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.

Morphine promotes migration and lung metastasis of mouse melanoma cells

Abstract Background Morphine is an analgesic agent used for cancer pain management. There have been recent concerns that the immunosuppressant properties of morphine can also promote cancer metastasis. Morphine is an agonist for toll like receptor 4 (TLR4) that has a dual role in cancer development. The promotor or inhibitor role of morphine in cancer progression remains controversial. We investigated the effects of morphine on migration and metastasis of melanoma cells through TLR4 activation. Methods Mouse melanoma cells (B16F10) were treated with only morphine (0, 0.1, 1, and 10 μM) or in combination with a TLR4 inhibitor (morphine10 μM +CLI-095 1μM) for either 12 or 24 hours. Migration of cells was analyzed by transwell migration assays. Twenty C57BL/6 male mice were inoculated with B16F10 cells via the left ventricle of the heart and then randomly divided into two groups (n = 10 each) that received either morphine (10 mg.kg−1, sub-q) or PBS injection for 21 days (control group). Animals were euthanized and their lungs removed for evaluation of metastatic nodules. Results Morphine (0.1, 1, and 10 μM) increased cell migration after 12 hours (p < 0.001) and after 24 hours of treatment with morphine (10 μM) (p < 0.001). Treatment with CLI-095 suppressed migration compared to cells treated with morphine alone (p < 0.001). Metastatic nodules in the morphine-treated group (64 nodules) were significantly higher than in the control group (40 nodules) (p < 0.05). Conclusion Morphine increases the migration and metastasis of mouse melanoma cells by activating TLR4.

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 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.

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.

Study on Mechanism of Linggan Wuwei Jiangxintang in Treating Acute Lung Injury / 中国实验方剂学杂志

ObjectiveTo explore the action mechanism of Linggan Wuwei Jiangxintang on the treatment of lipopolysaccharide （LPS）-induced acute lung injury （ALI） in mice. MethodTraditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， GeneCards， DisGeNET， and Herb databases were combined with clinical data from Gene Expression Omnibus （GEO） to screen the key targets of Linggan Wuwei Jiangxintang in the treatment of ALI. The protein-protein interaction （PPI） network was constructed to screen the core targets， and gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） pathway enrichment analyses were performed. The mouse ALI model was established by LPS induction to verify the effect and key targets of Linggan Wuwei Jiangxintang on the treatment of ALI. The expression levels of Toll-like receptor 4 （TLR4）， nuclear transcription factor-κB p65 （NF-κB p65）， and phosphorylated NF-κB p65 （NF-κB p-p65） in lung tissue were detected by Western blot. ResultThe analysis showed that the treatment of ALI with Linggan Wuwei Jiangxintang was related to 10 core targets such as interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， and JUN， involving TNF signaling pathway， Toll-like receptor signaling pathway， NF-κB signaling pathway， etc. The animal experimental results show that Linggan Wuwei Jiangxintang can reduce lung injury， improve the pathological state of ALI mice， significantly reduce the expression of TNF-α and IL-6 in serum， increase the activity of total superoxide dismutase （T-SOD） and catalase （CAT） in lung tissue， and reduce the expression levels of JUN， TLR4， NF-κB p65， and NF-κB p-p65 proteins in lung tissue. ConclusionLinggan Wuwei Jiangxintang can inhibit LPS-induced inflammation and oxidative damage in ALI mice， and its mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway and the reduction of inflammatory factors such as TNF-α and IL-6.

