Protective effect and mechanism of TLR4/NF-κB pathway regulated by miR-146a in intracerebral hemorrhage model rats / 中国免疫学杂志

Objective:To explore role of miR-146a in regulating TLR4/NF-κB pathway on inflammatory injury and neuropro-tection in intracerebral hemorrhage model rats and its possible mechanism.Methods:A total of 40 rats were selected and randomly divided into sham,model,over-expressing miR-146a adenovirus and negative virus injection groups,with 10 rats in each group.Garcia score was used for neurological function;HE staining was used to observe changes of brain tissues.ELISA was used to detect inflammatory factors levels.TLR4,NF-κB protein and gene expressions in brain tissues were detected by Western blot and RT-PCR.Results:Compared with model group,neural function score of overexpressed miR-146a adenovirus injection group was increased(P<0.05).Model group had abnormal cell morphology,edema and inflammation.Cell morphology,edema and inflammation were alleviated in overexpressed miR-146a adenovirus injection group.Inflammatory factors levels in model group were higher than sham group(P<0.05).Inflammatory factors levels in overexpressed miR-146a adenovirus injection group were lower than model group(P<0.05).TLR4,NF-κB protein and mRNA expressions in model group were increased than sham group(P<0.05).TLR4,NF-κB protein and mRNA expressions in overexpressed miR-146a adenovirus injection group were decreased than model group(P<0.05).Conclusion:miR-146a can improve neural function and reduce inflammatory injury in rats with intracerebral hemorrhage,possibly by inhibiting activation of TLR4/NF-κB signaling pathway and reducing inflammatory factors levels of brain tissues.

Research progress on the association between sleep deprivation and inner ear disease / 重庆医学

Inner ear diseases are common in the field of otolaryngology,including hearing loss,tinnitus and peripheral vestibular dysfunction.Their pathogenesis is relatively complex,which is one of the hot spots in current research.A large number of studies have demonstrated that sleep disorder is an important inducement of inner ear diseases.This paper reviews the impact of sleep deprivation on inner ear diseases in order to pro-vide a theoretical basis for the mechanisms of sleep deprivation on inner ear diseases.

Saponin from Panax japonicus IVa alleviates lipopolysaccharide-induced acute lung injury in rats via its anti-inflammatory and antioxidative ef-fects / 中国病理生理杂志

AIM:To investigate the effects of saponin from Panax japonicus IVa(SPJ IVa)on acute lung inju-ry in rats and to explore its possible protective mechanism.METHODS:Sixty SD rats were randomly divided into four groups,15 rats in each group:the control group,model group,low-dose SPJ IVa group,and high-dose SPJ IVa group.A rat model of ALI was established via intratracheal instillation of lipopolysaccharide(LPS,2 mg/kg).Rats in the low-and high-dose SPJ IVa groups were intraperitoneally injected with 15 and 45 mg/kg SPJ IVa,respectively,30 min after model-ing.Serum,bronchoalveolar lavage fluid(BALF),and lungs were collected 24 h after modeling.Pathomorphological changes in lung tissues were assessed using HE staining.The wet weight/dry weight ratio of lung tissues was measured us-ing the weighing method,whereas ELISA was used to measure the levels of interleukin-1β(IL-1β),IL-6,and tumor ne-crosis factor-α(TNF-α)in the serum and BALF.The levels of malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)were assessed using the kit method.Cell apoptosis in lung tissues was evaluated by immunohisto-chemical staining of cleaved caspase-3 and TUNEL.Western blot was used to measure the expression of nuclear factor E2-related factor 2(Nrf2),heme oxygenase-1(HO-1),nuclear factor-κB(NF-κB)p65,and Toll-like receptor 4(TLR4)in lung tissues.RESULTS:Compared with control group,the lung tissues of the model group were significantly damaged,and the lung injury scores(0.21±0.22 vs 2.98±0.46)and lung wet/dry weight ratios(3.09±0.41 vs 6.36±0.61)were significantly increased(P<0.01).Compared with model group,the lung injury scores(1.80±0.31 and 1.05±0.25 vs 2.98±0.46)and lung wet/dry weight ratios(5.25±0.44 and 3.89±0.35 vs 6.36±0.61)in low-and high-dose SPJ IVa groups were significantly reduced(P<0.01).The administration of LPS resulted in elevated levels of pro-inflammatory cy-tokines(IL-1β,IL-6,and TNF-α)as well as the oxidative marker MDA in both serum and BALF(P<0.01).Additional-ly,it led to a decrease in antioxidant markers SOD and GSH(P<0.01).However,treatment with both low and high doses of SPJ IVa effectively attenuated the LPS-induced production of pro-inflammatory factors and oxidative markers MDA(P<0.01),while also increasing SOD and GSH levels(P<0.05 or P<0.01).In the model group,evident apoptosis was ob-served in lung tissues,whereas treatment with low and high doses of SPJ IVa significantly suppressed TUNEL-positive cells and the expression of cleaved caspase-3(P<0.01).The expression levels of Nrf2,HO-1,NF-κB p65,and TLR4 in lung tissues were significantly higher in the model group than in the control group(P<0.01);in turn,after treatment with low and high doses of SPJ IVa,Nrf2 and HO-1 were further upregulated(P<0.01),whereas NF-κB p65 and TLR4 were downregulated(P<0.01).CONCLUSION:The inhibitory effect of SPJ IVa on LPS-induced ALI in rats may be attribut-ed to its ability to suppress the TLR4/NF-κB-and Nrf2/HO-1-mediated inflammatory response and oxidative stress.

