Aryl hydrocarbon receptors regulate pyocyanin-induced inflammatory factor expression in macrophages via p38MAPK/p65NF-κB signaling pathway / 中国药理学通报

Aim To investigate the effect of the aryl hydrocarbon receptor (AhR) on the expression of inflammatory factors in macrophages RAW264. 7 induced by pyocyanin (PCN) and the regulatory mechanism of its signaling pathway. Methods RAW264. 7 cells were treated with different concentrations of PCN for 24 h, respectively, and the effect of PCN on cell activity was detected by CCK8 assay to determine the optimal PCN concentration for manufacturing infection models. The cells were divided into the control group (given 0. 1% dimethyl sulfoxide DMSO), PCN group, PCN + AhR inhibitor (CH223191) group, and PCN + AhR agonist (FICZ) group, and the expression of AhR was detected by immunofluorescence. The expression levels of inflammatory factors (IL-6, IL-1β, and TNF-α) were detected by ELISA. The protein expression of AhR, pp38 MAPK and p-p65NF-κB, was detected by Western blotting. Results PCN induced a significant quantitative effect on AhR expression in RAW264. 7 cells. CH223191 increased PCN-induced inflammatory factor secretion and enhanced the phosphorylation of p38MAPK and p65NF-κB compared with the control group. FICZ decreased PCN-induced inflammatory factor production and reduced the phosphorylation of p38MAPK and p65NF-κB phosphorylation capacity. Conclusions AhR can regulate PCN-induced inflammatory factor expression in RAW264. 7 cells, and the p38MAPK/p65NF-κB signaling pathway may be an essential pathway for the involvement of AhR in immune regulation.

Effect of cangfudaotan decoction on TLR4/NF-κB p65 signaling pathway in obese PCOS-IR rats / 中国药理学通报

Aim To explore the regulatory effect of Cangfudaotan Decoction on the ovarian Toll receptor 4 (TLR4)/nuclear transcription factor kBp65 (NF-κB p65) signaling pathway in obese PCOS-IR rats. Methods Forty-eight female rats were randomly divided into normal group (n = 8) and model group (n = 40). The obese PCOS-IR rats were established by letrozole (1 mg · kg

Tongxie Yaofang Regulates T Lymphocyte Subsets to Improve Immune Microenvironment of Colorectal Cancer Under Chronic Stress / 中国实验方剂学杂志

ObjectiveTo explore the effect of Tongxie Yaofang on the immune microenvironment of colorectal cancer in mice under chronic stress and the underlying mechanism. MethodA total of 40 male SPF BABL/C mice were randomized into normal group， stress group， Tongxie Yaofang group （13.65 g·kg-1）， and Tongxie Yaofang-stress group （13.65 g·kg-1）， with 10 in each group. Chronic restraint stress was induced in mice and administration （ig） of Tongxie Yaofang began after 7 days of stress. On the 14th day， forced swim and tail suspension tests were used to examine the behavioral changes of mice after stress and the subcutaneous colorectal tumor was implanted in each group of mice. The effect of this prescription on the body mass and tumor volume of mice was observed. After the last administration， mouse serum and tumor samples were collected. The content of T lymphocytes （CD3+， CD4+， CD8+， and CD4+/CD8+） in tumor was detected by immunohistochemistry and flow cytometry and levels of corticosterone （CORT） in peripheral blood， and interleukin （IL）-2， interferon-γ （IFN-γ）， IL-6， and IL-10 in the serum were determined by enzyme-linked immunosorbent assay （ELISA）. The protein expression of inhibitor of nuclear factor-κB（IκB） kinase α/β （IKKα/β）， nuclear factor-κB （NF-κB）α （IκBα）， NF-κB p65， and phosphorylated （p）-NF-κB p65 was measured by Western blot. ResultCompared with the normal group， the stress group had large tumor volume （P<0.05）， low content of CD3+， CD4+， and CD4+/CD8+ （P<0.05， P<0.01）， high content of CD8+， low content of T helper 1 （Th1）-secreted IFN-γ （P<0.05）， high content of T helper 2 （Th2）-secreted IL-10 （P<0.05） and CORT （P<0.05）， high protein expression of p-NF-κB p65， NF-κB p65， and IKKα/β （P<0.05）， and low protein expression of IκBα （P<0.05）. Compared with the normal group， the Tongxie Yaofang group showed slow tumor growth， high content of CD3+， CD4+， and CD4+/CD8+ （P<0.01）， low content of CD8+ （P<0.05）， high content of Th1-secreted IL-2 and IFN-γ （P<0.05）， low content of Th2-secreted IL-6 and IL-10 （P<0.05）， low content of CORT， low protein expression of p-NF-κB p65， NF-κB p65， and IKKα/β （P<0.05）， and high protein expression of IκBα （P<0.01）. Tongxie Yaofang-stress group demonstrated slower tumor growth， higher content of CD3+， CD4+， and CD4+/CD8+ （P<0.01）， smaller content of CD8+ （P<0.05）， higher content of IL-2 and IFN-γ （P<0.05）， lower content of IL-6， IL-10 （P<0.05）， and CORT （P<0.05）， lower protein expression of p-NF-κB p65， NF-κB p65， and IKKα/β （P<0.05，P<0.01）， and higher protein expression of IκBα （P<0.01） than the stress group. ConclusionTongxie Yaofang can delay the growth of colorectal cancer under chronic stress and alleviate the deterioration of the immune microenvironment， possibly by inhibiting NF-κB signaling pathway， regulating the function of T lymphocyte subsets， and thus suppressing the secretion of pro-inflammatory factors.

