Anti-inflammatory effect and mechanism of the ethanol extract and the drug-containing serum of Zhuang medicine Stahlianthus involucratus on LPS-induced RAW264.7 cell inflammation / 中国药房

OBJECTIVE To study the anti-inflammatory effect and mechanism of the ethanol extract and the drug-containing serum of Zhuang medicine Stahlianthus involucratus on lipopolysaccharide （LPS）-induced RAW264.7 cell inflammation. METHODS The drug-containing serum or blank serum was obtained by intragastrical administration of ethanol extract of S. involucratus （75.35 g/kg） or purified water. Using RAW264.7 cells as objects， RAW264.7 cells were divided into normal control group， LPS group （1 μg/mL）， S. involucratus ethanol extract high-dose， medium-dose and low-dose groups （50， 25， 12.5 μg/mL）， 4% or 15% blank serum groups， 4% or 15% blank serum+LPS groups， 4% or 15% drug-containing serum groups， 4% or 15% drug-containing serum+LPS groups. After culturing for 24 h， cell viability， the contents of nitric oxide tumor necrosis factor α （TNF-α）， interleukinand IL-6 as well as mRNA expressions of Toll-like eceptor 4 （TLR4） and nuclear factor κB （NF- κB） and protein expressions of nitric oxide synthase （NOS） and cyclooxygenase 2 （COX-2） were all detected in each group. 0771-4953513。E-mail：zhuhuagx@163.com RESULTS After culturing for 24 h， there was no statisticalsignificance in the difference of cell viability. Compared with normal control group， the contents of NO， TNF-α， IL-1β and IL-6， mRNA expressions of TLR4 and NF-κB， and protein expressions of NOS and COX-2 were increased significantly in LPS group （P＜0.05）. Compared with 4% or 15% blank serum groups， the levels of above indexes were increased significantly in 4% or 15% blank serum+LPS groups （P＜0.05）. Compared with LPS group， the levels of above indexes were decreased significantly in S. involucratus ethanol extract groups （P＜0.05）. Compared with 4% or 15% blank serum+LPS groups， the levels of above indexes were decreased significantly in 4% or 15% drug-containing serum+LPS groups （P＜0.05）. CONCLUSIONS The ethanol extract and the drug-containing serum of S. involucratus can significantly alleviate LPS-induced inflammatory reaction， the mechanism of which may be associated with inhibiting the activity of TLR4/NF-κB signaling pathway， down-regulating the protein expressions of COX-2 and NOS， and reducing the release of inflammatory factors.

Role of TLR4/NF-κB signaling pathway in hydrogen-rich water improving cognitive impairment induced by ionizing radiation in rats / 环境与职业医学

Background In the process of radiotherapy, when radiation kills tumor cells, it inevitably damages normal tissue cells. Objective To investigate the role of Toll-like receptor 4 (TLR4)/nuclear factor−kappa B (NF-κB) signaling pathway in the improvement of cognitive impairment induced by ionizing radiation by hydrogen-rich water before and after whole brain irradiation in rats. Methods Fifteen male SD rats were randomly divided into three groups: control group, irradiated group (IR group), and hydrogen-rich water intervention group (IR+HRW group), with 5 rats in each group. The control group was not irradiated, but was given purified water (20 mL·kg−1) by gavage every day, while the IR group and the IR+HRW group were irradiated with a single dose of 20 Gy. Three days before, 10 min before, and 30 days after irradiation, purified water/hydrogen-rich water (20 mL·kg−1) was given by continuous gavage every day. The general condition of the rats was observed every day, and the body weight were measured on the 7th, 14th, 21st, and 30th days after irradiation. On the 30th day after irradiation, the learning and memory ability of the rats was tested by Morris water maze; the pathological changes of hippocampus were detected by hematoxylin-eosin (HE) staining after sacrificing the rats; the contents of glutathione (GSH), malondialdehyde (MDA), interleukin-1β (IL-1β), and hydroxyl radicals in brain tissues were detected by enzyme linked immunosorbent assay (ELISA); the mRNA and protein expression levels of TLR4, NF-κB, NOD-like receptor pyrin domain 3 (NLRP3), and cysteinyl aspartate specific proteinase 1 (Caspase 1) were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in the hippocampus of rats. Results After irradiation, the rats in the IR group showed symptoms such as head hair removal and salivation, while the symptoms of the rats in the IR+HRW group were milder. No animal died in the control and the IR+HRW groups, while one rat died in the IR group. From day 14 to day 30 after irradiation, the body weight of the rats in the IR+HRW group tended to be higher than that in the IR group, but the difference was not statistically significant (P>0.05). The Morris water maze results showed that the escape latency of the IR+HRW group was shortened compared with that of IR group from day 1 to day 5 except day 3, but the difference was not statistically significant (P>0.05). For the rats in the IR+HRW group, it took less time to reach the original location of the platform after removing the platform on day 6 and the number of crossing the platform and the residence time in the original platform quadrant increased (P<0.05). The HE staining showed that the number of hippocampal cells in the IR+HRW group was slightly reduced and arranged neatly, without obvious nuclear hyperchromatic and pyknotic phenomenon. The ELISA results showed that the MDA and hydroxyl radical levels were decreased in the IR+HRW group compared with the IR group (P<0.05), the GSH content was increased, and the IL-1β concentration was decreased, but the differences were not statistically significant (P>0.05). The results of qRT-PCR showed that the mRNA expression levels of TLR4 and Caspase 1 in the hippocampus of the IR+HRW group were decreased compared with the IR group (P<0.05), and the mRNA expression levels of NF-κB and NLRP3 were also decreased, but the differences were not statistically significant (P>0.05). The results of Western blotting showed that the expression levels of TLR4 and Caspase 1 protein in the hippocampus of the IR+HRW group were decreased compared with the IR group (P<0.05), and the expression levels of NF-κB p65 and NLRP3 protein were also decreased, but the differences were not statistically significant (P>0.05). Conclusion Hydrogen-rich water can improve cognitive impairment induced by ionizing radiation in rats, and its mechanism may be related to regulating TLR4/NF-κB signaling pathway, inhibiting inflammatory factors, and attenuating oxidative stress.