Effects of Zhuangyao Shuanglu Tongnao Formula on neuronal apoptosis of rats with ischemia-reperfusion induced injury / 中成药

AIM To investigate the effects of Zhuangyao Shuanglu Tongnao Formula on neuronal apoptosis in rats with cerebral ischemia-reperfusion injury based on the study of oxidative stress and inflammatory response.METHODS The rats were randomly divided into the sham operation group,the model group,the edaravone group(3.0 mg/kg),the low,medium and high dose groups(9.0,18.0,36.0 g/kg)of Zhuangyao Shuanglu Tongnao Formula,with 18 rats in each group.The middle cerebral artery occlusion/reperfusion was conducted by thread embolism method to simulate cerebral ischemia reperfusion injury in rats followed by 6 days corresponding drugs administration.Subsequently,the rats had their neurological function deficit scored by Zeal Longa scoring method;their sizes of cerebral infarction areas measured by TTC staining;their pathological damage and apoptosis of neurons in hippocampal CA1 area of ischemic penumbra of the brain tissue detected by HE staining and TUNEL staining;their SOD activity and levels of GSH,MDA,IL-6,IL-1β,TNF-α in brain tissue detected by kits;and their protein expressions of Bax,Bcl-2,caspase-3,cleaved-capase-3,TLR4,NF-κB p65,Nrf2,HO-1 in rat brain tissue determined by Western blot.RESULTS Compared with the model group,the groups intervened with edaravone,medium and high dose of Zhuangyao Shuanglu Tongnao Formula displayed improvements in the scores of nerve function defects,the rate of cerebral infarction,the rate of neuronal apoptosis,the levels of IL-6,IL-1β,TNF-α and MDA in the ischemic penumbra of brain tissues,the protein expressions of Bax and TLR4,the ratio of cleaved-capase-3/caspase-3 and p-NF-κB p65/NF-κB p65(P＜0.05),the levels of GSH,the activity of SOD and the protein expressions of Bcl-2,Nrf2 and HO-1(P＜0.05).CONCLUSION Being an inhibitor of oxidative stress and inflammatory response,Zhuangyao Shuanglu Tongnao Formula can alleviate brain injury in rats with cerebral ischemia reperfusion injury through the inhibition of neuronal apoptosis and improvement of neural function mediated by the inhibition of TLR4/NF-κB signal pathway and activation of Nrf2/HO-1 signal pathway.

Effect and mechanism of arctigenin on ventricular remodeling and inflammatory reaction in rats with chronic heart failure / 中华老年心脑血管病杂志