Derris eriocarpa Extract Treats Gout via TLR4/NF-κB/NLRP3 Pathway / 中国实验方剂学杂志

ObjectiveTo explore the anti-gout effect and mechanism of Derris eriocarpa extract by network pharmacological analysis combined with in vivo and in vitro experimental verification. MethodThe chemical components and candidate targets of D. eriocarpa were obtained from the database. The key targets and potential active components of D. eriocarpa in the treatment of gout were screened by the protein-protein interaction analysis， and then the Gene Ontology （GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment were performed for the key targets. A mouse model of hyperuricemia was established by intraperitoneal injection of hypoxanthine to observe the effect of D. eriocarpa alcohol extract on hyperuricemia. A rat model of gouty inflammation induced by the injection of microcrystalline sodium urate crystals into the foot and plantar was used to observe the effect of D. eriocarpa alcohol extract on gouty inflammation. A xylene-induced acute inflammation model was established to observe the anti-inflammatory effect of D. eriocarpa alcohol extract. The hot plate test and twisting test were performed to observe the pain-relieving effect of D. eriocarpa. The lipopolysaccharide （LPS）-induced RAW264.7 cells were used to study the anti-gout effect and mechanism of D. eriocarpa alcohol extract. ResultA total of 12 key targets and 15 potential active components were obtained from the D. eriocarpa-component-gout target network. The emodin， betulinic acid， and medicarpin endowed D. eriocarpa with anti-hyperuricemia， anti-inflammatory， and pain-relieving effects by acting on Toll-like receptor 4 （TLR4）， NOD-like reception protein 3 （NLRP3）， and nuclear factor （NF）-κB. Compared with the control group， the model groups showed elevated serum uric acid level in mice （P<0.01）， increased swelling degree of rats （P<0.05， P<0.01）， alleviated the auricular swelling of mice （P<0.05）， reduced the twisting times of mice （P<0.05， P<0.01）， and increased the hot plate pain threshold （P<0.05）. Moreover， the model group showed up-regulated mRNA level of TLR4 and protein levels of TLR4， NF-κB， and NLRP3 in cells （P<0.01）， and elevated levels of TLR4 and NF-κB in the cell supernatant （P<0.05， P<0.01）. Compared with the model group， the alcoholic extracts （20， 10， 5 g·kg-1） of D. eriocarpa lowered the serum uric acid level in hyperuricemic mice （P<0.01）， inhibited foot and plantar swelling in rats （P<0.05， P<0.01）， down-regulated the mRNA level of TLR4 and the protein levels of TLR4， NF-κB， NLRP3 in cells， and lowered the levels of TLR4， TNF-α， NF-κB， and IL-6 in cell supernatants （P<0.05， P<0.01）. ConclusionD. eriocarpa alcohol extract may exert the anti-gout， anti-inflammatory， and pain-relieving effects by regulating the TLR4/NF-κB/NLRP3 signaling pathway.

Dendrobium Polysaccharides Mitigate CSE-induced Inflammatory Damage of Human Bronchial Epithelial Cells via TLR4/NF-κB Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of Dendrobium polysaccharides on the secretion of inflammatory cytokines and Toll-like receptor 4 （TLR4）/nuclear factor （NF）-κB pathway in 16HBE cells exposed to cigarette smoke extract （CSE）. MethodThe 16HBE cells were classified into the control， CSE， and CSE+ Dendrobium polysaccharides （100， 200， 400 mg·L-1） groups. The cell-counting kit-8 （CCK-8） assay was employed to measure the cell viability， and a microscope was used to observe the cell morphology. The enzyme-linked immunosorbent assay was employed to measure the levels of interleukin （IL）-8， IL-1β， IL-4， IL-13， and transforming growth factor （TGF）-β in cell culture supernatants. Real-time PCR was carried out to determine the mRNA levels of Toll-like receptor 4 （TLR4）， nuclear factor-κB （NF-κB）， and IL-4. Western blot was employed to determine the protein levels of interleukin-4 receptor （IL-4R）， TLR4， myeloid differentiation primary response protein 88 （MyD88）， NF-κB， phosphorylated nuclear factor-κB （p-NF-κB）， and nucleoproteins nuclear factor-κB （NEs-NF-κB）. The immunofluorescence assay was employed to measure the nuclear translocation of NF-κB. ResultCompared with the control group， the CSE group showed elevated levels of IL-8， IL-1β， IL-4， IL-13， and TGF-β in the cell culture supernatants （P<0.05， P<0.01）， up-regulated expression levels of TLR4， MyD88， NF-κB， p-NF-κB， NEs-NF-κB， and IL-4 （P<0.01）， and significant nuclear translocation of NF-κB. Compared with the CSE group， Dendrobium polysaccharides increased the cell survival rate， recovered the cell activity， lowered the levels of IL-8， IL-1β， IL-4， IL-13， and TGF-β， down-regulated the expression of TLR4， MyD88， NF-κB， p-NF-κB， NEs-NF-κB， and IL-4 （P<0.05， P<0.01）， and reduced the nuclear translocation of NF-κB. ConclusionDendrobium polysaccharides showed significant protective effects on the 16HBE cells exposed to CSE by inhibiting the TLR4/NF-κB signaling pathway.

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.