Huanglian Jiedutang Treats Acute Gouty Arthritis via NLRP3 Inflammasome and TLR4/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Huanglian Jiedutang on the inflammatory injury in the mouse model of acute gouty arthritis （AGA） and to explore the mechanism of Huanglian Jiedutang in treating AGA. MethodForty male C57BL/6J mice were randomized into blank， model， colchicine （0.83 mg·kg-1）， and Huanglian Jiedutang （5 g·kg-1） groups. The mouse model of AGA was established by injecting monosodium urate （MSU） crystals into the ankle joint. The swelling degree of the right ankle joint of each mouse was measured every day for 7 days， and the pathological changes of the ankle joint were detected by hematoxylin-eosin （HE） staining after 7 days. The other 40 C57BL/6J mice were grouped as above. After 18 hours of modeling， the right ankle joint was collected， and real-time polymerase chain reaction was employed to measure the mRNA levels of interleukin （IL）-1β， tumor necrosis factor （TNF）-α， IL-6， NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein containing a CARD （ASC）， and Caspase-1. The expression levels of IL-1β， TNF-α， and IL-6 were measured by the enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of NLRP3 inflammasome， Toll-like receptor 4 （TLR4）， and nuclear factor-κB （NF-κB）. ResultCompared with the blank group， the model group showed swelling right ankle joint （P<0.01）， obvious foreign body granuloma in the ankle joint with inflammatory cell infiltration. After the treatment with Huanglian Jiedutang， the ankle joint swelling was relieved （P<0.05， P<0.01）， and the size of foreign body granuloma was reduced. Compared with the blank group， the model group showed up-regulated mRNA levels of IL-1β， TNF-α， and IL-6 in the ankle joint tissue （P<0.01）， up-regulated mRNA levels of NLRP3 and Caspase-1 in the NLRP3 inflammasome （P<0.05， P<0.01）， and up-regulated protein levels of NLRP3， Caspase-1， TLR4， and NF-κB （P<0.05， P<0.01）. Huanglian Jiedutang down-regulated the mRNA levels of IL-1β， TNF-α， IL-6， NLRP3， and Caspase-1 （P<0.05， P<0.01） and the protein levels of IL-1β， TNF-α， IL-6， NLRP3， Caspase-1， TLR4， and NF-κB （P<0.05 or P<0.01）. ConclusionInjecting MSU crystal resulted in local inflammatory injury of the joints in the mouse model of AGA. The treatment with Huanglian Jiedutang may alleviate the inflammatory injury by regulating the NLRP3 inflammasome and TLR4/NF-κB signaling pathway.

Research on the regulation of TLR4/NF-κB pathway and reparaive effect of Shen’an Decoction on cisplatin-induced kidney injuried rat / 国际中医中药杂志