Objective To investigate the influences of arctigenin(ATG)on ventricular remodeling and inflammatory reaction in chronic heart failure(CHF)rats,and to analyze its potential mecha-nism.Methods A total of 79 SD rats were randomly divided into sham operation group(n=12),and the remaining rats were inflicted with abdominal aortic coarctation to establish a rat CHF model.After modeling,60 CHF rats were randomly divided into CHF group,low and high dose ATG group(ATG-L and ATG-H groups,10 and 20 mg/kg,respectively),ATG+NC group[20 mg/kg ATG+100 μl high mobility group protein B1(HMGB1)negative control plasmid],and ATG+HMGB1 group(20 mg/kg ATG+100 pl HMGB1 overexpression plasmid),with 12 rats per group.After 4 weeks of corresponding intervention,heart function,levels of B-type brain na-triuretic peptide(BNP),N-terminal B-type brain natriuretic peptide precursor(NT-proBNP)andIL-6 and TNF-α,heart mass index(HMI)and left ventricular mass index(LVMI),pathological changes of myocardial tissue,cross-sectional area of myocardial cells and myocardial collagen vol-ume fraction(CVF)and protein expression of HMGB1/Toll-like receptor 4(TLR4)/NF-κB sig-naling pathway in left ventricular myocardial tissue were measured.Results Compared with the sham operation group,myocardial tissue HMGB1(0.42±0.05 vs 0.15±0.02)and TLR4(0.70± 0.09 vs 0.21±0.04)protein levels,and phosphorylated NF-κB p65(p-NF-κB p65)/NF-κB p65(0.73±0.09 vs 0.26±0.05)protein ratio were obviously increased in the CHF group,while the left ventricular ejection fraction(LVEF)and left ventricular short-axis fractional shortening(LVFS)were obviously decreased(P＜0.05).Myocardial tissue HMGB1(0.33±0.04、0.24±0.04 vs 0.42±0.05)and TLR4(0.56±0.06、0.41±0.05 vs 0.70±0.09)protein levels,and p-NF κB p65/NF-KB p65(0.61±0.08、0.49±0.06 vs 0.73±0.09)protein ratio were decreased,and the LVEF and LVFS were increased in the ATG-L group and ATG-H group than the CHF group(P＜0.05).Overexpression of HMGB1 obviously attenuated the inhibitory effects of ATG on HMGB1/TLR4/NF-κB signaling pathway,ventricular remodeling,and inflammatory reaction in CHF rats(P＜0.05).Conclusion ATG may suppress ventricular remodeling in CHF rats by in-hibiting HMGB1/TLR4/NF-κB signaling inflammatory pathway.

Protective effect and mechanism of Longshengzhi capsules on cerebral ischemia-reperfusion injury in rats / 中国药房

OBJECTIVE To explore the protective effect and mechanism of Longshengzhi capsules on cerebral ischemia- reperfusion injury in rats. METHODS The model of middle cerebral artery occlusion （MCAO） was established by using the improved thread occlusion method. The experiment was divided into six groups： sham surgery group （only separating blood vessels without inserting thread plugs， given the same volume of normal saline）， model group （modeling， given the same volume of normal saline）， nimodipine group （positive control， modeling， dose of 20 mg/kg）， and low-dose， medium-dose， and high-dose groups of Longshengzhi capsules （modeling， doses of 0.72， 1.44 and 2.88 g/kg， respectively）， with 10 mice in each group. Each group was given corresponding medication solution/normal saline by gavage， once a day， for 7 consecutive days. One hour after the last administration， the Zea Longa scoring method was used to score the neurological deficits in each group of rats， and the ABC enzyme-linked immunosorbent assay was used to detect the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rats； TTC staining was used to observe the volume of cerebral infarction in rats and calculate the cerebral infarction volume ratio. Hematoxylin eosin staining was used to observe the pathological changes in the brain tissue of rats. Immunohistochemical staining was used to detect the positive expression of NLRP3 protein in the brain tissue of rats. Real-time fluorescence quantitative PCR was used to detect mRNA relative expressions of Toll-like receptor 4 （TLR4） and nuclear factor-κB （NF-κB） in the brain tissue of rats. Western blot assay was adopted to detect the relative expressions of TLR4， NLRP3 and phosphorylated NF-κB （p-NF-κB） protein in the brain tissue of rats and its intracellular NF-κB protein. RESULTS Compared with the sham surgery group， the neural dysfunction score， serum levels of TNF-α and IL-6， cerebral infarction volume ratio， relative expression levels of NF-κB and TLR4 mRNA， as well as protein relative expressions of TLR4， NLRP3 and p-NF-κB in the brain tissue， and relative protein expression of intracellular NF-κB were increased significantly in the model group （P＜0.01）； the enlarged gap and significant edema were observed in cortical nerve cells of brain tissue in rats， with a large amount of inflammatory cell infiltration； the positive expression of NLRP3 protein in brain tissue of rats obviously increased. Compared with the model group， the levels of the above indicators in the medium-dose and high-dose groups of Longshengzhi capsules， as well as the Nimodipine group， were reversed to varying degrees， and most differences were statistically significant （P＜0.05 or P＜0.01）； the pathological morphology observation showed a significant improvement， and the positive expression of NLRP3 protein in the brain tissue of rats was obviously reduced. CONCLUSIONS Longshengzhi capsules may inhibit TLR4/NF-κB/NLRP3 signaling pathway and neuroinflammatory response， thereby achieving a protective effect against cerebral ischemia-reperfusion injury in rats.

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.

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.