Traditional Chinese Medicine Intervention in Ulcerative Colitis Based on TLR4 Signaling Pathway： A Review / 中国实验方剂学杂志

Ulcerative colitis （UC） is a disease that affects the mucosal and submucosal layers of the colon and is characterized by inflammation of the intestinal mucosa. The incidence of UC is increasing year by year， and it is complex and refractory， severely impacting the physical and mental health of patients. The pathological mechanism of this disease is complex， with immune responses and uncontrollable inflammatory reactions in the intestine being important physiopathologic mechanisms. Toll-like receptor 4 （TLR4）， as a transmembrane signaling receptor， plays a key role in mediating immune responses and inflammatory reactions in the development of UC. Currently， the treatment of UC mainly relies on salicylic acids， glucocorticoids， and other agents to reduce intestinal inflammation. While these drugs can partially inhibit the progression of the disease， they often come with significant adverse effects and the potential for relapse upon discontinuation. Traditional Chinese medicine （TCM） offers multiple pathways， effects， and targets for regulating the TLR4 pathway， suppressing inflammatory responses， and effectively intervening in the progression of UC. This approach has become a hot topic in the prevention and treatment of UC. Numerous studies have shown that TCM treatment of UC has unique advantages. TCM can enhance immune defenses， suppress inflammatory responses， promote intestinal mucosal healing， and maintain the balance of the intestinal microbiota by regulating the TLR4 signaling pathway， thereby effectively treating UC， with substantial progress achieved. However， there is currently a lack of comprehensive reviews on the role of TCM in regulating the TLR4 signaling pathway for the treatment of UC. Therefore， this article systematically summarized the relationship between the TLR4 signaling pathway and UC， as well as the role of TCM in this context， by reviewing relevant literature from recent years， aiming to provide new insights into the potential treatment and new drug development for UC.

Effects of salidroside on lung injury in rats with severe pneumonia and the possible mechanism / 中华微生物学和免疫学杂志

Objective:To investigate whether salidroside (SAL) improves lung tissue injury in rats with severe pneumonia (SP) through mediating toll-like receptor 4/nuclear transcription factor-κB/NOD-like receptor protein 3 (TLR4/NF-κB/NLRP3) signaling pathway.Methods:Seventy-five Wistar rats were used in this study. Fifteen of them were randomly selected as the sham operation group, and the others were induced by endotracheal infusion of Klebsiella pneumoniae ( Kp) suspension to construct a rat model of SP. After modeling, the rats were randomly divided into four groups with 15 rats in each group: model group, low-dose SAL group (30 mg/kg), high-dose SAL group (60 mg/kg) and dexamethasone (DXMS, 15 mg/kg) group. The sham operation group and the model group were given the same amount of normal saline for seven consecutive days. The wet-dry weight ratio (W/D) of lung tissues in each group was detected. HE and TUNEL staining methods were used to observe the morphology of lung tissues and cell apoptosis. The levels of TNF-α, IL-1β, IL-6, IL-18 and IL-10 in bronchoalveolar lavage fluid (BALF) were detected by ELISA. The expression of TLR4, myeloid differentiation factor (MyD88), NF-κBp65, phosphorylated NF-κBp65 (p-NF-κBp65) and NLRP3 at protein level in lung tissues was detected by Western blot. Results:The rat model of SP was successfully constructed by endotracheal infusion of Kp suspension. Compared with the sham operation group, the model group showed more severe edema of lung tissues, increased W/D value ( P<0.05), loose and incomplete alveolar structure, edema of alveolar wall and thickened alveolar wall, massive inflammatory cell infiltration, increased apoptosis rate as well as higher levels of TNF-α, IL-1β, IL-6 and IL-18 and lower lover of IL-10 in BALF. Moreover, the relative expression of TLR4, MyD88, NF-κBp65, p-NF-κBp65 and NLRP3 at protein level in lung tissues was increased in the model group ( P<0.05). Gradually improved pathological injury of lung tissues, decreased W/D value ( P<0.05), recovered alveolar structure, reduced alveolar wall edema and decreased cell apoptosis rate were observed in the low-dose and high-dose SAL groups as well as the DXMS group as compared with those of the model group. Besides, the three groups also showed decreased levels of TNF-α, IL-1β, IL-6 and IL-18 and increased level of IL-10 in BALF, and inhibited expression of TLR4, MyD88, NF-κBp65, p-NF-κBp65 and NLRP3 at protein level in lung tissues ( P<0.05). DXMS performed better in improving lung injury in rats with SP, followed by high and low doses of SAL ( P<0.05). Conclusions:SAL could reduce cell apoptosis and improve the Kp-induced lung injury in rats. The mechanism might be related to the blockage of TLR4/NF-κB/NLRP3 signaling pathway activation and inhibition of inflammatory factor expression.