Effect of Shaoyaotang on Expressions of TLR4, NF-κB p65 and IL-6 in Rats with Damp-heat Ulcerative Colitis / 中国实验方剂学杂志

Objective:To explore the mechanism of Shaoyaotang in the treatment of ulcerative colitis (UC) based on toll-like receptor 4 (TLR4)/nuclear factor kappaB (NF-κB) signaling pathway. Method:A total of 50 Wistar rats were selected, including half male and half female. The damp-heat UC rat model was replicated by the methods of the combination of diseases and syndromes and the combination of 2, 4, 6-nitrobenzene sulfonic acid (TNBS) and ethanol. After the successful modeling, the model rats were randomly divided into model group, salazulesulfonate group, and low, medium and high-dose Shaoyaotang groups, and 10 rats (half male and half female) were selected as the blank control group. Low, medium and high-dose Shaoyaotang groups were given 6, 12, 24 g·kg-1 by gavage, and salazonyl arsenic group was given 1 g·kg-1 by gavage. Blank control group was given the equal volume of normal saline for 21 consecutive days. Colon samples were collected after the last administration, and the expressions of TLR4, NF-κB p65 and IL-6 mRNA in colon tissues were detected by fluorescent quantitative polymerase chain reaction (Real-time PCR）, and the expressions of TLR4, NF-κB p65 and IL-6 protein in colon tissues were detected by Western blot. Result:Compared with the blank control group, the relative expressions of TLR4, NF-κB p65, IL-6 mRNA and protein in the model group were significantly increased (P<0.05). Compared with the model group, the expression levels of TLR4, NF-κB p65 and IL-6 mRNA and protein in the salazopyridine group and Shaoyaotang groups were significantly decreased (P<0.05). Conclusion:Shaoyaotang can inhibit the development of UC by regulating the expressions of TLR4, NF-κB p65 and IL-6 mRNA and proteins in the TLR4/NF-κB pathway.

Effect of Huangqi Guizhi Wuwutang on AGEs/RAGE/NF-κB Signaling Pathway in Diabetic Peripheral Neuropathy Rats / 中国实验方剂学杂志