Paeoniflorin ameliorates diabetic cognitive dysfunction by inhibiting TLR4/NF-κB signaling in ovariectomized mice / 中华内分泌代谢杂志

Objective:To investigate the effect of paeoniflorin on toll-like receptor 4(TLR4)/nuclear transcription factor(NF-κB) signaling pathway of streptozotocin combined with ovariectomized mice, and to explore whether it can improve the cognitive impairment of ovariectomized diabetic mice.Methods:Ninety female C57BL/6J mice were divided into SHAM group, ovariectomy group, diabetes group(intraperitoneal injection of STZ 50 mg·kg -1·d -1 for 5 consecutive days), dual model group(DM modeling and OVX operation), paeoniflorin low-dose intervention group(OVX+ STZ+ L-PF 50 mg·kg -1·d -1), paeoniflorin high-dose intervention group(OVX+ STZ+ H-PF 100 mg·kg -1·d -1; all groups n=15). After 8 weeks of paeoniflorin intervention, their cognitive function was tested by behavioral experiments(Morris water maze and Y maze). The pathological changes of hippocampal tissue were observed by HE and Nissl staining. The mRNA expressions of TLR4, tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) in hippocampal tissues were detected by real-time fluorescence quantitative PCR. The expression of TLR4, NF-κB P65, TNF-α, IL-6, IL-1β, β-amyloid protein(Aβ), tau proteins, and p-tau proteins were detected by Western blot. Results:Compared with SHAM group, the learning and memory ability of ovariectomy group, diabetes group and dual model group decreased, hippocampal cells were damaged, and the expression of related gene mRNA and protein were increased, especially in dual model group; Compared with dual model group, paeoniflorin intervention could delayed the learning and memory impairment, improve cognitive function, reduce the degree of hippocampal injury, and decrease the expression levels of related gene mRNA and protein, The above changes were the most pronounced at paeoniflorin high-dose intervention group.Conclusion:Paeoniflorin improves cognitive dysfunction in ovariectomized diabetic mice by inhibiting TLR4/NF-κB signaling pathway.

The role of TLR4/NF-κB signaling pathway in sleep deprivation induced Meniere's disease / 临床耳鼻咽喉头颈外科杂志

Objective:By detecting the levels of proteins in the Toll-like receptor-4/nuclear factor-κB （TLR4/NF-κB） signaling pathway and downstream proinflammatory cytokines in peripheral blood of patients with Meniere's disease （MD）, Pittsburgh Sleep Quality Index （PSQI） scores were collected to investigate the correlation between sleep disorders and MD and the role of TLR4/NF-κB signaling pathway in mediating sleep disorders inducing MD. Methods:Thirty-two MD patients and 20 family members of patients without middle ear and inner ear related diseases were selected. Basic data, PSQI and fasting peripheral blood of all subjects were collected. Enzyme linked immunosorbent assay.The levels of interleukin-1β（IL-1β）, tumor necrosis factor-α（TNF-α）, monocyte chemokine-1（MCP-1）, Toll-like receptor 4（TLR4） and nuclear factor-κB（NF-κB） in peripheral blood were detected by ELISA, and the data were statistically analyzed. Results:①PSQI score of MD group was higher than that of normal control group, and the difference was statistically significant（P<0.01）; The scores of every factors of PSQI in MD group were higher than those in normal control group, and the scores of factors 2, 4 and 6 were significantly different from those in normal control group. ②In the MD group, there were 18 patients with sleep disorders, with a prevalence rate of 56.25%, including 6 males with a prevalence rate of 50.00% and 12 females with a prevalence rate of 60.00%. ③The levels of five test indexes in MD group, sleep disorder group and non-sleep disorder group were higher than those in control group, and the levels of TLR4 and NF-κB in MD group were significantly different from those in control group（P<0.05）. The levels of IL-1β, TNF-α, TLR4 and NF-κB in sleep disorder group were significantly different from those in control group（P<0.05）. The levels of five test indexes in non-sleep disorder group were not statistically significant compared with those in control group. The levels of five test indexes in the MD sleep disorder group were higher than those in the MD group and the non-sleep disorder group, with no statistical significance. The levels of five test indexes in MD group were higher than those in non-sleep disorder group, with no statistical significance（P>0.05）. Conclusion:①Sleep disorders may be one of the important predisposing factors of some MD, and the effects of sleep disorders on MD are different between the sexes. ②Sleep disorders may activate TLR4/NF-κB signaling pathway to induce MD. The selection of TLR4/NF-κB signaling pathway related proteins and downstream pro-inflammatory factor inhibitors to intervene MD may provide a new idea for protecting the hearing balance function of MD.

Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway / 中国中药杂志

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-β-galactosidase(SA-β-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1β(IL-1β), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1β and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1β and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

Yifei Jianpi recipe improves cigarette smoke-induced inflammatory injury and mucus hypersecretion in human bronchial epithelial cells by inhibiting the TLR4/NF-κB signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore the mechanism of Yifei Jianpi recipe for improving cigarette smoke- induced inflammatory injury and mucus hypersecretion in cultured human bronchial epithelial cells.@*METHODS@#Serum samples were collected from 40 SD rats treated with Yifei Jianpi recipe (n=20) or normal saline (n=20) by gavage. Cultured human bronchial epithelial 16HBE cells were stimulated with an aqueous cigarette smoke extract (CSE), followed by treatment with the collected serum at different dilutions. The optimal concentration and treatment time of CSE and the medicated serum for cell treatment were determined with CCK-8 assay. The expressions of TLR4, NF-κB, MUC5AC, MUC7, and muc8 at both the mRNA and protein levels in the treated cells were examined with RT- qPCR and Western blotting, and the effects of TLR4 gene silencing and overexpression on their expressions were assessed. The expressions of TNF-α, IL-1 β, IL-6 and IL-8 in the cells were detected using ELISA.@*RESULTS@#At the optimal concentration of 20%, treatment with the medicated serum for 24 h significantly lowered the mRNA and protein expressions of TLR4, NF- κB, MUC5AC, MUC7, and MUC8 in CSE- exposed 16HBE cells, and these effects were further enhanced by TLR4 silencing in the cells. In 16HBE cells with TLR4 overexpression, the expressions of TLR4, NF-κB, MUC5AC, MUC7, and MUC8 were significantly increased after CSE exposure and were lowered following treatment with the medicated serum (P < 0.05). The medicated serum also significantly lowered the levels of TNF-α, IL-1β, IL-6 and IL-8 in CSE-exposed 16HBE cells (P < 0.05).@*CONCLUSIONS@#In the 16HBE cell model of chronic obstructive pulmonary disease (COPD), treatment with Yifei Jianpi recipe-medicated serum improves inflammation and mucus hypersecretion possibly by reducing MUC secretion and inhibiting the TLR4/NF-κB signaling pathway.

Study on the Effect and Mechanism of Hederagenin on Dextran Sodium Sulfate-Induced Ulcerative Colitis in mice / 中药新药与临床药理

Objective To study the effect and its mechanism of hederagenin(hed)on dextran sulfate sodium(DSS)-induced ulcerative colitis(UC)in mice.Methods(1)In vitro experiments:after treating RAW264.7 cells with different concentrations(0,2.5,5,10,20,40 μmol·L-1)of hed for 24 hours,the cell survival rate was detected by MTT assay.RAW264.7 cells were divided into:blank group,lipopolysaccharide(LPS)group(1 μg·L-1),LPS+2.5 μmol·L-1 hed group,LPS+5 μmol·L-1 hed group and LPS+10 μmol·L-1 hed group;an in vitro cellular inflammation model was established using LPS intervention for 24 hours and co-incubated with hed for 24 hours.The levels of interleukin 1β(IL-1β),IL-6 and tumor necrosis factor α(TNF-α)in the cell supernatant were determined by ELISA;the expression levels of TLR4/NF-κB pathway-related proteins in the cells were detected by Western Blot.(2)In vivo experiments:C57BL/6 mice were randomly divided into a blank group,a model group,a Salazosulfapyridine group(200 mg·kg-1),and an hed low-,medium-,and high-dosage groups(12.5,25,and 50 mg·kg-1),with 5 mice in each group.Mice were induced to establish UC model by drinking 3%DSS solution freely for 7 days.The UC model was then established by gavage once a day for 7 days.At the end of the administration,the Disease Activity Index(DAI)was evaluated;pathological changes in the colonic tissues of mice were observed by HE staining;the levels of IL-1β,IL-6,and TNF-α in the colonic tissue were measured by ELISA;and the expression levels of proteins related to the TLR4/NF-κB pathway in the colonic tissue were detected by Western Blot.Results(1)In vitro experiments:compared with the blank group(0 μ mol·L-1 group),there was no significant change in the cell survival rate in the 2.5-10 μmol·L-1 hed group(P＞0.05),and there was no significant toxicity effect on RAW264.7 cells.Compared with the blank group,the expression levels of IL-1β,IL-6,and TNF-α in RAW264.7 cells in the LPS group were significantly increased(P＜0.01);and the protein expression levels of TLR4 and p-NF-κ B/NF-κ B were significantly increased(P＜0.01).Compared with the LPS group,the expression levels of IL-1β and TNF-α in RAW264.7 cells in the hed 2.5,5,and 10 μmol·L-1 concentration groups were significantly decreased(P＜0.05,P＜0.01),and the protein expression levels of TLR4,p-NF-κB/NF-κB were significantly decreased(P＜0.05,P＜0.01);the IL-6 expression level of RAW264.7 cells in the hed 5 and 10 μmol·L-1 concentration groups was significantly reduced(P＜0.05,P＜0.01).(2)In vivo experiments:compared with the blank group,the body mass of mice in the model group was consistently reduced(P＜0.01),the DAI score was significantly elevated(P＜0.01),and the length of the colon was significantly shortened(P＜0.01);the colonic tissue showed obvious epithelial cell damage,and the histopathological scores were significantly elevated(P＜0.01);and the expression levels of the pro-inflammatory cytokines IL-1β,IL-6 and TNF-α were significantly increased(P＜0.01);protein expression levels of TLR4 and p-NF-κB/NF-κB were significantly increased(P＜0.01)in colon tissue.Compared with the model group,the body mass of mice in the low-,medium-and high-dose groups of hed were significantly increased(P＜0.05,P＜0.01),the DAI score was significantly decreased(P＜0.05,P＜0.01),the pathological damage of colon tissue improved to different degrees,and the protein expression levels of TLR4,p-NF-κB/NF-κB in the colonic tissue were significantly decreased(P＜0.05,P＜0.01);the colon length of mice in the medium-and high-dose groups of hed were significantly increased(P＜0.05,P＜0.01),and the expression levels and histopathological scores of IL-1β,IL-6,and TNF-α in colon tissue were significantly reduced(P＜0.05,P＜0.01).Conclusion Hed were able to effectively ameliorate colonic histopathological injury and reduce the levels of inflammatory factors in DSS-induced UC mice,and their mechanism of action may be related to the inhibition of the TLR4/NF-κB pathway.

