Protective Effect of Zingiberis Rhizoma Recens on LPS-induced Acute Lung Injury in Mice Through TLR4/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the protective effect and mechanism of Zingiberis Rhizoma Recens alcohol extract on lipopolysaccharide （LPS）-induced acute lung injury in mice. MethodBalb/c mice were randomly divided into normal group， model group， dexamethasone group， and low- and high-dose Zingiberis Rhizoma Recens groups. Mice in the normal group were instilled with normal saline through the nose， and the other groups were instilled with normal saline containing LPS （50 μg）. After 30 minutes of modeling， the dexamethasone group was gavaged with 5 mg·kg-1 of dexamethasone acetate solution， the low- and high-dose Zingiberis Rhizoma Recens groups were gavaged with different doses of （7， 14 g·kg-1） of Zingiberis Rhizoma Recens alcohol extract， and the normal group and the model group were gavaged with the same volume of water. After 24 hours of modeling， the total number of white blood cells in bronchoalceolar lavage fluid （BALF） was detected by cell counter， and the levels of the inflammatory factors including tumor necrosis factor （TNF）-α， interleukin （IL）-1β， IL-6， and superoxide dismutase （SOD）， and myeloperoxidase （MPO） was detected by enzyme-linked immunosorbent assay （ELISA）. Haematoxylin-eosin （HE） staining method was used to observe the pathological changes of lung tissue in each group， and the Western blot was used to detect the protein expression of nuclear transcription factor （NF）-κB p65， phosphorylation （p）-NF-κB p65， and Toll-like receptor 4 （TLR4） in lung tissue. ResultCompared with the normal group， the white blood cell count in BALF and the levels of TNF-α， IL-1β， IL-6， and MPO in the model group was increased （P<0.01）， and the level of SOD was decreased （P<0.05）. Pathological damage of lung tissue was obvious， and the protein expression of NF-κB p65， p-NF-κB p65， and TLR4 in lung tissue was increased （P<0.01）. Compared with the model group， the white blood cell count in BALF and the levels of TNF-α， IL-1β， IL-6， and MPO in the treatment group was decreased （P<0.05，P<0.01）， and the level of SOD was increased （P<0.05，P<0.01）. Pathological damage of lung tissue was alleviated， and the protein expression of NF-κB p65， p-NF-κB p65， and TLR4 in lung tissue was decreased （P<0.01）. ConclusionZingiberis Rhizoma Recens alcohol extract may play a protective role in LPS-induced acute lung injury in mice by inhibiting the TLR4/NF-κB signaling pathway.

Toll-like receptor 2-mediated downstream cytokine levels as determinant of malaria pathogenesis

Background & objectives: Toll-like receptors (TLRs) are transmembrane proteins that recognize specific molecular patterns and activate downstream cytokine production usually for the eradication of invading pathogens. The objective of this study was to evaluate the genetic polymorphism of TLR2 Arg753Gln (rs 5743708) and soluble cytokines and TLR2 expression levels in malaria disease cases. Methods: The study included prospectively collected 2 ml blood samples from 153 individuals clinically suspected for malaria and confirmed by microscopy and RDT from Assam. Stratification of the study groups was done as healthy control (HC, n=150), uncomplicated malaria (UC-M, n=128) and severe malaria (SM, n=25). The PCR-restriction fragment length polymorphism (RFLP) method was applied for the analysis of TLR2 Arg753Gln polymorphism and following the ELISA for soluble serum TLR2 (sTLR2) and its associated downstream cytokines, viz. tumour necrosis factor (TNF)-? and interferon (IFN)-? levels. Results: Variation in TLR2 Arg753Gln gene showed no association with the susceptibility and the severity of malarial infection. Soluble TLR2 expression was significantly higher in uncomplicated malaria (UC-M) cases compared to healthy controls (P=0.045) and in terms of SM cases, the expression was also found to be higher in UC-M cases (P=0.078). The TNF-? expression was significantly higher in SM cases compared to both UC-M and control (P=0.003 and P=0.004). Similarly, significantly elevated expression of IFN-? was noted in SM cases compared to both UC-M (P=0.001) and healthy controls (P<0.001). Interpretation & conclusions: The present study suggests the association of deregulated TLR2 pathway that leads to the deleterious downstream immune response in the development of malarial pathogenicity.

Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-κB/NLRP3 Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Buyang Huanwutang in regulating macrophage polarization based on the Toll-like receptor 4 （TLR4） / nuclear factor-κB （NF-κB） / nucleotide-binding oligomerization domain-like receptor 3 （NLRP3） pathway. MethodRAW264.7 macrophages were intervened with lipopolysaccharide （LPS） of different concentrations （0， 1.25， 2.5， 5， 10， 20， 40， and 80 mg·L-1） for 24 hours. Cell Counting Kit-8 （CCK-8） assay was used to determine the cell viability of RAW264.7 macrophages. The optimal concentration was chosen to establish an in vitro inflammation model induced by LPS. Cells were divided into a blank group （20% blank serum）， a model group （20% blank serum + 10 mg·L-1 LPS）， a model control group （20% FBS + 10 mg·L-1 LPS）， low-， medium-， and high-dose （5%， 10%， and 20%） Buyang Huanwutang-containing serum groups， a high-dose （20%） Buyang Huanwutang combined with NLRP3 inhibitor MCC950 （50 μmol·L-1） group， a high-dose （20%） Buyang Huanwutang combined with reactive oxygen species （ROS） inhibitor NAC （10 μmol·L-1） group， and a high-dose （20%） Buyang Huanwutang combined with NF-κB inhibitor PDTC （10 μmol·L-1） group. Enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， and tumor necrosis factor-α （TNF-α） in RAW264.7 macrophages. Flow cytometry was employed to measure ROS levels in macrophages. Western blot was used to determine the protein expression of M1-type macrophage-related factors inducible nitric oxide synthase （iNOS） and TNF-α， M2-type macrophage-related factors arginase-1 （Arg-1） and interleukin-10 （IL-10）， as well as the proteins in the TLR4/NF-κB/NLRP3 pathway. ResultCCK-8 results indicated that under 10 mg·L-1 LPS stimulation， RAW264.7 macrophages exhibited the highest cell viability （P<0.01）. Compared with the blank group， the model group showed significantly increased levels of IL-1β， IL-18， and TNF-α （P<0.05，P<0.01）， increased ROS expression （P<0.05，P<0.01）， increased protein expression of M1-type macrophage factors iNOS and TNF-α （P<0.01）， decreased protein expression of M2-type macrophage factors Arg-1 and IL-10 （P<0.05，P<0.01）， and upregulated expression levels of TLR4， myeloid differentiation factor 88 （MyD88）， phosphorylated inhibitor of NF-κB （p-IκB）/NF-κB inhibitor （IκB）， phosphorylated NF-κB （p-NF-κB） p65/NF-κB p65， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， and pro-Caspase-1 （P<0.05， P<0.01）. Compared with the model group， all Buyang Huanwutang-treated groups and inhibitor groups significantly reduced levels of IL-1β， IL-18， and TNF-α （P<0.01）， suppressed the expression of inflammatory factors in RAW264.7 macrophages， decreased cellular ROS expression levels （P<0.01）， downregulated M1-type macrophages iNOS and TNF-α protein expression （P<0.01）， upregulated M2-type macrophages Arg-1 and IL-10 protein expression （P<0.01）， and lowered protein expression levels of TLR4， MyD88， p-IκB/IκB， p-NF-κB p65/NF-κB p65， NLRP3， ASC， and pro-Caspase-1 （P<0.05， P<0.01）. ConclusionBuyang Huanwutang can improve macrophage inflammation， potentially by reducing macrophage ROS levels， inhibiting RAW264.7 macrophage polarization， and downregulating the protein expression levels of the TLR4/NF-κB/NLRP3 pathway.