Objective:To explore the protective effect of Shen’an decoction on cisplatin induced renal injury in rats by regulating TLR4/NF-κB signaling pathway. Methods:Sixty SD rats were randomly divided into blank group, model group, positive control group, high dose Shen’an Decoction group, medium dose Shen’an Decoction group and low dose Shen’an Decoction group, with 10 rats in each group. Except the blank group, the other groups were intraperitoneally injected with cisplatin 7.5 mg/kg to prepare the acute kidney injured rat model. After the modeling, the high, medium and low dose groups of Shen’an Decoction were gavaged with 0.698, 1.395 and 2.790 g/ml Shen’an Decoction respectively, and the positive control group was gavaged with benazepril hydrochloride suspension of 5 mg/kg, once a day for 14 days. The kidney histopathological changes of rats in each group were observed by HE staining. The content of BUN, creatinine(CR), Cystatin C (Cys-C), TNF-α, and IL-6 in serum were detected by ELISA. The expression of TLR-4, NF-κB p65, myeloid differentiation factor (MyD88), and tumor necrosis factor receptor related factor 6 (TRAF-6) in renal tissue were detected by Western blot. Results:Compared with the model group, the growth condition of rats in each dose group of Shen’an Decoction was significantly improved ( P<0.05), and the kidney coefficient was significantly decreased ( P<0.05). The serum levels of BUN, Cr, Cys-C, TNF-α, IL-6 of rats in each dose group of Shen’an Decoction were significantly decreased ( P<0.05). The expression of TLR-4 (0.54 ± 0.07, 0.52 ± 0.09, 0.41 ± 0.04 vs. 0.86 ± 0.06), NF-κB (0.74 ± 0.02, 0.72 ± 0.06, 0.67 ± 0.05 vs. 0.93 ± 0.03), MyD88 (0.86 ± 0.02, 0.82 ± 0.03, 0.61 ± 0.02 vs. 1.04 ± 0.03), and TRAF-6 (0.65 ± 0.04, 0.58 ± 0.08, 0.54 ± 0.07 vs. 0.90 ± 0.06) in kidney tissue of rats in each dose group of Shen’an Decoction was significantly decreased ( P<0.05). Conclusion:Shen’an Decoction can protect the renal function of rats by inhibiting TLR4/NF-κB signaling pathway and alleviating the pathological changes of renal injury induced by cisplatin.

Anti-inflammatory effect and mechanism of ethanol extract from Saposhnikoviae Radix in LPS-induced inflammation mouse model / 中国中药杂志

This paper aimed to explore the anti-inflammatory effect of ethanol extract from Saposhnikoviae Radix in a lipopolysaccharide(LPS)-induced inflammation mouse model and its regulation of TLR4/NF-κB signaling pathway. The ethanol extract from Saposhnikoviae Radix was separated and purified on the macroporous adsorption resin and its main chemical components were identified by UPLC-QE/MS. The identification results showed that the top ten components of ethanol extract from Saposhnikoviae Radix were mainly chromones and coumarins. A mouse model of inflammation induced by intraperitoneal injection of LPS was used to investigate the anti-inflammatory effects of ethanol extract from Saposhnikoviae Radix after intragastric administration for seven successive days. Mice in all groups except for the control group were treated with intraperitoneal injection of LPS(0.015 g·kg~(-1)) one hour after the last administration, and twelve hours later, the blood was sampled and separated and the broncoalveolar lavage fluid(BALF) was collected. The levels of nitric oxide(NO), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) in mouse serum and BALF were detected by ELISA. The harvested lung tissue was stained with hematoxylin-eosin(HE) for observing the pathological changes, followed by the detection of protein expression levels of related molecules in TLR4/NF-κB signaling pathway by Western blotting. The results showed that the ethanol extract from Saposhnikoviae Radix significantly ameliorated the pathological conditions in lung tissue of model mice, reversed the increase in NO, TNF-α, IL-6, and IL-1β levels of mouse serum and BALF, down-regulated the protein expression levels of Toll-like receptor 4(TLR4), myeloid differentiation factor(MyD88), and phosphorylated nuclear transcription factor κB-p65/nuclear transcription factor κB-p65(P-NF-κB p65/NF-κB p65), and up-regulated the NF-κB inhibitory protein α(IκBα). The ethanol extract from Saposhnikoviae Radix exhibited a good anti-inflammatory effect in the LPS-induced acute inflammation muse model, which might be related to the inhibition of the activation of TLR4/NF-κB inflammatory signaling pathway. Chromones and coumarins have been proved to be the active components for its anti-inflammatory effects.

Study on Mechanism of the Improvement Effects of Xinmaikang on Atherosclerosis in Rabbits Based on TLR 4/ NF-κB Signaling Pathway / 中国药房