Effects of Xiaokeshu recipe on levels of serum inflammatory factors in type2 diabetic rats and its mechanism / 中国临床药理学与治疗学

AIM: To explore the possible mechanism of Xiaokeshu recipe in the treatment of type 2 diabetes mellitus (T2DM) by observing the effects of Xiaokeshu Recipe on serum inflammatory factors. METHODS: Male SPF-grade SD rats were fed with high-fat and high-sugar fodder combined with intraperitoneal injection of streptozotocin (STZ) to prepare the model of T2DM. The model rats were randomly divided into a model group,a metformin group, low and high-dose Xiaokeshu recipe groups, and a normal group was set up. After successful modeling, the metformin group and the Xiaokeshu recipe groups were treated with metformin and Xiaokeshu recipe by gavage respectively, normal saline was given by gavage in the normal group and the model group. The general living status of rats before and after treatment was observed. After 4 weeks of drug intervention, serum samples and ileum tissue of rats were collected for biochemical and Western blot. RESULTS: As compaed with the model group,the polydipsia and polyuria in the low and high-dose Xiaokeshu recipe and the metformin groups could be improved. As compaed with the model group, the levels of fasting blood glucose (FBG), fasting insulin (FINS) and interleukin-1β (IL-1β) of rats in the low-dose Xiaokeshu recipe group were decreased, but the differences were statistically insignificant (P0.05), the levels of FBG, FINS and IL-1β of rats in the high-dose Xiaokeshu recipe and the metformin groups were significantly decreased as compared with the the model group (P 0.05 or P0.01). As compaed with the model group, the levels of Lipopolysaccharide (LPS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the high-dose Xiaokeshu recipe and the metformin groups were decreased (P<0.05 or P<0.01). As compared with the model group, the expressions of toll-like receptor 4 (TLR4) and nuclear transcription factor (NF-κB) protein in ileal tissue were down-regulated in the low and high-dose Xiaokeshu Recipe and the metformin groups (P<0.05 or P<0.01). CONCLUSION: Xiaokeshu recipe may reduce the level of serum LPS, inhibit the TLR4/NF-κB signaling pathway, and reduce the release of inflammatory factors, thus improving insulin resistance and reducing the blood sugar of the body.

Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway / 中国中药杂志

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-β1(TGF-β1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-β1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Effect of Erjing Pills on alleviating neuroinflammation of AD rats based on TLR4/NF-κB/NLRP3 pathway and its mechanism / 中国中药杂志