High mobility group box-1 inhibition reduces expression of matrix metalloproteinase-9 in astrocytes in rats after spinal cord injury / 中华创伤骨科杂志

Objective:To investigate the role and underlying mechanisms of inhibiting high mobility group box-1 (HMGB1) in the expression of matrix metalloproteinase-9 (MMP-9) in spinal cord astrocytes (AS) in rats after spinal cord injury (SCI).Methods:After an SCI model was established in Sprague-Dawley (SD) rats using a modified Allen's Weight-Dropping method and ethyl pyruvate (EP) or glycyrrhizin (GL) was used to inhibit the effect of HMGB1, the rats were divided into a sham group, an SCI group, an SCI+EP (50 mg/kg) group, and an SCI+GL (100 mg/kg) group. The expression levels of glial fibrillary acid protein (GFAP) and MMP-9 in spinal cord AS were observed. After the spinal cord AS in SD rats was cultured and incubated by the oxygen-glucose deprivation/reoxygenation (OGD/R) procedure, the expression of MMP-9 protein was detected at 6 h/R 6 h, 12 h, 24 h, and 48 h after OGD. The time point with the highest expression was chosen in the subsequent experiments as an OGD/R group. HMGB1 was inhibited by HMGB1 shRNA or EP to observe the effect of HMGB1 on the expression of MMP-9 protein in AS treated with OGD/R. Then, toll-like receptor 4 (TLR4) inhibitor, TIR-domain-containing adaptor inducing interferon- β (TRIF) inhibitor, and nuclear factor-kappa B (NF- κB) inhibitor were used to investigate the effects of TLR4/TRIF/NF- κB signaling pathway during the regulation of HMGB1 on MMP-9 in vitro. Results:Western blot showed that the expression of MMP-9 protein in the spinal cord was significantly increased in rats at 1 d after SCI, and the expression of MMP-9 protein in the SCI+EP group and the SCI+GL group was significantly lower than that in the SCI group ( P<0.001). Immunofluorescence showed that GFAP and MMP-9 proteins were co-localized in the spinal cord after SCI, and the expression of GFAP and MMP-9 proteins in the SCI+EP and SCI+GL groups was significantly lower than that in the SCI group ( P<0.05). Since the expression of MMP-9 protein in the spinal cord AS cultured in vitro was significantly higher in the OGD 6h/R 12h group than that in the normal group and the OGD 6h/R 6h, 24, and 48 h groups, the OGD 6h/R 12h was taken as the OGD/R group. The MMP-9 protein expression in AS in the OGD/R+HMGB1 shRNA group and the OGD/R+EP group was significantly lower than that in the OGD/R group ( P<0.001). In the cultured AS, moreover, inhibiting TLR4, TRIF, and NF- κB reduced MMP-9 protein expression after OGD 6 h/R 12 h when compared with that in the OGD/R group ( P<0.001). Conclusions:HMGB1 inhibition may result in a reduction in MMP-9 expression both in the spinal cord AS in SCI rats and in AS after OGD/R treatment in vitro. HMGB1 may regulate MMP-9 protein expression in AS after OGD/R treatment via the TLR4/TRIF/NF- κB signal pathway.

The effect of SIRT1 activator on inflammatory response in chronic Periodontal disease mice through TLR4/NF-κB signaling pathway / 中国医师杂志

Objective:To explore the effect of silent information regulator 1 (SIRT1) activator SRT1720 on inflammatory response in chronic periodontal disease mice and whether its mechanism is related to the toll like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling pathway.Methods:Forty 8-week-old male C57BL/6 mice were selected and divided into a blank control group ( n=8) and an experimental group ( n=32). The experimental group mice were ligated with periodontal pockets and fed with high sugar drinking water. The experimental group was randomly divided into a model group ( n=8) and an SRT1720 group ( n=24). The blank control group and the model group were given physiological saline orally every day. The SRT1720 group was further divided into a low dose group [20 mg/(kg·d), n=8], a medium dose group [50 mg/(kg·d), n=8], and a high dose group [100 mg/(kg·d), n=8] based on the different doses of SRT1720. Four weeks later, the expression levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) in gingival crevicular fluid of mice in each group were detected by enzyme linked immunosorbent assay (ELISA); The real-time quantitative polymerase chain reaction (RT-qPCR) method was used to detect the mRNA expression levels of IL-6, IL-1β, MCP-1, SIRT1, TLR4, NF-kB p65 in the gingival tissue of mice in each group; Western blot was used to determine the expression levels of SIRT1, TLR4, and NF-κB p65 proteins in mouse gingival tissue. Results:Compared with the blank control group, the expression levels of IL-6, IL-1β, and MCP-1 inflammatory factors in the gingival crevicular fluid of experimental group mice increased, while the expression levels of IL-6, IL-1β, MCP-1, TLR4, NF-κB p65 mRNA in gingival tissue increased. The expression levels of TLR4, NF-κB p65 protein in gingival tissue increased, while the expression levels of SIRT1 mRNA and protein in gingival tissue decreased, with statistical significance (all P<0.05). Compared with the model group, the expression levels of IL-6, IL-1β, and MCP-1 inflammatory factors in the gingival crevicular fluid, IL-6, IL-1β, MCP-1, TLR4, NF-κB p65 mRNA expression levels in gingival tissue, and TLR4, NF-κB p65 protein expression levels in the gingival tissue of SRT1720 group mice showed a dose-dependent decrease. The expression levels of SIRT1 mRNA and protein in gingival tissue showed a dose-dependent increase, and the differences were statistically significant (all P<0.05). Conclusions:SIRT1 activator SRT1720 can improve the inflammatory response of chronic periodontal disease mice, which may be related to the inhibition of TLR4/NF-kB signaling pathway.

