模完成后,各组分别连续28 d灌胃给予相应药物或生理盐水,每天1次。给药前和给药后间隔1周分别检测各组大鼠空腹血糖(FBG)含量;28 d后,称大鼠体质量,生化分析法检测24 h尿蛋白量(24 h-Pro)和血清尿素氮(BUN)、肌酐(SCr)含量,HE染色法观察肾组织病理学改变,Masson染色法观察肾组织纤维化状况,免疫组化检测各组大鼠肾组织相关蛋白表达,Western blot法检测肾组织蛋白TLR4、MAPK、NF-κB、Ⅰ型胶原蛋白(Col-Ⅰ)、Ⅲ型胶原蛋白(Col-Ⅲ)表达。结果与对照组相比,模型组大鼠FBG含量、24 h-Pro和血清BUN、SCr含量均明显升高(P<0.01),体质量明显降低(P<0.01);肾组织出现肌束紊乱、炎性细胞浸润、弥漫性水肿、肾小球体积增大、系膜增生,肾小管扩张,肾间质大量炎性细胞浸润等病理学改变及肾小球、肾小管间质纤维化,胶原容积分数(CVF)升高(P<0.01);TLR4、MAPK、NF-κB、Col-Ⅰ、Col-Ⅲ蛋白表达明显上调(P<0.01);与模型组相比,汉黄芩素各剂量组和二甲双胍组大鼠FBG含量、24 h-Pro和血清BUN、SCr含量均明显降低(P<0.01),体质量明显升高(P<0.01);肾组织纤维化明显改善,CVF明显降低(P<0.01);TLR4、MAPK、NF-κB、Col-Ⅰ、Col-Ⅲ蛋白表达明显下调(P<0.01)。结论汉黄芩素可对DN大鼠肾纤维化有明显保护作用,其作用机制可能与TLR4/MAPK/NF-κB信号通路有关。

益肾通癃汤通过上调miR-145-5p抑制TLR4/p38 MAPK/NF-κB信号通路抗前列腺癌作用机制研究

目的探讨益肾通癃汤(YSTLD)通过上调miR-145-5p抑制TLR4/p38 MAPK/NF-κB信号通路抗前列腺癌的作用机制。方法借助miRNA芯片技术检测YSTLD处理前列腺癌PC-3细胞后的miRNA表达谱的变化,筛选miRNA芯片结果中差异显著的miRNA,并通过实时荧光定量聚合酶链反应(qRT-PCR)进行验证。慢病毒转染miR-145-5p入前列腺癌PC-3细胞,CCK8法及划痕实验检测miR-145-5p对前列腺癌PC-3细胞增殖与迁移作用;qRT-PCR法及Wstern blot法检测miR-145-5p对TLR4/p38 MAPK/NF-κB信号通路及凋亡相关基因caspase3、TNF-α、Bax、Bcl-2表达的影响;qRT-PCR及Wstern blot法检测YSTLD含药血清对miR-145-5p、TLR4/p38 MAPK/NF-κB信号通路及凋亡相关基因caspase3、TNF-α、Bax、Bcl-2表达的影响。结果miRNA基因芯片检测发现YSTLD处理前列腺癌PC-3细胞后存在35种miRNA表达水平与对照组存在显著差异,其中以miR-145-5p差异最为显著,同时qRT-PCR验证发现YSTLD处理的PC-3细胞中的miR-145-5p水平显著高于DMSO对照组(P<0.05)。慢病毒转染miR-145-5p入前列腺癌PC-3细胞后,发现miR-145-5p能抑制前列腺癌PC-3细胞的增殖与迁移。过表达miR-145-5p可上调前列腺癌PC-3细胞caspase3、TNF-α及Bax mRNA表达水平,下调p38 MAPK、p65 NF-κB及Bcl-2的mRNA表达水平(P<0.05),同时上调前列腺癌PC-3细胞caspase3蛋白表达水平,下调TLR4、p38 MAPK、p65 NF-κB的蛋白表达水平(P<0.05);YSTLD含药血清在干预前列腺癌PC-3细胞后,可上调前列腺癌PC-3细胞caspase3、TNF-α及Bax mRNA表达水平,下调p38 MAPK、p65 NF-κB、Bcl-2、TRAF1的mRNA表达水平(P<0.05),同时上调前列腺癌PC-3细胞caspase3蛋白表达水平,下调TLR4、p38 MARK、p65 NF-κB、TRAF1的蛋白表达水平(P<0.05)。结论YSTLD可通过上调miR-145-5p的表达水平,抑制TLR4/p38 MAPK/NF-κB信号通路促进前列腺癌PC-3细胞凋亡,这可能是YSTLD抗前列腺癌的重要机制。

基于TLR4/MAPKs/NF-κB信号通路探讨蠲痹汤对寒湿痹阻证类风湿关节炎模型大鼠的治疗作用及机制研究

目的:观察蠲痹汤对寒湿痹阻证模型类风湿关节炎(RA)模型大鼠的保护作用,并探讨其可能的作用机制。方法:将75只SPF级雄性SD大鼠,随机分为5组(n=15),正常对照组、模型组及蠲痹汤低(10 g/kg)、中(20 g/kg)、高(30 g/kg)剂量组。经左后足跖皮内注射完全弗氏佐剂(0.1 mL/只)持续刺激14 d,以建立佐剂性类风湿关节炎大鼠病理模型,后开始给予相应剂量的蠲痹汤,连续28 d。采用Elisa试剂盒检测血清TNF-α、IL-1β、IL-6、IL-10水平,免疫组化分析滑膜组织中COMP蛋白表达,Western blot检测各组大鼠COMP、TLR4、NF-κBP65、p-NF-κBP65、P38、p-P38蛋白的表达。结果:蠲痹汤能显著改善RA模型大鼠的精神状态、饮食、饮水量和滑膜组织的病理学改变,降低足肿胀率;调节血清中IL-1β、IL-6、TNF-α、IL-10水平;免疫组化和Western blot试验结果表明蠲痹汤能显著调节COMP、TLR4、NF-κBP65、p-NF-κBP65、P38、p-P38蛋白的表达。结论:蠲痹汤能改善类风湿关节炎,其机制与调节炎症和Toll样受体4/丝裂原活化蛋白激酶/核转录因子κB信号通路密切相关。