This paper aimed to study the effect of Erjing Pills on the improvement of neuroinflammation of rats with Alzheimer's di-sease(AD) induced by the combination of D-galactose and Aβ_(25-35) and its mechanism. SD rats were randomly divided into a sham group, a model control group, a positive drug group(donepezil, 1 mg·kg~(-1)), an Erjing Pills high-dose group(9.0 g·kg~(-1)), and an Erjing Pills low-dose group(4.5 g·kg~(-1)), with 14 rats each group. To establish the rat model of AD, Erjing Pills were intragastrically administrated to rats for 5 weeks after 2 weeks of D-galactose injection. D-galactose was intraperitoneally injected into rats for 3 weeks, and then Aβ_(25-35) was injected into the bilateral hippocampus. The new object recognition test was used to evaluate the learning and memory ability of rats after 4 weeks of intragastric administration. Tissues were acquired 24 h after the last administration. The immunofluorescence method was used to detect the activation of microglia in the brain tissue of rats. The positive expressions of Aβ_(1-42) and phosphory protein Tau~(404)(p-Tau~(404)) in the CA1 area of the hippocampus were detected by immunohistochemistry. The levels of inflammatory factors interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in the brain tissue were determined by enzyme-linked immunosorbent assay(ELISA). Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptors 3(NLRP3) pathway-associated proteins in the brain tissue were determined by Western blot. The results showed that as compared with the sham group, the new object recognition index of rats in the model control group decreased significantly, the deposition of Aβ_(1-42) and p-Tau~(404) positive protein in the hippocampus increased significantly, and the levels of microglia activation increased significantly in the dentate gyrus. The levels of IL-1β, TNF-α, and IL-6 in the hippocampus of the model control group increased significantly, and the expression levels of TLR4, p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus increased significantly. Compared with the model control group, the Erjing Pill groups enhanced the new object recognition index of rats, decreased the deposition of Aβ_(1-42) and the expression of p-Tau~(404) positive protein in the hippocampus, inhibited the activation of microglia in the dentate gyrus, reduced the levels of inflammatory factors IL-1β, TNF-α, and IL-6 in the hippocampus, and down-regulated the expression levels of TLR4, p-NF-κB P65/NF-κB P65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus. In conclusion, Erjing Pills can improve the learning and memory ability of the rat model of AD presumably by improving the activation of microglia, reducing the expression levels of neuroinflammatory factors IL-1β, TNF-α, and IL-6, inhibiting the TLR4/NF-κB/NLRP3 neuroinflammation pathway, and decreasing hippocampal deposition of Aβ and expression of p-Tau, thereby restoring the hippocampal morphological structure.

Investigation of the Tumor Suppression Effect and Immune Function of Shenqi Yiliu Decoction Combined with Cisplatin on H22 Liver Cancer Tumor-Bearing Mice Based on HMGB1/TLR4/NF-κB Pathway / 世界科学技术-中医药现代化

Objective Based on HMGB1/TLR4/NF-κB pathway,to investigate the effects of Shenqi Yiliu decoction combined with cisplatin on H22 liver cancer tumor mice and the effects of related immune indicators.Methods 50 SPF grade male KM mice,10 mice were taken as blank group by random number table method,and the other 40 mice were replicated as H22 hepatocellular carcinoma tumor-bearing mice model.After successful replication of the model,the model mice were randomly divided into model group,cisplatin group(2.5×10-3 g·kg-1),Shenqi Yiliu decoction TCM group(27.03 g·kg-1),and Shenqi Yiliu decoction TCM(27.03 g·kg-1)combined with cisplatin(2.5×10-3 g·kg-1),10 mice in each group were treated for 13 d.Determine tumor suppression rate,spleen index and thymus index;HE observes changes in oncology pathology;streaming cells detect the level of CD4+T,CD8+T cells in the spleen tissue;PT-PCR and WB method detect genes and protein expression related to HMGB1/TLR4/NF-κB signaling pathways in tumor tissues.Results ①Compared with the blank group,the mean body mass and mouse spleen index,thymus index,CD4+ T cell level and CD4+T/CD8+T value were significantly lower and CD8+T cell level was higher in the model group(P<0.05);②Compared with the model group,the mean tumor mass decreased(P<0.05),tumor volume decreased(P<0.05),and body mass increased(P<0.05)in each treatment group,and the spleen index,thymus index,CD4+T cell level and CD4+T/CD8+T ratio increased and CD8+T cell level decreased in both the Chinese medicine group and the combination group,and the treatment effect was significant in the Chinese medicine group(P<0.05),and HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression in tumor tissues of mice were reduced,and the effect was significant in the combined group(P<0.05).③Compared with the cisplatin group,HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression were reduced in the tumor tissues of mice in the combination group(P<0.05).④HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression in tumor tissues of mice in the combined group were reduced compared with those in the Chinese medicine group(P<0.05).Conclusion Shenqi Yiliu decoction combined with cisplatin can effectively inhibit tumor growth and improve related immune indexes in H22 hepatocellular carcinoma tumor-bearing mice,and the mechanism may be related to the inhibition of HMGB1/TLR4/NF-κB signaling pathway activation.