Role of TLR4/NF-κB signaling pathway in long-term cognitive impairment induced by multiple exposures to sevoflurane anesthesia in neonatal rats / 中华麻醉学杂志

Objective:To evaluate the role of Toll-like receptor 4 (TLR4)/nuclear transcription factor κB (NF-κB) signaling pathway in long-term cognitive impairment induced by multiple exposures to sevoflurane anesthesia in neonatal rats.Methods:Seventy-five SPF healthy newborn Sprague-Dawley rats of either sex, aged 6 days, weighing 12-20 g, were divided into 3 groups ( n=25 each) using a random number table method: control group (group C), multiple exposures to sevoflurane anesthesia group (group S) and TLR4 inhibitor plus multiple exposures to sevoflurane anesthesia group (group I+ S). The rats in group S and group I inhaled 3% sevoflurane for 2 h at 6, 7 and 8 days after birth. TLR4 inhibitor TAK-242 10 mg/kg was intraperitoneally injected before each exposure to sevoflurane in group I, and the equal volume of normal saline was given instead in the other two groups. The spontaneous activity was evaluated by open field test on day 29 after birth, and the cognitive function was assessed by Morris water maze test on days 30-34 after birth. After the behavioral test, the blood samples from the abdominal aorta were collected, and then the rats were sacrificed under deep anesthesia to isolate the hippocampal tissues for measurement of the levels of S100β and neuron-specific enolase (NSE) in serum and hippocampal interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) (by enzyme-linked immunosorbent assay), expression of TLR4, NF-κB p65 and phosphorylated NF-κB p65 (p-NF-κB p65) (by Western blot) and for microscopic examination of the pathological changes of hippocampal CA1 region after HE staining. Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the TLR4 expression was up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was increased, the levels of serum S100β protein and NSE and hippocampal IL-1β, IL-6 and TNF-α were increased ( P<0.05), and the pathological changes in the hippocampal CA1 region were aggravated in group S. Compared with group S, the escape latency was significantly shortened, the number of crossing the original platform was increased, TLR4 expression was down-regulated, the ratio of p-NF-κB p65/NF-κB p65 was decreased, the levels of S100β and NSE in serum and hippocampal IL-1β, IL-6 and TNF-α were decreased ( P<0.05), and the pathological changes in hippocampal CA1 area were significantly attenuated in group P. Conclusions:The mechanism by which multiple exposures to sevoflurane anesthesia induces long-term cognitive impairment is related to activation of TLR4/NF-κB signaling pathway and increase in hippocampal inflammatory responses in neonatal rats.

Research on cell membrane-coated drug delivery nanovesicles in inducing the polarization of tumor-associated macrophages and treating osteosarcoma / 中华骨科杂志