OBJECTIVE：To explore t he mechanism of Xinmaikang improving atherosclerosis （AS）in rabbits. METHODS ：A total of 50 male Zealand rabbits were randomly divided into sham operation group ，model group ，simvastatin group [positive control ， 2.60 mg/（kg·d）] and Xinmaikang low-dose and high-dose groups [ 0.21，0.84 g/（kg·d）]，with 10 rabbits in each group. Rabbits in sham operation group were fed with ordinary diet ，and only femoral artery was separated and ligated ，and abdominal aortic endothelium was not strained ；the other groups were given high-fat diet and received abdominal aortic intimal balloon injury to induce AS model. Ten weeks after operation ，sham operation group and model group were given intragastric administration of normal saline ，and administration groups were given corresponding drug solution intragastrically （normal saline as solvent ）with the volume of 100 mL，once a day ，for consecutive 12 weeks. After last administration ，the pathological changes of abdominal aorta and inner wall in rabbits were observed in each group. The serum contents of triglyceride （TG），total cholesterol （TC），low density lipoprotein cholesterol （LDL-C），high density lipoprotein cholesterol （HDL-C），interleukin-6（IL-6）and IL- 1β were detected，and the contents and protein expression of Toll-like receptor 4（TLR4）and nuclear factor-κB p65（NF-κB p65）in abdominal aortic tissue were determined. RESULTS ：Compared with sham operation group ，the intima of abdominal aorta in model group was rich in lipids ，the thickness of vessel wall and plaque area were increased obviously ，and there was obvious vascular endothelial injury. The contents of TG ，TC，LDL-C，IL-6 and IL- 1β in serum，the contents and protein expression of TLR 4 and NF-κB p65 in abdominal aorta tissue were significantly increased ，while the content of HDL-C was decreased significantly （P＜0.05 or P＜ 0.01）. Compared with model group ，the lesion of rabbit abdominal aorta were alleviated ，and no obvious damage was found on the inner wall. The contents of TG ，TC，LDL-C，IL-6，IL-1β of Xinmaikang high-dose group and simvastatin group as well as the content of NF-κB p65 and protein expression of TLR4 and cnd- NF-κB p65 were improved significantly （P＜0.05 or P＜0.01）. CONCLUSIONS：Xinmaikang can improve AS in rabbits ， and its mechanism may be assicated with inhibiting the expression of TLR 4，NF-κB p65 and inhibiting inflammatory response.

Study on the Action Mechanism of 6-Shogaol in the Treatment of Ulcerative Colitis from the Signaling Pathway of Notch and TLR4/ NF-κB / 中国药学杂志

OBJECTIVE: To explore the effect and mechanism of 6-gingerenol on ulcerative colitis based on TLR4/NF-κB signaling pathway related to immune inflammation and Notch signaling pathway related to mucosal repair. METHODS: Forty Kunming mice were randomly divided into the control group (n=10), the model group (n=10), the sulfasalazine group (n=10) and the 6-gingerenol group (n=10). Ulcerative colitis model mice were induced by oral administration of 2% dextran sulfate sodium(DSS) except those in the control group. The mice in the sulfasalazine group and the 6-gingerenol group were given sulfasalazine and 6-gingerenol (ig 100 mg•kg-1) respectively, and mice in the other groups were given normal saline. The serum and colon were isolated after 20-days treatment. The expressions of Hes-1, Math-1, MUC2 and olfm4 proteins in colonic tissues were detected by immunofluorescence. The expression levels of notch-1, Hes-1, Math-1, TLR4, NF-κBp65 mRNA and proteins in colonic tissues were detected by RT-PCR and Western blot. The levels of TNF-α and IL-1β in serum were detected by ELISA. RESULTS: Compared with the model group, the expressions of Notch-1, Hes-1, TLR4 and NF-κB p65 proteins and the relative expressions of mRNA in colon epithelial tissue of mice in sulfasalazine group and 6-gingerenol group were significantly decreased, while the relative expression of mRNA in Math-1 protein was significantly increased, and the contents of TNF-α and IL-1β in serum were significantly decreased. The expression of olfm4 protein was significantly decreased and the expression of MUC2 protein was significantly increased in 6-gingerenol group, but there was no significant change in olfm4 and MUC2 proteins expressions in sulfasalazine group. Compared with sulfasalazine group, the expressions of Notch-1 and Hes-1 proteins and the relative expression of mRNA in colon epithelial tissue in the 6-gingerenol group were significantly decreased, the relative expression of mRNA and protein of Math-1 were significantly increased, the expression of olfm4 protein was significantly decreased, and the expression of MUC2 protein was significantly increased, but the expressions of TLR4 and NF-κBp65 proteins and mRNA, and the TNF-α and IL-1β in serum were not significantly changed in 6-gingerenol group. CONCLUSION: 6-Gingerenol could treat ulcerative colitis though inhibiting intestinal immune inflammation and promoting the repair of damaged colonic mucosa. The mechanism is related to its inhibition on activation of Notch and TLR4/NF-κB signaling pathway.

Overexpression of miR-31 regulates TLR4/NF-κB signaling pathway and apoptotic protein in colitis model mice / 中国应用生理学杂志