The impact and interaction of TLR2 and IRF-5 gene polymorphisms on the susceptibility to neonatal sepsis / 中国医师杂志

Objective:To investigate the impact and interaction of Toll like receptor 2 (TLR2) and interferon regulatory factor 5 (IRF-5) gene polymorphisms on the susceptibility to neonatal sepsis.Methods:A total of 78 cases of neonatal septicemia patients admitted to Baoding Children′s Hospital from July 2018 to August 2021 were prospectively selected as the study group, and 78 cases of healthy newborns in the same period were selected as the control group. The TLR2 and IRF-5 gene polymorphisms and the levels of inflammatory markers [C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in different genotypes of infants were compared between the two groups. We evaluated the relationship between TLR2 and IRF-5 genotypes, inflammatory markers, and susceptibility to neonatal sepsis, and analyzed the interaction between their gene polymorphisms and susceptibility to neonatal sepsis.Results:There were significant differences in the distribution of TLR2 (rs3804099) and IRF-5 (rs2004640) loci genotype and Allele frequency between the two groups (all P<0.05); The serum CRP, TNF-α, and IL-6 levels in children with TLR2 (rs3804099) genotype TT genotype [(111.12±30.87)mg/L, (77.50±20.02)pg/ml, (40.27±11.31)pg/ml] were higher than those in children with CC/CT genotype [(72.46±24.51)mg/L, (54.18±17.65)pg/ml, (28.34±9.05)pg/ml], and the differences were statistically significant (all P<0.05). The serum CRP, TNF-α, and IL-6 levels [(113.90±28.94)mg/L, TNF-α (79.84±19.82)pg/ml, IL-6 (41.05±11.49)pg/ml] in children with the IRF-5 (rs2004640) TT genotype were higher than those in children with the GG/GT genotype [(70.88±22.16)mg/L, (52.27±16.73)pg/ml, (27.96±9.75)pg/ml], and the differences were statistically significant (all P<0.05). The TT genotypes at TLR2 (rs3804099) and IRF-5 (rs2004640) loci were positively correlated with serum CRP, TNF-α, and IL-6 levels (all P<0.05); The TT genotypes at TLR2 (rs3804099) and IRF-5 (rs2004640) loci were independent risk factors for susceptibility to neonatal sepsis (all P<0.05); The TT genotype at the TLR2 (rs3804099) locus and the TT genotype at the IRF-5 (rs2004640) locus exhibited a positive interaction in susceptibility to neonatal sepsis ( OR=7.467, γ=1.728). Conclusions:TLR2 (rs3804099) TT genotype and IRF-5 (rs2004640) TT genotype significantly increase the susceptibility to neonatal sepsis, and there is a positive interaction between the two.

Expression of TLR3 in human adenocarcinoma of the lung cells induced by respiratory syncytial virus and its significance in the diagnosis of pneumonia in children / 中国医师杂志

Objective:To study the expression of Toll like receptor 3 (TLR3) in human adenocarcinoma of the lung cells induced by respiratory syncytial virus (RSV) and its significance in the diagnosis of pneumonia in children.Methods:A549 cells were divided into RSV infection group [added 1 μg/ml Lipopolysaccharide (TLR3 agonist) transfected RSV virus after 150 μl intervention], Lipopolysaccharide stimulation group (added 1 μg/ml Lipopolysaccharide 150 μl intervention) and normal control group (normal culture). The mRNA expressions of tumor necrosis factor-α, interleukin 8, TLR3 protein and TLR3 in A549 Cells of different groups were compared. We prospectively selected 80 children with RSV infectious pneumonia admitted to Baoding Second Central Hospital from August 2019 to October 2021 as the RSV pneumonia group, and sixty children with common pneumonia were taken as the common pneumonia group, and 60 healthy children in our hospital were taken as the control group. The mRNA expression of serum TLR3 in different groups was compared, and the diagnostic efficacy of serum TLR3 in RSV pneumonia was evaluated by receiver operating characteristic.Results:There was a statistically significant difference in the expression of TLR3 protein among different groups of A549 cells ( P<0.001). The expression differences of TLR3 mRNA in different groups of A549 cells at different time points were statistically significant(all P<0.001). There was significant difference in the expression of tumor necrosis factor-α and interleukin 8 of A549 cells at different time points in different groups (all P<0.05). There was a statistically significant difference in the expression of serum TLR3 mRNA among the three groups of subjects ( F=155.237, P<0.001). The critical value for TLR3 gene diagnosis was 66.87, with corresponding sensitivity of 73.75%, specificity of 70.83%, and the area under curve (AUC) of 0.803(95% CI: 0.753-0.855). Conclusions:Respiratory syncytial virus induces human lung cancer cells and promotes disease progression through TLR3 expression; Serum TLR3 can be used for the diagnosis of RSV pneumonia.

The role of Toll-like receptor 4-mediated immune inflammation in inorganic arsenic-induced liver fibrosis in rats / 中华地方病学杂志