Modulating effects of Astragalus polysaccharide on immune disorders via gut microbiota and the TLR4/NF-κB pathway in rats with syndrome of dampness stagnancy due to spleen deficiency / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The syndrome of dampness stagnancy due to spleen deficiency (DSSD) is relatively common globally. Although the pathogenesis of DSSD remains unclear, evidence has suggested that the gut microbiota might play a significant role. Radix Astragali, used as both medicine and food, exerts the effects of tonifying spleen and qi. Astragalus polysaccharide (APS) comprises a macromolecule substance extracted from the dried root of Radix Astragali, which has many pharmacological functions. However, whether APS mitigates the immune disorders underlying the DSSD syndrome via regulating gut microbiota and the relevant mechanism remains unknown. Here, we used DSSD rats induced by high-fat and low-protein (HFLP) diet plus exhaustive swimming, and found that APS of moderate molecular weight increased the body weight gain and immune organ indexes, decreased the levels of interleukin-1β (IL-1β), IL-6, and endotoxin, and suppressed the Toll-like receptor 4/nuclear factor-‍κB (TLR4/NF-‍κB) pathway. Moreover, a total of 27 critical genera were significantly enriched according to the linear discriminant analysis effect size (LEfSe). APS increased the diversity of the gut microbiota and changed its composition, such as reducing the relative abundance of Pseudoflavonifractor and Paraprevotella, and increasing that of Parasutterella, Parabacteroides, Clostridium XIVb, Oscillibacter, Butyricicoccus, and Dorea. APS also elevated the contents of short-chain fatty acids (SCFAs). Furthermore, the correlation analysis indicated that 12 critical bacteria were related to the body weight gain and immune organ indexes. In general, our study demonstrated that APS ameliorated the immune disorders in DSSD rats via modulating their gut microbiota, especially for some bacteria involving immune and inflammatory response and SCFA production, as well as the TLR4/NF-κB pathway. This study provides an insight into the function of APS as a unique potential prebiotic through exerting systemic activities in treating DSSD.

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.

Reduction of inflammation response and transition of M1 toward M2 phenotypes of macrophages in response to SiO2 challenge by inhibition of TLR4 / 环境与职业医学

Background The mechanisms of silicon dioxide (SiO2)-induced inflammation and cell injury in pulmonary macrophages are not fully characterized. Objective To investigate the potential roles of inhibition of toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling in inflammation and macrophage polarization in mouse Raw264.7 cells in response to SiO2 stimulation. Methods Sixteen 6- to 8-week-old C57BL/6 mice, half male and half female, were intratracheally instilled with 50 µL of SiO2 (50 mg·mL−1 in saline) or normal saline via oropharyngeal route, and the lungs of mice were harvested at 14 d and 28 d post the first challenge of SiO2. HE staining of mouse lung was used for histopathological analysis. The expressions of TLR4 signaling-related proteins were detected by Western blotting (WB) and immunofluorescent (IF) assay, including TLR4, myeloid differentiation factor 88 (Myd88), and TNF receptor associated factor 6 (TRAF6). Raw264.7 cells were stimulated with SiO2 (100 μg·cm2) for 12 h in absence or presence of TLR4 inhibitor M62812 for 13 h before the culture supernatants and cell lysates were harvested for analysis. The expressions of key components of TLR4 signaling cascade including TLR4, Myd88, and phosphorylated nuclear factor-kappa B P65 (P-NF-κB P65), P-1NF-kappa-B inhibitor α (P-1κbα), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), M1 phenotype markers inducible nitric oxide synthase (iNOS) and cluster of differentiation 86 (CD86), as well as M2 phenotype arginase-1 (Arg-1) were accessed by WB and IF. The expressions of inflammation factors IL-6 and TNF-α in supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Results After SiO2 intratracheal instillation for 14 d, the HE staining results showed obvious fibrotic nodules in the lung tissues of mice. The results of WB analysis revealed more abundant TLR4, Myd88, and TRAF6 in the silicosis mouse lung samples than in the controls. The results of IF assay showed an increased abundance of TLR4 and Myd88 proteins in the lung samples of silicosis mice at 14 d post the silica challenge, compared to the controls, indicating TLR4 signaling activation. As seen in the in vitro experiment, significant upregulations after the exposure to 100 μg·cm2 SiO2 were observed in TLR4 and P-1κbα at 6, 12, and 24 h (P<0.05); Myd88 at 12 and 24 h (P <0.05); and P-NF-κB P65 at 12 h (P<0.05). The inhibitor significantly suppressed the expressions of TLR4, Myd88, TRAF6, P-NF-κB P65, TNF-α, and IL-6 in Raw264.7 cells. In addition, the SiO2-induced M1 phenotype marker iNOS was significantly suppressed, but the M2 phenotype marker Arg-1 was increased in the Raw264.7 cells. Conclusion The inhibition of TLR4/NF-κB signaling could result in a reduction of the inflammation response and the transition of M1 toward M2 phenotypes of macrophages in response to SiO2 challenge.