To investigate the effects of miR-31 on TLR4/NF-κB signaling pathway and apoptosis-related proteins in dextran sulfate sodium (DSS) induced mouse colon colitis. Methods: ① Mouse model of colon colitis: 1% DSS was used to induce mouse ulcerative colitis (UC). Fourteen FVB non-transgenic mice were randomly divided into control group (n= 6), DSS group (n= 8), and 16 FVB miR-31 transgenic mice were randomly divided into miR-31 overexpression group (n= 8), miR-31 overexpression +DSS group (n= 8). DSS was dissolved in water and administered to mice by drinking water. The DSS group and miR-31+DSS group drank 1% DSS water in the first week, normal sterilized water in the second week, and 1% DSS water in the third week, after 5 weeks, the modeling was completed, then the colon tissues of the mice were collected. Western blot and IHC were used to detect the expressions of NF-κB p65, TLR4, Bax and Bcl-2 proteins in mouse colon tissue, TUNEL was used to detect apoptosis of mouse colon tissues. ② Cell culture experiments: Transfection of miR-31mimic and inhibitor by lipofectamine resulted in overexpression or knockdown of miR-31 in human colon epithelial cell line HCT 116 cells, each group was repeated three times and cells were collected 48 h later, Western blot was used to detect the expressions of NF-κB p65 and TLR4 protein. ① In animal experiments, compared with the control group, the expression levels of NF-κB p65, TLR4 protein and apoptotic cell index in the DSS group and miR-31 overexpression group in mouse colon tissue were significantly increased (P＜0.05 or P＜0.01), and the Bcl-2 / Bax ratio was significantly reduced (P＜0.05 or P＜0.01); and compared with the DSS group, the expression levels of NF-κB p65, TLR4 protein and apoptotic cell index in the miR-31+DSS group were significantly increased (P＜0.01), while the Bcl-2/Bax ratio was significantly decreased (P＜0.01). ② In cell experiments, compared with the control group, the expression levels of NF-κB p65 and TLR4 protein in the over-expressed miR-31 group of HCT 116 cells were significantly increased (P＜0.05 or P＜0.01), the expressions of NF-κB p65 and TLR4 protein in miR-31 knockdown group were decreased (P＜0.05). miR-31 promotes the development of colitis by promoting TLR4/NF-κB signaling pathway and mediating apoptosis of intestinal epithelial cells.

Quercetin alleviates lipopolysaccharide-induced acute kidney injury in mice by suppressing TLR4/NF-κB pathway / 南方医科大学学报

OBJECTIVE@#To investigate the protective effect of quercetin against lipopolysaccharide (LPS)- induced acute kidney injury (AKI) in mice and explore its mechanism.@*METHODS@#Forty male BALB/c mice were randomly divided into control group (with saline treatment), 15 mg/kg LPS group, and quercetin-treated groups with intragastric quercetin treatment (once daily for 3 consecutive days) at low (25 mg/kg) and high (50 mg/kg) dose prior to 15 mg/kg LPS injection. LPS was administered by intraperitoneally injection 1 after the last gavage of quercetin. The mice were sacrificed 24 h after LPS injection for analysis of kidney pathologies, blood urea nitrogen (BUN) and creatinine levels; serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 were detected by ELISA, and the expressions of Toll-like receptor-4 (TLR4), MyD88, TRAF-6 and NF-κBp65 in the kidney were detected by Western blotting.@*RESULTS@#Quercetin significantly lessened renal pathologies, lowered BUN and creatinine levels ( < 0.05) and inhibited TNF-α, IL-1β, and IL-6 production in mice with LPS-induced AKI ( < 0.05). Pretreatment with quercetin also significantly inhibited TLR4, MyD88, and TRAF-6 expressions and NF-κBp65 activation in the kidneys of the rats with LPS challenge ( < 0.05).@*CONCLUSIONS@#Quercetin pretreatment can protect mice against LPSinduced AKI by inhibiting TLR4/NF-κB signaling pathway.

Protective effect of phellodendrine on bacterial vaginosis mice / 中草药

Objective To investigate the impact of phellodendrine on treating bacterial vaginosis (BV) mice. Methods BV model mice were induced by vaginal injecting with EPEC and MRSA. After the mice in control and model groups being administrated with phellodendrine (10, 20, and 40 mg/kg) for 7 d, the mice were sacrificed under deep anesthesia. The mice vaginal pathology changes were observed using HE staining method. IL-1β, IL-6, IL-8, and TNF-α secretion in vagina and PGE2, PGF2α levels in uterus were detected using ELISA assay. Uterine MMP-9, TLR4, and NF-κB expression levels were measured using western blotting. Results Vaginal pathology changes were improved by phellodendrine. Specifically, phellodendrine could reduce IL-1β, IL-6, IL-8, and TNF-α secretion in vagina, it could also reduce PGE2 and PGF2α content, and inhibit MMP-9, TLR4, and NF-κB expression levels in uterus tissue. Conclusion Phellodendrine could effectively treat BV and reduce the rise of pre-term birth by simultaneously inhibiting endovaginal inflammatory response and regulating intrauterine TLR4/NF-κB signal pathway in BV mice.