Objective:To observe the expression levels of Toll-like receptor 4 (TLR4) signaling pathway-related proteins and their phosphorylation in the liver tissues of rats with inorganic arsenic poisoning, and to explore the role of TLR4-mediated inflammatory signaling pathway in arsenic-induced liver fibrosis injury.Methods:Eighteen healthy weanling SD rats were divided into 3 groups according to their body weight (80 - 100 g) using a random number table (6 rats in each group, half males and half females). The control group was given 10 ml/kg of normal saline by gavage. The sodium arsenite (NaAsO 2) exposure group was given 10 mg/kg of NaAsO 2 by gavage. The TAK-242 intervention group was given 10 mg/kg of NaAsO 2 by gavage, and 0.5 mg/kg of TAK-242 was also administered intraperitoneally to inhibit TLR4 after 12 weeks. All rats were administered 6 days a week for 36 weeks. At the end of the treatment, the liver tissues and serum of the rats in each group were collected. HE and Masson staining were used to observe the pathological and fibrotic changes of the liver tissues. Automatic biochemical analyzer was used to detect serum liver function indexes of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Western blot was used to detect the expression changes of rat liver fibrosis protein α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), Vimentin and TLR4 signaling pathway-related proteins TLR4, nuclear factor κB (NF-κB)-p65 subunit (p65), NF-κB-p50 subunit (p50) and their phosphorylation p-p65 and p-p50 expression levels. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of inflammatory related factors interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and IL-10. Results:HE and Masson staining results showed that compared with the control group, the NaAsO 2 exposure group showed significant inflammatory cell infiltration, hepatocyte necrosis and collagen fibrous deposition, while the TAK-242 intervention group showed improvement of the inflammatory cell infiltration and reduction of collagen fibrous deposition compared with the NaAsO 2 exposure group. The results of serum liver function indexes showed that ALT, AST and ALP in NaAsO 2 exposure group were increased compared with the control group, but the TAK-242 intervention group was significantly decreased compared with the NaAsO 2 exposure group ( P < 0.05). Western bolt results showed that in NaAsO 2 exposure group, the expression levels of fibrosis protein α-SMA, TGF-β1 and Vimentin (1.04 ± 0.19, 0.92 ± 0.14, 1.20 ± 0.21) and TLR4 signaling pathway-related proteins and their phosphorylation TLR4, p50, p-p50 and p-p65 (1.16 ± 0.21, 0.95 ± 0.16, 1.24 ± 0.23, 1.56 ± 0.25) were higher than the control group (0.44 ± 0.08, 0.42 ± 0.08, 0.72 ± 0.07, 0.69 ± 0.15, 0.71 ± 0.11, 0.46 ± 0.07, 0.54 ± 0.11, P < 0.05), and the TAK-242 intervention group (0.60 ± 0.13, 0.59 ± 0.16, 0.49 ± 0.11, 0.47 ± 0.08, 0.86 ± 0.09, 0.79 ± 0.14, 1.02 ± 0.17) were lower than the NaAsO 2 exposure group ( P < 0.05). There was no significant difference in the expression level of TLR4 signal pathway-related protein p65 among the three groups ( F = 14.29, P = 0.053). ELISA results showed that the secretion levels of IL-6 and TNF-α [(98.89 ± 4.58), (83.25 ± 4.57) ng/g] in rats liver tissues of the NaAsO 2 exposure group were higher than the control group [(27.30 ± 3.92), (27.77 ± 1.83) ng/g, P < 0.05], while the secretion level of IL-10 [(36.88 ± 3.86) ng/g] was lower than the control group [(77.96 ± 7.87) ng/g, P < 0.05]. In TAK-242 intervention group, IL-6 and TNF-α secretion levels [(44.32 ± 3.60), (36.51 ± 2.93) ng/g] were lower and IL-10 secretion level [(60.40 ± 4.94) ng/g] was higher compared with the NaAsO 2 exposure group ( P < 0.05). Conclusion:TLR4-mediated inflammatory signaling pathway-related proteins and their phosphorylation are highly expressed in the liver tissues of rats with inorganic arsenic poisoning, and inhibition of TLR4 signaling pathway could significantly reduce the degree of liver fibrosis injury caused by inorganic arsenic in rats.

Mechanism of Flemiphilippinin D Regulating Inflammatory Response in CIA Rats Through TLR2/MyD88/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Flemiphilippinin D on collagen-induced arthritis （CIA） in rats and explore its mechanism. MethodForty rats were randomly divided into normal group， CIA group， methotrexate （MTX） group （1.35 mg·kg-1）， low-dose Flemiphilippinin D group （1.5 mg·kg-1）， and high-dose Flemiphilippinin D group （3.0 mg·kg-1）， with eight rats in each group. Except for the normal group， the CIA model was induced by type Ⅱ collagen. Each group was given corresponding liquid medicine or normal saline， once a week in the MTX group， and once a day in the Flemiphilippinin D groups for a total of 28 days. The arthritis score and joint swelling degree of rats were experimentally recorded. Pathological changes in the ankle joint of rats were observed by hematoxylin-eosin （HE） staining. Serum levels of inflammatory cytokines interleukin （IL）-1β， IL-6， IL-8， and tumor necrosis factor （TNF）-α were detected by enzyme-linked immunoabsorbent assay （ELISA）， and the mRNA expression of Toll-like receptor 2 （TLR2）， myeloid differentiation factor 88 （MyD88）， and nuclear transcription factor-κB （NF-κB） p65 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the protein expressions of TLR2， MyD88， and NF-κB p65 were detected by Western blot. ResultCompared with the normal group， the ankle joint of the CIA group was significantly swollen， and the clinical score of arthritis and the degree of joint swelling were significantly increased （P<0.01）. The ankle joint tissue structure was significantly damaged， and the levels of inflammatory factors IL-1β， IL-6， IL-8， and TNF-α in serum were significantly increased （P<0.01）. The mRNA levels and protein levels of TLR2， MyD88， and NF-κB p65 were significantly increased（P<0.01）. Compared with the CIA group， arthritis clinical score and joint swelling of rats in each administration group were significantly reduced （P<0.05， P<0.01）， and the pathological changes in the ankle joint were significantly improved. The contents of serum IL-1β， IL-6， IL-8， and TNF-α were significantly decreased （P<0.05， P<0.01）. The mRNA levels and protein levels of TLR2， MyD88， and NF-κB p65 in the ankle joint were significantly decreased （P<0.05， P<0.01）. ConclusionTo a certain extent， Flemiphilippinin D can reduce the expression of inflammatory factors in rheumatoid arthritis rats and play a good therapeutic effect. It works perhaps by inhibiting the activation of the TLR2/MyD88/NF-κB signaling pathway and thus shows an anti-inflammatory effect.

Effect of Toll-like receptor 4 on liver regeneration during acetaminophen-induced liver injury in mice / 临床肝胆病杂志