Objective:To study the effect of Huangqi Guizhi Wuwutang on receptor of advanced glycation end products(AGEs)/advanced glycation endproducts (RAGE)/nuclear transcription factor-kappa B p65 (NF-κB p65) signaling pathway in the diabetic peripheral neuropathy rats through an animal modeling experiment, and discuss the mechanism of Huangqi Guizhi Wuwutang in alleviating diabetic peripheral neuropathy. Method:Rat model of diabetic peripheral neuropathy was established by high-fat diet and intraperitoneal injection with streptozotocin (STZ). After successful modeling, Huangqi Guizhi Wuwutang intervention began in the fifth week. The patients in high-dose group (19.40 g∙kg-1∙d-1), middle-dose group (4.85 g∙kg-1∙d-1) and low-dose group (2.43 g∙kg-1∙d-1) were given by gavage continuously for 12 weeks. The western medicine control group was given 25 mg∙kg-1∙d-1 by gavage. After the experiment, serum interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect RAGE and NF-κB p65 mRNA expressions in sciatic nerve tissue. The expressions of RAGE, NF-κB and phosphorylation(p)-NF-κB p65 proteins in sciatic nerve tissues were detected by Western blot (WB). Result:Compared with the normal group, serum IL-1β and TNF-α protein levels, RAGE mRNA and NF-κB p65 mRNA levels, RAGE protein, NF-κB p65 protein and p-NF-κB p65 protein levels were significantly increased in the model group (P<0.01), the ratio of p-NF-κB p65 to NF-κB p65 was increased, and the phosphorylation of NF-κB p65 was enhanced (P<0.01). After the intervention of Huangqi Guizhi Wuwutang, compared with the model group, serum IL-1β and TNF-α protein levels, RAGE and NF-κB p65 mRNA levels, RAGE protein, NF-κB p65 protein and p-NF-κB p65 protein levels were all decreased (P<0.01), the ratio of p-NF-κB p65 to NF-κB p65 was decreased in high-dose group (P<0.01). The effect was obvious with the increase of dose of astragalus cassia twig. Conclusion:Huangqi Guizhi Wuwutang can alleviate diabetic peripheral neuropathy, and its mechanism may be related to blocking the expression of RAGE on tissue cell surface in AGEs/RAGE/NF-κB signaling pathway, inhibiting the activation of NF-κB and inducing TNF-α triggered oxidative stress and excessive inflammatory response, so as to avoid cell damage and dysfunction.

Effect of Betulic Acid on RAGE/NF-κB/iNOS Signaling Pathway Expression in Steatosis Cell / 中国实验方剂学杂志

Objective::To investigate the effect of betulic acid(BA) on steatosis LO2 cells. Method::LO2 cells were intervened with BA at different gradient concentrations (0, 10, 20, 40, 80, 160, 250 μmol·L-1) for 24 hours. methyl thiazolyl tetrazolium(MTT) staining was used to observed cell viability to determine the final concentration of BA. The cells were divided into control, model, dimethylsulfoxide (DMSO) and BA groups, as well as BA groups intervened with low, middle and high concentrations. First, model, DMSO and BA group's cells were cultured in 10% Lipid Mix 1 medium for 24 hours to establish a nonalcoholic fatty liver model. Then, DMSO group and low, medium and high-concentration groups were separately cultured with 0.1%DMSO medium and 20, 40, 80 μmol·L-1 BA medium for 24 hours. And control and model groups were cultured in drug-free medium for 24 hours. Oil red O staining and Nile red staining were used to observe the intracellular lipid droplets. Immunofluorescence was used to detect the protein expression of inducible nitric oxide synthase (iNOS). Western blot was used to detect the protein expression levels of receptor for advanced glycation end-products (RAGE), nuclear factor κB p65 (NF-κB p55) and iNOS. Result::BA within the concentration of 80 μmol·L-1 had no significant toxicity on LO2 cells. Compared with control group, the intracellular lipid droplets were significantly increased in the model group, and the expressions of oxidative stress-related proteins RAGE, NF-κB p65 and iNOS also increased significantly(P<0.05). Compared with model group, the intracellular lipid droplets in DMSO group were similar to those in model group, with no significant difference in the three protein expressions between the two groups. However, the intracellular lipid droplets deposition in the BA group was significantly decreased. And the expressions of RAGE, NF-κB p65 and iNOS proteins in high-concentration BA group were significantly decreased(P<0.05, P<0.01). Conclusion::BA can significantly improve the intracellular fat deposition in LO2 cells, which was probably related to the inhibition of the expressions of oxidative stress-related proteins RAGE, NF-κB p65 and iNOS.

Effect of Xiao Xumingtang Combined with Ultra-acupuncture Along Governor Meridian on Autophagy-related Protein NF-κB p65 in Cerebral Ischemia Reperfusion Model / 中国实验方剂学杂志