Inhibitory effects of right cervical sympathetic trunk transection on in-flammatory response after acute myocardial infarction in rats and its in-fluence on HMGB1/TLR4/NF-κB signaling pathway / 中国病理生理杂志

AIM:To observe the effects of transection of right cervical sympathetic trunk(TCST)on inflam-matory response and expression of high mobility group box 1(HMGB1)and TLR4/NF-κB signaling pathway in the rats af-ter acute myocardial infarction(AMI).METHODS: AMI model was established by ligation of left anterior descending coronary artery in SD rats,then the model rats were randomly divided into MI group and MI +TCST group.MI+TCST model was performed by transection of right cervical sympathetic trunk after left anterior descending coronary artery ligation. The rats in MI group and MI+TCST group were divided into 1,3,7,14 and 28 d subgroups,and another sham operation group threading without ligation, with 8 rats in above each group.After modeling for 4 weeks, the cardiac function was measured by echocardiography.All rats were killed to harvest the hearts for mesuring cardiac hypertrophy index.The myo-cardial tissue close to infarction was observed with HE staining.The relative mRNA expression levels of HMGB1, tumor necrosis factor α(TNF-α)and interleukin(IL)-6 at different time points were detected by real-time PCR.The protein ex-pression of HMGB1 and TLR4 at different time points after AMI was determined by Western blot.The effect of transection of right cervical sympathetic trunk on the expressions of HMGB 1 and TLR4/NF-κB signaling pathway was also analyzed. RESULTS:Compared with the MI group,left ventricular ejection fraction(LVEF)and left ventricular shorterning fraction (LVFS)were significantly higher(P<0.05),left ventricular end-diastole dimension(LVEDd),left ventricular end-sys-tole dimension(LVESd)and cardiac hypertrophy index were significantly lower(P<0.05), and the mRNA levels of HMGB1,TNF-αand IL-6 decreased significantly in MI +TCST group(P<0.05).Western blot results revealed that the protein expression level of HMGB1 increased in the infarct border zone at 3 d,and reached its peak at 7 d,then gradually decreased,and at 28 d after MI in MI group was still significantly higher than that in sham group(P<0.05).The protein expression of TLR4 was consistent with that of HMGB1.Transection of right cervical sympathetic trunk reduced protein ex-pression of HMGB1 and TLR4/NF-κB signaling pathway-related proteins(P<0.05).CONCLUSION: Transection of right cervical sympathetic trunk improves ventricular remodeling and maintaining cardiac function.The mechanism may be related to inhibiting HMGB1/TLR4/NF-κB signaling pathway to reduce inflammatory response.

Effect of Kudiezi Injection on inflammation and TLR-4/NF-κB signal in cerebral ischemia reperfusion rats / 药物评价研究

Objective To investigate the protective effects of Kudiezi (KDZ) Injection on cerebral ischemia reperfusion injury in rats and to explore its protective mechanism.Methods The rat model of middle cerebral artery occlusion (MCAO) was established by modified suture method,and cerebral blood flow was monitored using laser Doppler flowmetry (LDF).Male SD rats were randomly divided into control group,model group,Kudiezi Injection high and low dose groups.After ischemia-reperfusion for 24 h,the neurological scores were evaluated.After anesthesia,the blood samples and brain tissues were collected,and the expression of inflammatory was detected by Elisa.Western blotting was used to detect the expression of TLR-4 and NF-κB protein.Results Behavioral scores showed that neural function defect was serious in model group compared with control group (P ＜ 0.01).In model group,cerebral index and cerebral infarction area were significantly higher than those of the control group;After KDZ intervention,the symptoms of neurological deficit was alleviated (P ＜ 0.01),the cerebral index and cerebral infarction area of mode were decreased,and the neuronal necrosis was reduced.Kudiezi Injection could significantly reduce the cerebral homogenate and serum levels of TNF-α (P ＜ 0.05) and increase IL-10 level (P ＜ 0.05).Westem blotting showed that Kudiezi Injection could reduce the expression of TLR-4 and NF-κB protein (P＜0.05).Conclusion Kudiezi Injection has protective effect on cerebral ischemia reperfusion rats.After ischemia-reperfusion,Kudiezi Injection could reduce the levels of TNF-α and raise IL-10.Its mechanism may be associated to the down-regulation of TLR-4/NF-κB signaling pathway.

Alleviation of insulitis of NOD mice by DPP-4 inhibitor through suppressing TLR4/NF-κB signaling path-way / 实用医学杂志

Objective To explore the effects of DPP-4 inhibitor sitagliptin on insulitis of NOD mice and the potential mechanism. Methods 223-month-old female NOD mice were randomly divided into Sitagliptin(Si-ta)group(n=11)gavaged with Sita(30 mg/Kg)daily for 12 weeks and control(Con)group with equal volume of normal saline(NS). The body weight,food-intake,water-intake and blood glucose were recorded weekly. Intraperi-toneal glucose tolerance test(IPGTT)was performed at the end of treatment and the blood sample was collected. Then mice were execute. The serum insulin level was measured by ELISA. Pancreas morphology and insulitis were evaluated by hematoxylin-eosin staining. The protein expression level of TLR4 ,MyD88 and NF-κB was analyzed by Western Blot. Results Compared with that of Con group ,the insulitis of Sita group was significantly alleviative(P < 0.05)and the serum insulin level was increased significantly(P < 0.05). The protein expression level of TLR4(P<0.05),MyD88(P<0.01)and NF-κB(P<0.05)in Sita group was significantly decreased. Conclusion Sitagliptin alleviates insulitis in NOD mice and increases the serum insulin level ,probably owing to the suppression of TLR4/MyD88/NF-κB signaling pathway.