Objective To investigate whether Toll-like receptor 4 (TLR4) inhibition affects liver regeneration during acetaminophen (APAP)-induced liver injury in mice, as well as the mechanism of TLR4 involved in liver regeneration. Methods A total of 78 male CD-1 mice were divided into nine groups using a random number table, i.e., three control groups (normal control group, solvent control group, inhibitor control group) with 6 mice in each group and six experimental groups (APAP 24-hour group, TAK-242+APAP 24-hour group, APAP 48-hour group, TAK-242+APAP 48-hour group, APAP 72-hour group, TAK-242+APAP 72-hour group) with 10 mice in each group. The mice in the experimental groups were given a single dose of intraperitoneally injected APAP (300 mg/kg), and TAK-242 was intraperitoneally injected at a dose of 3 mg/kg at 3 hours before APAP administration. Serum and liver tissue samples were collected at different time points. The biochemical method was used to measure the serum level of alanine aminotransferase (ALT); HE staining was used to observe liver pathological changes; RT-PCR, Western blot, and immunohistochemistry were used to measure the expression levels of Cyclin D1, PCNA, Ki-67, STAT3, and p-STAT3. The t -test was used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t -test was used for further comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups, and the Kruskal-Wallis H test was used for comparison between multiple groups and further comparison between two groups. Results Compared with the normal control group, the APAP 24-hour and 48-hour groups had a significantly higher serum level of ALT (both P < 0.05), and the TAK-242+APAP 24-hour and 48-hour groups had a significantly higher serum level of ALT than the APAP group at the same time point (both P < 0.05). HE staining showed typical central lobular necrosis in the liver of APAP-treated mice, and the TAK-242+APAP 24-hour and 48-hour groups had a significantly larger necrotic area than the APAP group at the same time point (both P < 0.05). RT-PCR, Western blot, and immunohistochemistry showed that the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had significantly lower mRNA and protein expression levels of Cyclin D1 than the APAP group at the same time point (all P < 0.05); the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had a significantly lower mRNA expression level of PCNA than the APAP group at the same time point (all P < 0.05), and the TAK-242+APAP 24-hour and 48-hour groups had a significantly lower protein expression level of PCNA than the APAP group at the same time point (all P < 0.05); the TAK-242+APAP 24-hour and 72-hour groups had a significantly lower mRNA expression level of Ki-67 than the APAP group at the same time point (all P < 0.05), and the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had a significantly lower protein expression level of Ki-67 than the APAP group at the same time point (all P < 0.05). In addition, the TAK-242+APAP 24-hour and 48-hour groups had a significantly lower phosphorylation level of STAT3 than the APAP group at the same time point (both P < 0.05). Conclusion TLR4 may promote liver regeneration by increasing the phosphorylation level of STAT3 during APAP-induced liver injury in mice.

Mechanism of salidroside preventing myocardial fibrosis based on TLR4-mediated pyroptosis pathway / 中国药房

OBJECTIVE To investigate the effects of salidroside （Sal） on myocardial fibrosis and pyroptosis and its potential mechanism. METHODS The mice were randomly divided into control group， model group and Sal low-dose， medium-dose and high-dose groups， with 10 mice in each group. Except for the control group， the mice in other groups were injected subcutaneously with isoproterenol 5 mg/（kg·d）to prepare the myocardial fibrosis model. Since modeling， mice in the Sal low-dose， medium-dose and high-dose groups were given 10， 30 and 50 mg/kg of Sal by intragastric administration every day； control group and model group were given 10 mL/kg of normal saline by intragastric administration every day， for 14 consecutive days. After the last medication， the mice were sacrificed； hematoxylin-eosin staining was used to observe pathological change of myocardial tissue and calculate the diameter of myocardial cell； Masson and Sirius Red staining were used to observe the degree of myocardial fibrosis in mice and calculate the collagen volume fraction （CVF）； quantitative real-time PCR was performed to detect the mRNA expressions of collagen type Ⅰ （Col Ⅰ）， α-smooth muscle actin （α-SMA）， Toll-like receptor 4 （TLR4）， NOD-like receptor pyrin domain containing 3 （NLRP3）， caspase-1 andgasdermin D （GSDMD） in myocardial tissues. The total protein expressions of Col Ⅰ， α-SMA， TLR4， NLRP3，caspase-1 and GSDMD in myocardial tissues and protein-positive cell score were measured by Western blot assay and immunohistochemistry. RESULTS Compared with control group， the myocardial cells in the model group were enlarged， the arrangement of myocardial fibers was disordered， the matrix metabolism was significantly increased， the CVF in myocardial tissue was significantly increased， and the mRNA and protein expression levels of Col Ⅰ， α-SMA， TLR4， NLRP3， caspase-1 and GSDMD were elevated and protein-positive cell score was increased significantly （P＜0.01）. Compared with model group， the myocardial cell morphology was clearer， myocardial fibrosis was alleviated， and the levels of the above indicators in myocardial tissue of Sal medium-dose and high-dose groups had been reversed to varying degrees， especially in Sal high-dose group（P＜0.05 or P＜0.01）. In addition， the Sal low-dose group also reversed some fibrosis and pyroptosis-related indicators to some extent. CONCLUSIONS Sal can significantly prevent the occurrence and development of myocardial fibrosis， and the mechanism of action may be related to the inhibition of TLR4-mediated pyroptosis pathway in myocardial tissue.

Lycium barbarum Polysaccharide Regulates Activation of RAW264.7 Macrophages Through MGL/TLR Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Lycium barbarum polysaccharides （LBP） in promoting the activation of RAW264.7 macrophages. MethodRAW264.7 macrophages were stimulated with LBP at different concentrations （50， 100， 200 mg·L-1）， and those stimulated with lipopolysaccharide （LPS） at 100 μg·L-1 and galactose （Gal） at 100 mg·L-1 as positive controls. After 24 h of LBP stimulation， the cell counting kit-8 （CCK-8） was used to detect the survival rate of RAW264.7 macrophages treated with LBP （0， 50， 100， 200， 400， 800 mg·L-1）. The levels of interleukin-6 （IL-6） and interleukin-12 （IL-12） in cell culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. The protein and mRNA expression of p38 mitogen-activated protein kinase （MAPK）， extracellular signal-regulated kinase （ERK）， c-Jun N-terminal kinase （JNK）， and nuclear factor κB （NF-κB） in Toll-like receptor 4 （TLR4）/Toll-like receptor 2 （TLR2）/macrophage galactose-type lectin （MGL） pathway of RAW264.7 macrophages was detected by Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultCCK-8 results showed that compared with the results in the blank group， the survival rate of RAW264.7 macrophages decreased in the 400， 800 mg·L-1 LBP groups （P<0.05）. ELISA results showed that compared with the blank group， 50 mg·L-1 LBP could promote the secretion of IL-12 in RAW264.7 macrophages （P<0.01）. Compared with the blank group， 100 mg·L-1 LBP and 200 mg·L-1 LBP could promote the secretion of IL-6 in RAW264.7 macrophages （P<0.05， P<0.01）. Western blot results showed that compared with the blank group， the LBP groups （50， 100， 200 mg·L-1） enhanced protein expression levels of MAPK key molecules （p-p38 MAPK， p-ERK， p-NF-κB， and p-JNK） in TLR4， TLR2， and MGL pathways （P<0.05， P<0.01）. Compared with the model group， the 200 mg·L-1 LBP group could promote the expression level of p-NF-κB protein in RAW264.7 macrophages （P<0.01）. Real-time PCR results showed that compared with the blank group， the LBP groups （50， 100， and 200 mg·L-1） enhanced the mRNA expression levels of MAPK key molecules （p38 MAPK， ERK， NF-κB， and JNK） in TLR4 and TLR2 pathways （P<0.05， P<0.01）. Compared with the model group， the 50 and 200 mg·L-1 LBP groups could promote the mRNA expression levels of JNK and ERK2 in RAW264.7 macrophages （P<0.05， P<0.01）. ConclusionLBP can regulate the activation of RAW264.7 macrophages and participate in the immune response through the TLR2/TLR4/MGL pathway.