Milled flaxseed-added diets ameliorated hepatic inflammation by reducing gene expression of TLR4/NF-κB pathway and altered gut microbiota in STZ-induced type 1 diabetic mice

Flaxseed has displayed the potential beneficial as functional foods.However,most studies focused on effects of flaxseed extracts or ingredients in flaxseed.Besides,few studies showed that flaxseed extracts contributed to anti-type 1 diabetes(T1D),yet the underlying mechanism is still unknown.In the present study,16.7% of milled flaxseed(MF)-added diet was given to diabetic mice induced by streptozocin for 6 weeks.The results showed that MF feeding 1)slightly decreased blood glucose levels and improved the ability of glucose tolerance by oral glucose tolerance test,2)decreased liver tumor necrosis factor-αlevels and increased liver glycogen levels with significance via down-regulating TLR4/NF-κB pathways,3)and significantly altered some beneficial bacteria in gut microbiota.In conclusion,the present study showed that milled flaxseed showed the potential on anti-T1D through anti-inflammation via TLR4/NF-κB and altering the gut microbiota in STZ-induced diabetic mice.

Bioinformatic Analysis and Experimental Verification of QJHGD on Caerulein-induced Inflammatory Response in SAP Model Rats Based on TLR4/NF-κB/My D88 Pathway

[Objectives]To conduct bioinformatic analysis and experimental verification of Qingjie Huagong Decoction(QJHGD)on caerulein-induced inflammatory response in severe acute pancreatitis(SAP)model rats based on TLR4/NF-κB/MyD88 pathway.[Methods]The effective component groups and potential targets of QJHGD were collected by the network pharmacology method.A drug-component-target network was constructed.The GO and KEGG of targets were enriched and analyzed with the aid of Metascape database,and the target pathway related to SAP inflammation was screened.The SAP rat model was established by caerulein combined with lipopolysaccharide,and QJHGD was intragastrically administered.Pancreatic tissue was observed by HE staining.In addition,enzyme-linked immunosorbent assay and immunohistochemistry were used to verify the anti-inflammatory effect of QJHGD on SAP rats and its regulatory effect on TLR4/NF-κB/MyD88 target pathway.[Results]A total of 105 active components of QJHGD and 148 key targets of SAP were predicted and screened;KEGG was enriched in 320 different pathways including toll-like receptor and NF-κB classical pathways.Animal experiment verified that QJHGD reduced serum amylase,serum lipase activity,IL-6,TNF-αlevels in SAP rats;HE staining showed the effect of QJHGD on the pathological changes of pancreas,and QJHGD inhibited the positive expression of key proteins of TLR4,NF-κB and MyD88 in the inflammatory transduction pathway.[Conclusions]The mechanism of QJHGD improving pancreatic injury in SAP rats may be related to down-regulating the expression of key proteins in the TLR4/NF-κB/MyD88 pathway.

补阳还五汤抑制TLR4信号通路减轻db/db糖尿病小鼠周围神经病变炎症反应

目的观察补阳还五汤对db/db糖尿病小鼠周围神经病变炎症反应中TLR4/MAPK/NF-κB信号通路以及炎症因子的作用,探讨其对周围神经小鼠炎症损伤的保护机制。方法将50只db/db小鼠随机分成5组:空白对照组、模型组、补阳还五汤低、中、高剂量组[1.6,3.2,6.4 g/(kg·d)],每组10只。空白对照和模型组以10 ml/kg体质量的去离子水灌胃,各给药组小鼠均以10 ml/kg体质量相应浓度的药物灌胃,每日1次,连续84 d。第84天最后一次给药后,检测小鼠足底热敏反应时间,采用ELISA法检测小鼠血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)及IL-1β的表达水平,采用Western blot法检测小鼠背根神经节中TLR4、p38 MAPK、p-P38、IκB-α、P65、p-IκB-α和p-P65的表达量。结果与空白对照组相比,模型组小鼠足底热敏反应时间显著增加(P<0.05),其血清中IL-6、IL-1β和TNF-α及背根神经节中TLR4、p-P38、p-IκB-α和p-P65蛋白的表达水平也显著增强,差异具有统计学意义(P<0.05)。与模型组对比,各给药组能显著缩短小鼠足底热敏反应时间(P<0.05);血清中IL-6、IL-1β和TNF-α及背根神经节中TLR4、p-P38、p-IκB-α和p-P65表达水平下调(P<0.05)。随着剂量的增加,db/db小鼠热敏反应时间及相关炎性因子的表达水平相应降低,其中以高剂量组db/db小鼠减少较为显著。结论补阳还五汤可通过抑制TLR4、p38 MAPK及NF-κB信号转导通路的异常激活,缓解db/db糖尿病小鼠周围神经病变的炎症反应,从而保护周围神经的功能。