Effect of Kudiezi Injection on inflammation and TLR-4/NF-κB signal in cerebral ischemia reperfusion rats / 药物评价研究

Objective To investigate the protective effects of Kudiezi (KDZ) Injection on cerebral ischemia reperfusion injury in rats and to explore its protective mechanism.Methods The rat model of middle cerebral artery occlusion (MCAO) was established by modified suture method,and cerebral blood flow was monitored using laser Doppler flowmetry (LDF).Male SD rats were randomly divided into control group,model group,Kudiezi Injection high and low dose groups.After ischemia-reperfusion for 24 h,the neurological scores were evaluated.After anesthesia,the blood samples and brain tissues were collected,and the expression of inflammatory was detected by Elisa.Western blotting was used to detect the expression of TLR-4 and NF-κB protein.Results Behavioral scores showed that neural function defect was serious in model group compared with control group (P ＜ 0.01).In model group,cerebral index and cerebral infarction area were significantly higher than those of the control group;After KDZ intervention,the symptoms of neurological deficit was alleviated (P ＜ 0.01),the cerebral index and cerebral infarction area of mode were decreased,and the neuronal necrosis was reduced.Kudiezi Injection could significantly reduce the cerebral homogenate and serum levels of TNF-α (P ＜ 0.05) and increase IL-10 level (P ＜ 0.05).Westem blotting showed that Kudiezi Injection could reduce the expression of TLR-4 and NF-κB protein (P＜0.05).Conclusion Kudiezi Injection has protective effect on cerebral ischemia reperfusion rats.After ischemia-reperfusion,Kudiezi Injection could reduce the levels of TNF-α and raise IL-10.Its mechanism may be associated to the down-regulation of TLR-4/NF-κB signaling pathway.

Alleviation of insulitis of NOD mice by DPP-4 inhibitor through suppressing TLR4/NF-κB signaling path-way / 实用医学杂志

Objective To explore the effects of DPP-4 inhibitor sitagliptin on insulitis of NOD mice and the potential mechanism. Methods 223-month-old female NOD mice were randomly divided into Sitagliptin(Si-ta)group(n=11)gavaged with Sita(30 mg/Kg)daily for 12 weeks and control(Con)group with equal volume of normal saline(NS). The body weight,food-intake,water-intake and blood glucose were recorded weekly. Intraperi-toneal glucose tolerance test(IPGTT)was performed at the end of treatment and the blood sample was collected. Then mice were execute. The serum insulin level was measured by ELISA. Pancreas morphology and insulitis were evaluated by hematoxylin-eosin staining. The protein expression level of TLR4 ,MyD88 and NF-κB was analyzed by Western Blot. Results Compared with that of Con group ,the insulitis of Sita group was significantly alleviative(P < 0.05)and the serum insulin level was increased significantly(P < 0.05). The protein expression level of TLR4(P<0.05),MyD88(P<0.01)and NF-κB(P<0.05)in Sita group was significantly decreased. Conclusion Sitagliptin alleviates insulitis in NOD mice and increases the serum insulin level ,probably owing to the suppression of TLR4/MyD88/NF-κB signaling pathway.

Immunoregulatory effect of BuyangHuanwu Decoction on experimental autoimmune encephalomyelitis mice / 中国免疫学杂志

Objective:To explore the anti-inflammatory therapeutic effect and possible immunoregulatory mechanism of Buyang Huanwu Decoction (BYHWD) on the development of experimental autoimmune encephalomyelitis (EAE). Methods:Female C57BL/6 mice were immunized subcutaneously with myelin oligodendrocyte glycoprotein peptides ( MOG35-55 ) ,and randomly divided into saline group,BYHWD group,with 13 mice in each group. At the 3th day,25 ml/kg of saline was orally given to each mouse of saline group,50 g/kg ig of crude BYHWD was orally given to each mouse in BYHWD group for 25 days. Clinical score and body mass were recorded every day. Inflammatory cell infiltrations of spinal cord were observed by HE staining Myelin staining observes the demyelination situa-tion. And the expression of ROCKⅡ in spleen was detected by immunofluorescence staining. The subtypes of CD4+ T cells were analyzed by flow cytometry. Western blot was used to detect the expression of TLR4,Myd88,NF-κB,COX-2,ROCKⅡ in spinal cord and ROCKⅡ in brain. Results: The neurologicalscore significantly decreased in EAE mouse of BYHWD group compared with the saline group (P<0. 001) . BYHWD inhibited the inflammatory cell infiltration and demyelination in the nervous centralis(P<0. 05). The treatment of BYHWD effectively reduced the increased the proportion of CD25+(P<0. 05),IL-10+(P<0. 05),TGF-β+(P<0. 01), IFN-γ+( P<0. 05 ) CD4+T cells , and inhibited the expression of peripheral and central ROCKⅡ( P<0. 05 ) ;BYHWD reduced the expression of TLR4,MyD88,NF-κB,COX-2 in spinal cord (P<0. 05). Conclusion:BYHWD can exert anti-inflammatory and immune regulation effect by the inhibition of ROCKⅡ/TLR4/ NF-κB signaling pathway and regulation of the proportion of peripheral T cell sub-sets.