Anti-inflammatory Effect and Mechanism of Duhuo Jishengtang on Rheumatoid Arthritis Rats Based on TLR2/p38 MAPK/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the anti-inflammatory effect of Duhuo Jishengtang （DHJST） on collagen-induced arthritis （CIA） model rats and its effect on the Toll-like receptor 2 （TLR2）/p38 mitogen-activated protein kinase （MAPK）/nuclear factor-κB （NF-κB） signaling pathway. MethodForty-eight male SD rats were randomly divided into the following six groups （n=8）： normal group， model group， methotrexate （MTX） group， low-dose DHJST （DHJST-L） group， medium-dose DHJST （DHJST-M） group， and high-dose DHJST （DHJST-H） group. The CIA model was established by injecting bovine type Ⅱ collagen into the rat tail root with the collagen antibody induction method. After model induction， rats were treated with drugs by gavage. The rats in the MTX group received MTX at 2.0 mg·kg-1， three times a week， and those in the DHJST groups received DHJST at 3.8， 7.6， 15.2 g·kg-1·d-1 for 28 days. The rats in the normal group and the model group were given the same dose of normal saline. The weight of the rats was recorded， and the paw swelling degree was observed. The arthritis index and immune organ index were measured， and the changes in the microcirculation indexes of the rats were detected with a microcirculation detector. Hematoxylin-eosin （HE） staining was used to detect the pathological morphologic changes in rat synovial tissues and the apoptosis rate of synovial cells was detected by flow cytometry to determine the therapeutic effect of DHJST on rheumatoid arthritis. Enzyme-linked immunosorbent assay （ELISA） was used to detect the changes in serum levels of tumor necrosis factor-α （TNF-α）， interleukin （IL）-1β， IL-17A， and interferon-γ （IFN-γ）. The protein expression of TLR2， NF-κB p65， phosphorylated NF-κB p65 （p-NF-κB p65）， p38 MAPK， and p-p38 MAPK was detected by Western blot. ResultCompared with the normal group， the model group showed reduced body weight （P<0.01）， increased paw swelling degree， arthritis index， and immune organ index （P<0.01）， increased comprehensive microvascular score and vascular resistance （P<0.01）， significant hyperplasia of synovial tissues and massive infiltration of inflammatory cells as revealed by pathological sections， and up-regulated expression levels of TNF-α， IL-1β， IL-17A， and IFN-γ in serum， and TLR2， p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK in synovial tissues （P<0.01）. Compared with the model group， the DHJST groups showed increased body weight of rats （P<0.01）， decreased paw swelling degree， arthritis index， and immune organ index （P<0.05， P<0.01）， reduced comprehensive microvascular score and vascular resistance （P<0.05， P<0.01）， improved synovial histopathological injury， increased apoptosis rate of synovial cells （P<0.01）， and down-regulated levels of TNF-α， IL-1β， IL-17A， and IFN-γ in serum （P<0.05， P<0.01） and TLR2， p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK in synovial tissues （P<0.05， P<0.01）. ConclusionDHJST may alleviate the inflammatory reaction in CIA rats by regulating the TLR2/p38 MAPK/NF-κB signaling pathway， thus exerting its anti-rheumatoid arthritis effect.

Value of bedside high-flow continuous blood purificationcombined with Xuebijing in the treatment of severe sepsis / 中国医师进修杂志

Objective:To investigate the effect of bedside high-flow continuous blood purification (CBP) combined with Xuebijing in the treatment of severe sepsis (SS) and the influence on the patient′s coagulation-fibrinolysis index, immunity index and expression of peripheral blood Toll-like receptor 4 (TLR4).Methods:Ninety-three patients with SS who were admitted and treated in the Lianyungang First People′s Hospitalfrom January 2017 to October 2019 were selected. They were divided into the combined group (51 cases, treatment with bedside high-flow CBP and Xuebijing injection based on bundle therapy) and the control group (42 cases, treatment with Xuebijing injection based on bundle therapy). The changes in coagulation and fibrinolysis index, immunity index, biochemical index such as TLR4 before treatment and after 1 week of treatment were compared between the two groups. The incidences of complications in both groups were statistically analyzed, and the discharge time from ICU, mechanical ventilation time and 28-day mortality were recorded.Results:After 1 week of treatment, the levels of prothrombin time (PT) and activated partial thromboplastin time (APTT) in the two groups were shortened, D-dimer (D-D) and fibrinogen (FIB) were decreased ( P<0.05); and the levels of PT and APTT in the combined group were shorter than those in the control group, the levels of DD and FIB were lower than those in the control group, there were statistical differences ( P<0.05). After 1 week of treatment, the levels of CD 4+ and CD 4+/CD 8+ ratio in both groups were increased ( P<0.05), and the levels of CD 4+ and CD 4+/CD 8+ ratio in the combined group were higher than those in the control group ( P<0.05). After 1 week of treatment, the levels of TLR4, C-reactive protein (CRP), procalcitonin (PCT), white blood cell count (WBC), blood lactate (Lac), blood urea nitrogen (BUN) and serum creatinine (Scr) in both groups were decreased ( P<0.05), meanwhile, the above indexes in the combined group were lower than those in the control group ( P<0.05). The incidence of multiple organ failure and the 28-day mortality rate in the combined group were lower than those in the control group: 3.92%(2/51) vs. 19.05%(8/42), 13.73%(7/51) vs. 30.95%(13/42), there were statistical differences ( P<0.05). The discharge time from ICU and mechanical ventilation time in the combined group were shorter than those in the control group: (12.35 ± 2.14) d vs. (14.17 ± 3.36) d, (7.12 ± 2.23) d vs. (8.51 ± 2.39) d, there were statistical differences ( P<0.05). Conclusions:Bedside high-flow CBP combined with Xuebijing injection in the treatment of SS can improve the patient′s condition, regulate the balance of coagulation and fibrinolysis, avoide the activation of coagulation, inhibite inflammatory response, reduce the expression of TLR4 in peripheral blood, improve immune function, protecte kidney function and promotethe patient′s recovery.