Objective:To prepare cell membrane-coated nanovesicles with targeted delivery of toll-like receptor 4 (TLR4) agonist, and to explore the effect and mechanism of inducing the polarization of tumor-associated macrophages (TAMs) and treating osteosarcoma.Methods:TLR4 agonist loaded nanovesicles were prepared by polycarbonate membrane extruders. The morphology and size of nanovesicles were detected by transmission electron microscopy (TEM) and particle size analyzer, and the drug loading performance of the nanovesicles to TLR4 agonist was investigated. TLR4 agonist loaded nanovesicles were co-incubated with macrophages in vitro, and the targeting ability of nanovesicles to macrophages and its role in regulating the function of macrophages were detected by confocal fluorescence microscopy. In vitro experiments, a cell co-culture system was established. After the upper layer macrophages were treated by the control group, the TLR4 agonist group and the TLR4 agonist loaded nanovesicle group, the lower layer osteosarcoma cells were collected for CCK-8 and cloning formation experiments to evaluate their effects on the proliferation and migration of osteosarcoma cells. In vivo experiments, an osteosarcoma subcutaneous graft tumor model was established, and mice were randomly divided into the control group, the TLR4 agonist group, and the TLR4 agonist loaded nanovesicle group. After the treatment by caudal vein, the tumor targeting ability of nanovesicles in vivo was explored through the in vivo imaging system, and the volume of tumor tissue was continuously detected. The subcutaneous tumors were stained to detect macrophage-related markers, and their effect on the polarization of macrophages was evaluated. The TUNEL fluorescence of tumor tissues was further detected.Results:TEM showed the round shape of TLR4 agonist loaded nanovesicle and the size was about 200 nm. The co-incubation of 0.05 mg TLR4 agonist with 0.1 mg nanovesicles was the best condition for the preparation of drug-loaded nanovesicles. The drug loading efficiency was about 35% and the drug loading content was about 0.11 mg/mg. The membrane-coated nanovesicles could efficiently load and deliver TLR4 agonist. TLR4 agonist loaded nanovesicles were labeled with DiD red fluorescent dye, and then the labeled nanovesicles were co-incubated with macrophages. It was found by confocal fluorescence microscopy that DiD labeled TLR4 agonist loaded nanovesicles significantly accumulated in macrophages, and the fluorescence of M1-type macrophage marker (iNOS) was significantly enhanced, which could induce M1 polarization of macrophages. In vitro experiments, it was found that the number of osteosarcoma cells in the TLR4 agonist loaded nanovesicle group was significantly reduced under the light microscope, and the cell morphology was wrinkled and rounded. CCK-8 and cloning formation experiments showed that the proliferation and migration ability of osteosarcoma cells in the TLR4 agonist loaded nanovesicle group was significantly reduced compared with the control group and the TLR4 agonist group. A subcutaneous graft tumor model was established. In vivo imaging experiments showed that TLR4 agonist loaded nanovesicles locally accumulated in tumor tissues in vivo, but were not distributed in other organs. The growth of tumor tissue was significantly inhibited in the TLR4 agonist loaded nanovesicle group. Moreover, the fluorescence of M1-type macrophage marker (iNOS) was significantly enhanced (relative fluorescence intensity: 3.27±0.19), while the fluorescence of M2-type macrophage marker (CD163) was significantly decreased (relative fluorescence intensity: 0.14±0.04). TUNEL fluorescence staining showed that the apoptosis level of osteosarcoma cells was significantly increased (relative fluorescence intensity: 9.53±0.21).Conclusion:Membrane-coated nanovesicles could targeted deliver TLR4 agonist to osteosarcoma, induce TAMspolarization, remodel tumor immunosuppressive microenvironment, promote cell apoptosis, and effectively kill osteosarcoma.

Improvement effect and mechanism of calycosin on acute inflammatory injury secondary to intracerebral hemorrhage / 中国药房

OBJECTIVE To investigate the improvement effect and mechanism of calycosin （CA） on acute inflammatory injury secondary to intracerebral hemorrhage. METHODS Male C57BL/6 mice were injected with type Ⅶ collagenase into the basal ganglia to establish an intracerebral hemorrhage model， which were divided into sham-operation group（phosphate buffered saline instead of collagenase）， model group， and different CA dose groups（15，30，60，120 mg/kg）. Based on the modified neurological severity score （mNSS） to screen the intervention doses， the volume of intracerebral hemorrhage， brain water content， the expressions of ionized calcium-binding adaptor molecule 1 （Iba1） in brain tissue， Toll-like receptor 4 （TLR4） and its downstream inflammatory factors [tumor necrosis factor-α （TNF-α）， inducible nitric-oxide synthase （iNOS）， interleukin-1β （IL- 1β）] in brain tissue， and the apoptosis of cells in brain tissue were detected. Primary microglia were cultured in vitro， and the expressions of TLR4 and its downstream inflammatory factors were detected. Primary neurons and primary microglia were co- cultured in vitro， and the apoptosis of neurons was detected. RESULTS The doses of 30 mg/kg and 60 mg/kg were selected as intervention doses of CA for subsequent experiments. Compared with the sham-operation group， the mice in the model group had cerebral hemorrhage， the volume of cerebral hemorrhage and brain water content were significantly increased （P＜0.05）； the positive expression rate of Iba1 protein in brain tissue was significantly increased， and the relative expression levels of TLR4， TNF-α， IL-1β and iNOS protein in brain tissue were up-regulated significantly. The apoptosis rate also increased significantly （P＜0.05）. Compared with model group， the above indexes of the mice in the 30 and 60 mg/kg CA groups were significantly improved （P＜0.05）. CA significantlyreduced the relative expression levels of TLR4 and its downstream inflammatory factors in microglia， and reduced the apoptosis of neurons in the co-culture system of primary neurons and primary microglia （P＜0.05）. CONCLUSIONS CA can exert a protective effect on the brain， which may be related to relieving the secondary acute inflammatory injury after intracerebral hemorrhage by inhibiting TLR4-mediated inflammatory response.

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.