The expressions of Toll-like receptor4/NF-κB and PI3K/AKT/NF-κB signaling pathways in ratulcerative colitis induced by the combined enema of trinitrobenzene sulphonic acid and ethano and the interventional effect of electroacupuncture on them / 西安交通大学学报(医学版)

ABSTRACT:Objective To explore the expressions of Toll-like receptor4/NF-κB and PI3K/AKT/NF-κB signa-ling pathways in rat ulcerative colitis (UC)induced by the combined enema of trinitrobenzene sulphonic acid and ethano and the interventional effect of electroacupuncture on them.Methods Totally 240 male Wistar rats were randomly divided into 6 groups:normal control group,model control group,electroacupuncture group,TLR4mAb group,LY294002 group,and TLR4mAb combined with LY294002 (T&L)group.The combined enema of trinitro-benzene sulphonic acid (TNB)and ethanol was intrarectally administered for 4 weeks to induce UC.At the same time of modeling ,Zusanli point was electro-acupunctured in electroacupuncture group while intraperitoneal injec-tion of TLR4mAb and LY294002 was given respectively to the corresponding group.Each rat was treated with the above-mentioned TLR4mAb injection and LY294002 injection in T&L group for 4 weeks.The disease activity index (DAI)of all the rats was evaluated daily.The rats were killed after 4 weeks.The colonic mucosa damage index (CMDI)and tissue damage index (TDI)were evaluated by a pathologic grading system.The expressions of P-Akt and active NF-κB protein in the colon mucosa were determined by Western blotting.TLR4 mRNA,PI3K mRNA, AKT mRNA,NF-κB mRNA,TNF-αmRNA and IL-1βmRNA expressions were measured with RT-PCR.Results Compared with those in normal control group,TLR4 mRNA,PI3K mRNA,P-AKT,active NF-κB,TNF-αmRNA and IL-1βmRNA expressions as well as DAI,CMDI and TDI were all increased obviously in model control group (P <0.01).Compared with those in model control group,TLR4mRNA expression was decreased obviously in TLR4mRNA group (P <0.01),the expressions of PI3KmRNA and P-AKT were decreased obviously in LY294002 group (P <0.01 ).Not only TLR4mRNA expression but also PI3KmRNA and P-AKT expressions were decreased significantly in electroacupuncture group and T&L group (P <0.01 ).Corresponding to the above-mentioned chan-ges,active NF-κB,TNF-αmRNA and IL-1βmRNA expressions as well as DAI,CMDI and TDI were decreased obvi-ously in all the treated groups compared with those in model control group (P <0.05 or P <0.01),but the six inde-xes were better in electroacupuncture group and T&L group than in TLR4mAb group and LY294002 group (P <0.05).There were obvious positive correlations of active NF-κB with TNF-αmRNA and IL-1β mRNA expressions (r 1 =0.579,P <0.05;r 2 =0.561,P <0.05).Conclusion Electroacupuncture can significantly decrease NF-κB activity and TNF-αmRNA and IL-1β mRNA expressions in UC rats,thus alleviating the severity of UC,which is closely correlated to its blocking both TLR4/NF-κB and PI3K/AKT/NF-κB signaling pathways.

Inhibition of astragaloside IV against isoproterenol-induced myocardial hypertrophy in neonatal rats and its mechanism / 中草药

Objective: To investigate the protective effect of astragaloside IV (As IV) on myocardial hypertrophy induced by isoproterenol (ISO) and its mechanism. Methods: The primary cultures of neonatal rat cardiac myocytes were cultured for 48 h in vitro. The cardiac myocytes were treated with As IV, IκBα phosphorylation inhibitor BAY11-7082, and β-blockers Propranolol for 30 min, followed by 48 h incubation with ISO 10 μmol/L. The cardiomyocyte volume was measured by computer photograph analysis system. Total protein contents were assayed by the method of Bradford. RT-PCR was used to quantify the mRNA expression of ANP and TLR4; Western blotting was used to quantify the expression of TLR4, p65, and IκBα proteins; ELISA was used to quantify TNF-α and IL-6. Results: Compared with the control group, the cell size, total protein content, ANP and TLR4 mRNA, TLR4, p65, TNF-α and IL-6 were increased, and expression of IκBα protein was decreased in ISO group (P < 0.01). As IV, BAY11-7082, and Propranolol could remarkably down-regulate the over-expression of the cell size, total protein content, ANP and TLR4 mRNA, TLR4, p65, TNF-α, and IL-6, and increase the expression of IκBα protein (P < 0.05). Conclusion: As IV has the protective effect on cardiac hypertrophy induced by ISO, which is partially referring to inhibiting the TLR4/NF-κB signaling pathway and more than attenuating inflammatory effect.