Traditional Chinese Medicine Intervenes in Atherosclerosis by Regulating TLR/NLRP3 Pathway via TRPV1/TRPA1 / 中国实验方剂学杂志

Atherosclerosis is a chronic inflammatory disease caused by lipid accumulation and vascular endothelial dysfunction. The Toll-like receptor （TLR）/nuclear transcription factor-κB （NF-κB） pathway and the NOD-like receptor protein 3 （NLRP3） inflammasome pathway play a proinflammatory role， while the transient receptor potential vanilloid subtype 1 （TRPV1） and transient receptor potential ankyrin 1 （TRPA1） play a protective role in the occurrence of atherosclerosis. We reviewed the relevant studies published in the last 10 years. The results showed that activation of TRPV1/TRPA1 could activate endothelial-type nitric oxide synthase （eNOS） and inhibit the generation of reactive oxygen species （ROS） and cholesterol crystal （CC） to modulate the TLR/NF-κB and NLRP3 inflammasome pathways， thereby inhibiting TLR/NLRP3-mediated inflammatory response. A variety of compound prescriptions and active components of Chinese medicinal materials can activate TRPV1/TRPA1 or its downstream pathway to regulate the TLR/NLRP3 pathway in atherosclerosis. This paper introduces the mechanisms of compound prescriptions and active components of Chinese medicinal materials in regulating the TLR/NLRP3 pathway via TRPV1/TRPA1 in atherosclerosis. This review provides new ideas for the research on the interactions between Chinese medicines in the treatment of atherosclerosis and provides a new strategy for the clinical treatment of atherosclerosis with traditional Chinese medicine.

Clinical diagnosis and prognostic value of serum TLR4 and VEGFA expression in diabetic retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the expression and clinical significance of Toll-like receptor 4(TLR4)and vascular endothelial growth factor A(VEGFA)in the serum of patients with diabetic retinopathy(DR).METHODS: A total of 183 patients with type 2 diabetes mellitus(T2DM)admitted to our hospital from January 2021 to January 2022 were collected as the study subjects. They were grouped into non diabetic retinopathy(NDR)group(n=54), proliferative diabetic retinopathy(PDR)group(n=68)and non proliferative diabetic retinopathy(NPDR)group(n=61). In the same period, 70 volunteers who underwent physical examination in our hospital were randomly stratified according to age and sex. After discharge, DR patients were followed up for 1a and grouped into a poor prognosis group(n=40)and a good prognosis group(n=89)based on whether they had visual impairment. Enzyme-linked immunosorbent assay(ELISA)was applied to detect the levels of TLR4 and VEGFA in serum; Logistic regression was applied to analyze the influencing factors of DR; receiver operating characteristic(ROC)curve was applied to analyze the clinical value of serum TLR4 and VEGFA levels in diagnosing DR and predicting prognosis.RESULTS: There were statistical significance in TLR4 and VEGFA levels among the control group, NDR group, PDR group, and NPDR group(F=935.753, 516.936, all P&#x003C;0.05), and further pairwise comparisons showed statistical significance(P&#x003C;0.05); the expression levels of TLR4 and VEGFA in the serum of patients with poor prognosis were higher than those of patients with good prognosis(P&#x003C;0.01); the results of Logistic regression analysis showed that TLR4, VEGFA, course of disease, and HbA1c were all risk factors for the occurrence of DR(P&#x003C;0.05); the ROC results showed that the AUC of serum TLR4, VEGFA levels, and their combination for predicting DR was 0.869, 0.862, and 0.931, respectively, the AUC of serum TLR4, VEGFA levels, and their combined prediction of visual disability in DR patients was 0.864, 0.863, and 0.938, respectively.CONCLUSION: The expression of TLR4 and VEGFA in serum of DR patients is up-regulated, and the combined detection of TLR4 and VEGFA can be used as a potential indicator to evaluate the occurrence and poor prognosis of DR.

Macrophages and kidney transplantation / 器官移植

Kidney transplantation is the optimal treatment for patients with end-stage renal disease, whereas long-term survival of renal allografts remains a challenging issue. Renal ischemia-reperfusion injury (IRI) and rejection of renal allografts are considered as important influencing factors of long-term survival of renal allografts, which are regulated by innate and adaptive immune cells. Macrophages are one type of innate immune cells that could assist initiating adaptive immunity and are divided into M1, M2 and regulatory macrophages. Previous studies have revealed that M1 macrophages may aggravate renal IRI and acute T cell-mediated rejection (TCMR). However, M2 macrophages may mitigate renal IRI and acute TCMR, whereas it is positively correlated with antibody-mediated rejection (AMR). Regulatory macrophages are a special subgroup of macrophages, which may induce immune tolerance in organ transplantation and have promising clinical application prospects and basic scientific research value. In this article, the relationship among macrophage typing, macrophages and renal IRI, rejection of renal allografts, regulatory macrophages and immune tolerance was reviewed, and the potential mechanism was analyzed, aiming to induce changes in macrophage subtypes or eliminate specific subtypes of macrophages, thereby improving clinical prognosis of the recipients and long-term survival of renal allografts.

Analysis of therapeutic mechanism of Liushen Wan against colitis-associated colorectal cancer based on network pharmacology and validation in mice / 南方医科大学学报

OBJECTIVE@#To explore the therapeutic mechanism of Liushen Wan (LSW) against colitis-associated colorectal cancer (CAC) by network pharmacology.@*METHODS@#TCMSP, BATMAN-TCM, CNKI, PubMed, Genecards, OMIM, and TTD databases were used to obtain the related targets of LSW and CAC. The common targets of LSW and CAC were obtained using Venny online website. The PPI network was constructed using Cytoscape 3.8.2 to screen the core targets of LSW in the treatment of CAC. GO and KEGG enrichment analysis were conducted using DAVID database. The therapeutic effect of LSW on CAC was evaluated in a C57BL/6J mouse model of AOM/DSS-induced CAC by observing the changes in body weight, disease activity index, colon length, and size and number of the tumor. HE staining and RT-qPCR were used to analyze the effect of LSW on inflammatory mediators. Immunohistochemistry and TUNEL staining were used to evaluate the effect of LSW on the proliferation and apoptosis of AOM/DSS-treated colon tumor cells. Immunohistochemistry and Western blotting were used to detect the effects of LSW on the expression of TLR4 proteins in CAC mice.@*RESULTS@#Network pharmacology analysis identified 69 common targets of LSW and CAC, and 33 hub targets were screened in the PPI network. KEGG pathway enrichment analysis suggested that the effect of LSW on CAC was mediated by the Toll-like receptor signaling pathway. In the mouse model of AOM/DSS-induced CAC, LSW significantly inhibited colitis-associated tumorigenesis, reduced tumor number and tumor load (P < 0.05), obviously improved histopathological changes in the colon, downregulated the mRNA levels of proinflammatory cytokines, and inhibited the proliferation (P < 0.01) and promoted apoptosis of colon tumor cells (P < 0.001). LSW also significantly decreased TLR4 protein expression in the colon tissue (P < 0.05).@*CONCLUSION@#LSW can inhibit CAC in mice possibly by regulating the expression of TLR4 to reduce intestinal inflammation, inhibit colon tumor cell proliferation and promote their apoptosis.