Objective:To investigate the effect of Xiao Xumingtang combined with super-acupuncture along governor meridian on autophagy-related protein nuclear factor-kappa B p65 (NF-κB p65) in cerebral ischemia-reperfusion model rats, so as to study the relationship between autophagy-related protein NF-κB p65 and brain protection mechanism, and look for the best intervention time point of acupuncture. Method:A total of 152 adult SD male rats were randomly divided into sham operation group, model group, high-dose Xiao Xumingtang group (high-dose drug group), low-dose Xiao Xumingtang group (low-dose drug group) and acupuncture group. There were seven groups including high-dose Xiao Xumingtang + acupuncture group (high acupuncture group) and low-dose Xiao Xumingtang + acupuncture (low acupuncture group). Model group, high-dose drug group, low-dose drug group, and acupuncture group were divided into 4 subgroups according to 30 minutes, 2, 4， 6 h of ischemia-reperfusion, with 6 animals in each group. After successful modeling, according to Zea Longa's neural function score, eligible rats were included into the corresponding groups. The sham operation group only received carotid artery dissection, the model group was only modeled without any treatment, high and low-dose Xiao Xumingtang groups were calculated based on the body surface area of the animal and given 60 g·kg-1·d-1 and 15 g·kg-1·d-1 drug by gavage for treatment, acupuncture was performed to smooth governor meridian and regulate the mind. After 14 days of consecutive treatment, neurological function was scored. Western blot was used to detect the expression of autophagy-related protein NF-κB p65 in rat brain tissue. Result:Compared with the sham operation group, the neurological impairment scores of the model group, the high-dose drug group, the low-dose drug group, the acupuncture group, the high acupuncture group, and the low acupuncture group were significantly increased at each time point (P<0.01). The neurological impairment scores were significantly lower at each time point than those of the high-dose drug group, low-dose drug group, acupuncture group, high acupuncture group, and low acupuncture group (P<0.01), compared with the sham operation group, NF-κB p65 in model group, high-dose drug group, low-dose group, acupuncture group, high acupuncture group and low acupuncture group was significantly increased in the brain tissue at each time point (P<0.01), compared with the model group, the expression of NF-κB p65 protein in the brain tissue of model group, high-dose drug group, low-dose group, acupuncture group, high acupuncture group and low acupuncture group was decreased at each time point (P<0.05), particular in the high acupuncture group (P<0.01). Conclusion:Xiao Xumingtang combined with ultra-early acupuncture along governor meridian can significantly alleviate neurological impairment in rats with cerebral ischemia-reperfusion model. Xiao Xumingtang combined with ultra-early acupuncture along governor meridian can inhibit cerebral ischemia-reperfusion model rats. The activity of autophagy-related protein NF-κB P65 protects the brain function. There is no significant difference in the brain protective effect of Xiao Xumingtang combined with ultra-early acupuncture along governor meridian within 6 hours.

Effects of Taoren Chengqitang on Intestinal Mucosal Barrier and TLR9 Signaling Pathway in Septic Rats / 中国实验方剂学杂志

Objective: To study the protective effect of Taoren Chengqitang on intestinal mucosal barrier in septic rats and its possible mechanism. Method: Rats were divided into sham operation group, model group (replication of septic rats with cecal ligation and perforation), low, middle and high-dose Taoren Chengqitang groups (2.85,5.70,8.55 g·kg-1), and dexamethasone group (0.01 g·kg-1),with 12 rats in each group. After last administration, rats were put to death, the morphological changes of intestinal mucosa were observed under electron microscope, the bacterial translocation rates in lymphoglandulae mesentericae, liver, kidney and spleen tissues were detected; the levels of serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and diamine oxidase (DAO) and intestinal fatty acid binding protein (i-FABP) levels in small intestine tissues were detected by enzyme-linked immunosorbent assay (ELISA).Expressions of Toll like receptor 9 (TLR9), myeloid differentiation factor 88 (MyD88) and nuclear factor-kappa B subunit p65 in small intestine tissues were detected by Western blot and immunohistochemistry. Result: Compared with sham operated group, the bacterial translocation rate, TNF-α and IL-1β levels in model group increased significantly, DAO, i-FABP, mucosal thickness and villus height decreased significantly, and protein expressions of TLR9, MyD88 and nucleus NF-κB p65 increased significantly (PPκB p65 protein decreased significantly. Compared with model group, the bacterial translocation rate, TNF-α and IL-1β levels in organs of low, medium and high-dose Taoren Chengqitang groups and dexamethasone group decreased significantly, DAO, i-FABP, mucosal thickness and villus height increased significantly, while the protein expressions of TLR9, MyD88 and nucleus NF-κB p65 decreased significantly (PκB p65 protein increased significantly. Conclusion: Taoren Chengqitang has a certain protective effect on intestinal mucosal barrier in septic rats, which may be related to the inhibition of TLR9 signaling pathway.