Mechanism of Baicalein in Inhibiting Microglial Activation and Protecting SH-SY5Y Nerve Cells / 中国实验方剂学杂志

ObjectiveTo investigate the effect of baicalein （BAI） on SH-SY5Y cell injury in lipopolysaccharide （LPS）-activated BV-2 cells conditioned medium and its mechanism. MethodThe BV-2 cells were activated with 1 mg∙L-1 of LPS to establish the conditioned medium of the LPS group， and a blank group and groups of BAI with low， medium， and high concentrations （4， 8， 16 μmol∙L-1） were established. SH-SY5Y cells were cultured with the conditioned medium of each group. The cell viability of BV-2 cells in each group after the intervention was determined by cell counting kit-8 （CCK-8）. The content of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β） in the supernatant of BV-2 cells in each group was determined by enzyme-linked immunosorbent assay （ELISA）. The protein expression of α-synuclein （α-syn） and tyrosine hydroxylase （TH） in SH-SY5Y cells was observed by immunohistochemical （IHC） staining， and the nuclear transfer of nuclear factor kappa-B p65 protein （NF-κB p65， p65） in SH-SY5Y cells was observed by immunofluorescence （IF）. The protein expression of Toll-like receptor 4（TLR4）， p65， phosphorylated p65 （p-p65）， and Myeloid differentiation factor 88 （MyD88） in SH-SY5Y cells was observed by Western blot. ResultAs compared with the blank group， the viability of BV-2 cells in the LPS group was significantly decreased （P<0.01）， and the content of TNF-α， IL-6， and IL-1β in the cell supernatant was significantly increased （P<0.01）. As compared with the LPS group， the cell viability was significantly increased in groups of BAI with low， medium， and high concentrations （P<0.01）， and TNF-α in the cell supernatant was significantly decreased （P<0.01）. The content of IL-6 in the cell supernatant was decreased in the BAI group with high concentration （P<0.05）， and the content of IL-1β in the cell supernatant was significantly decreased in the BAI groups with medium and high concentrations （P<0.01）. The results of conditioned medium cultured SH-SY5Y cells showed that as compared with the blank group， the protein expression of p65 in the LPS group entered into the nucleus and accumulated， and the protein expression of TH was significantly decreased （P<0.01）， while that of α-syn， TLR4， MyD88， and p-p65 was increased （P<0.05， P<0.01）. Compared with the LPS group， the protein expression of p65 in SH-SY5Y cells in BAI groups with low， medium， and high concentrations gradually dispersed into the cytoplasm and had the enhanced protein expression of TH （P<0.01） but the lowered protein expression of α-syn （P<0.01）. The protein expression of TLR4， MyD88， and p-p65 was decreased in the BAI group with high concentration （P<0.05， P<0.01）， the protein expression of p-p65 and MyD88 was decreased in the BAI group with medium concentration， and the protein expression of MyD88 was decreased in the BAI group with low concentration （P<0.05）. There was no significant difference in the protein expression of p65 among groups. ConclusionBAI can inhibit the activation of BV-2 cells， thereby inhibiting the inflammatory response caused by LPS and further inhibiting the damage of inflammation to SH-SY5Y cells. The mechanism may be related to the regulation of the TLR4/MyD88/NF-κB signaling pathway and reduction of the inflammatory response， thus playing a neuroprotective role.

Mechanism of Schisandra Chinensis-mediated microglia phenotypic transformation by regulation of the TLR4 pathway based on miR-124 / 药学学报

To investigate the mechanism by which Schisandra Chinensis mediates the phenotypic transformation of microglia via microRNA-124 (miR-124)-based regulation of the Toll-like receptor 4 (TLR4) pathway, a model was established using lipopolysaccharide (LPS) stimulation of BV2 cells. Cells were treated with different doses of Schisandra Chinensis extract (SCE). MiR-124 inhibitors and negative control sequences (NC inhibitor) were transfected into LPS-induced BV2 cells and treated with SCE. The MTT assay was used for cell activity detection; an NO kit was used to measure NO release; ELISA kits were used to measure the levels of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α). Microglia markers, including ionized calcium binding adapter molecule-1 (IBA-1) and arginase-1 (Arg-1), and the nuclear translocation of nuclear factor-kappa B (NF-κB) were evaluated by immunofluorescent staining. NF-κB p65, IBA-1, Arg-1, TLR4, myeloid differentiation primary factor 88 (MyD88), inhibitor of nuclear factor-kappa B kinases-α (IKK-α), IL-10, TNF-α were detected by immunoblot. SCE at concentrations ranging from 31.25 to 250 μg·mL-1 had no significant effect on cell activity. SCE treatment significantly inhibited NO release induced by LPS (P < 0.001, P < 0.01), increased the level of IL-10 (P < 0.05), and decreased the level of TNF-α (P < 0.001). In addition, SCE significantly reduced the expression of TNF-α, IBA-1, TLR4, and MyD88 (P < 0.01, P < 0.001) and elevated the expression of IL-10, Arg-1, NF-κB P65 and IKK-α (P < 0.001, P < 0.01, P < 0.05). SCE treatment could also promote the expression of miR-124 (P < 0.01). However, transfection with the miR-124 inhibitor increased TNF-α (P < 0.001), decreased the level of IL-10 (P < 0.05), increased the mRNA level and the protein expression of TNF-α and IBA-1 (P < 0.05, P < 0.01, P < 0.001), and decreased the mRNA level and protein expression of IL-10 and Arg-1 (P < 0.001, P < 0.01). In addition, the inhibition of TLR4 and MyD88 was attenuated. In conclusion, SCE appears to inhibit the activation of TLR4 signaling pathway by upregulating miR-124 so as to inhibit microglia M1 polarization and promote microglia M2 polarization.