Mechanism of Modified Erchentang on Signaling Pathway of TLR4/MyD88/NF-κB in Lung Tissue of Rats with Chronic Obstructive Pulmonary Disease / 中国实验方剂学杂志

Objective: To study the effect of modified Erchentang on expressions of Toll-like receptor 4 (TLR4), myeloid differentiation factor (MyD88) and nuclear factor-κB (NF-κB) genes in the lung tissue homogenate of rats with chronic obstructive pulmonary disease (COPD). Method: Forty SD rats were randomly divided into normal group, model group, modified Erchentang group and EVP4593 (NF-κB inhibitor) group. Rat COPD models were prepared through cigarette smoke and tracheal dripping with lipopolysaccharide (LPS). After the modeling, normal and model groups were intragastrically given normal saline solution, EVP4593 group was given EVP4593(1 mg · kg-1) through subcutaneous injection, and modified Erchentang group was given corresponding herbal drugs intragastrically (10 g · kg-1) for 14 days. The levels of high mobility group box 1(HMGB1), chemokines CXCL-2, CXCL-3 and monocyte chemoattractant protein-1 (MCP-1) in rats serum were detected by enzyme-linked immunosorbent assay in rats serum. The expressions of Toll-like receptors 4(TLR4), myeloid differentiation factor (MyD88) and nuclear factor-κB p65 (NF-κB p65) mRNA were detected by Real-time fluorescence quantitative PCR (Real-time PCR) method. Western blot were used to detect the levels of TLR4, MyD88, NF-κB p65 and p-NF-κB p65 protein. Immunohistochemistry (IHC) method was used to detect the localization and expressions of TLR4, MyD88 and p-NF-κB p65 protein in the lung tissue. Result: The mRNA and protein expressions of TLR4, MyD88 and NF-κB p65 were increased significantly (PPκB p65 mRNA and protein were decreased significantly (PConclusion: Modified Erchentang may inhibit the inflammatory response of COPD effectively. The mechanism may be related to the inhibition of the expressions of the signal molecule genes involved in the TLR4/MyD88/NF-κB pathway and the reduction of the release of HMGB1, CXCL-2, CXCL-3 and MCP-1.

Effects of electroacupuncture on inflammatory response of cardiac muscle tissue in mice with acute myocardial ischemia / 中国针灸

<p><b>OBJECTIVE</b>To observe the effects of electroacupuncture (EA) on inflammatory reaction of acute myocardial ischemia (MI) in mice, and to explore its action mechanism.</p><p><b>METHODS</b>Forty adult male C57BL/6 mice were randomly divided into a control group, a sham operation group, a model group and an EA group, 10 mice in each one. The model was established in the model group and EA group by ligating the left anterior descending branch of coronary artery. The mice in the EA group were treated with EA at "Neiguan" (PC 6) with 2 mA of intensity and 2 Hz /100 Hz of frequency; EA was given 30 min per treatment, once a day for totally 5 days. The mice in the control group and model group were treated with immobilization and no EA was given. The mice in the sham operation group were not treated with ligating at the left anterior descending branch of coronary artery, but the remaining procedure was identical to the model group. The electrocardiogram was recorded and △ST was calculated to evaluate the model. TTC and HE staining methods were applied to evaluate the infarct size and pathologic change of myocardial tissue, respectively. Western blot method was applied to test the protein expression levels of tumor necrosis factor-α (TNF-α), nuclear factor-κB p65 (NF-κB p65), interleukin-1β (IL-1β) and interleukin-8 (IL-8).</p><p><b>RESULTS</b>Compared with the sham operation group, the S-T segments in the model group and EA group were increased obviously after modeling (both <0.01), indicating the MI model was established successfully. The TTC and HE staining results indicated, compared with the sham operation group, the model group had larger infarction size (<0.01), more myocardial fibers injury and inflammatory infiltration; compared with the model group, the infarction size of the EA group was significantly reduced (<0.01), and the myocardial fibers injury and inflammatory infiltration were improved. Compared with the control group, the protein expression levels in the sham operation group were similar (all >0.05); compared with the sham operation group, the expression levels of TNF-α, NF-κB p65, IL-1β and IL-8 were significantly increased in the model group (<0.01, <0.05); compared with the model group, the expression levels of TNF-α, NF-κB p65, IL-1β and IL-8 were significantly reduced in the EA group (all <0.05).</p><p><b>CONCLUSION</b>EA might reduce the protein expression levels of TNF-α, NF-κB p65, IL-1β and IL-8 in cardiac muscle tissue to inhibit inflammatory reaction and achieve myocardial protective effect in mice with acute myocardial ischemia.</p>