ABSTRACT
Aim To investigate the effect of histamine H, receptor (HjR) on the immune responses in astrocytes induced by lipopolysaccharide (LPS) and the regulatory mechanism of its signaling pathway. Methods LPS was used to establish an in vitro astrocyte inflammation model. Rat primary astrocytes were divided into the control group, LPS group, LPS + Hj R agonist group (2-pyridylethlamine, Pyri), and HjR agonist group. Astrocytes were treated with Pyri 100 p,mol • L~ for 1 h, then stimulated with LPS at 100 p,g • L~ for 24 h. Cell viability was measured using the CCK-8 assay. The expression of GFAP and HjR was detected by immunofluorescence. Glial morphological changes were observed under a microscope. The levels of proinflammatory mediators (TNF-a and IL-6) were detected by ELISA. The protein expressions of p-Akt, Akt, p-NF-KB p65, and NF-KB p65 were detected by Western blot. Results Compared with the control group, more activated astrocytes with fewer cell processes and branches were observed in the LPS group. Besides, LPS enhanced the GFAP expression level, reduced the H,R expression level and stimulated the production of TNF-a and IL-6 from astrocytes. Pre treatment with Pyri for 1 h ameliorated the glial morphological changes stimulated by LPS, inhibited LPS-induced upregulation of GFAP level and the inflammatory factors secretion. In addition, LPS stimulated astrocytes showed a higher phosphorylation of Akt and NF-KB p65, which was also ameliorated by Pyri. Conclusions H, R agonist can inhibit LPS-induced astrocyte activation and inflammatory factor secretion, and the Akt/NF-KB signaling pathway may be an important pathway for the involvement of H,R in immune regulation.
ABSTRACT
Aim To investigate the effect of colchicine on lipopolysaccharide (LPS) induced endothelial to mesenchymal transition (EndMT) in human umbilical vein vascular endothelial cells (HUVECs) and its related mechanisms. Methods The EndMT model was established by treating HUVECs with LPS. Cell proliferation rate was detected by CCK-8 assay, cytotoxicity was detected by LDH assay, and the optimal drug concentration was screened. The cells were divided into the normal control group, the normal control + colchicine (10 nmol • L) group, the LPS (10 mg • L) model group, and the LPS + colchicine (10 nmol • L) group. The morphologic changes of the cells were observed under an inverted microscope, the cell migration ability was detected by Transwell assay, and the ability of tube formation was analyzed by tube formation assay. The expression of endothelial markers (CD31/ VE-cadherin) and mesenchymal cell markers (a-SMA/FSP-1) were detected by Western blot. NF-KB inhibitor was used to detect the changes in related signaling pathways. Results CCK-8 and LDH experiments showed that 10 nmol • L colchicine was the optimal concentration. LPS could induce morphological changes in HUVECs, and colchicine could reverse morphological changes in HUVECs to a certain extent. Transwell experiment showed that the migration ability of HUVECs in the LPS treatment group was significantly enhanced (P < 0. 05), and colchicine could significantly reverse this phenomenon (P < 0. 05) . Tube formation experiment showed that LPS decreased the endothelial tube formation ability of HUVECs (P < 0. 05), while colchicine treatment markedly improved LPS-induced tube formation defects (P < 0. 05) . Western blot assay showed that after colchicine co-cultured with LPS, the expression levels of CD31 and VE-cadherin significantly increased compared with the model group (P < 0. 05), while the expression levels of a-SMA and FSP-1 significantly decreased compared with the model group (P < 0. 05) . During the induction of EndMT by LPS, colchicine could inhibit the activation of the NF-KB/Snail signaling pathway. Conclusions Colchicine can effectively inhibit EndMT induced by LPS, and the mechanism may be related to the regulation of the NF-KB/Snail signaling pathway.
ABSTRACT
Aim To explore the mechanism of asiatic acid (AA) on alcoholic hepatitis (AH) based on the network pharmacology and experimental verification in vivo methods. Methods The potential mechanism of AA on AH was explored by data collection, network construction, and enrichment analysis. Meanwhile, the model of alcoholic hepatitis disease was induced by in-tragastric administration of edible alcohol every day in SD rats. The key related indicators were detected, including biochemical markers, inflammatory responses, alcohol metabolism, pathological changes in liver tissues, and the expression of proteins of the NF-kB pathway. Results A total of 24 overlapping targets of AA and AH were screened out, and 20 signaling path ways and 12 GO functional entries were obtained. This study focused on the first pathway, hsa05200; Pathways in cancer. The pathway contained NF-kB signaling pathway. In vivo results showed that AA significantly reduced the serum levels of ALT and AST, increased the levels of alcohol metabolism and decreased the liver content of TNF-a and GSH. Additionally, AA significantly inhibited p-IKKa/(3, p-Ii
ABSTRACT
Aim To investigate the effect of compound Q-L on MH7A based on NF-KB and MAPK signaling pathways and its mechanism. Methods Human rheumatoid arthritis fibroblast synovial cell line (MH7A) was selected as the experimental object. The effect of compound Q-l on the proliferation of MH7A cells was determined by CCK-8 method. The effect of compound Q-l on the migration ability of MH7A cells was detected by Transwell assay. TNF-α solution was used as inducer, and the content of TNF-α and IL-6 in cell supernatant was determined by ELISA. The protein expressions of p65, p-p65, IκBα, P-IκBα, p38, p-p38, ERK, p-ERK, JNK and p-JNK in the cells were determined by Western blot. Results Compound Q-l at different concentrations significantly inhibited the activity of MH7A cells. Compound Q-l significantly inhibited the migration of MH7A cells. Compound Q-l significantly reduced the contents of TNF-α and IL-6 in cell supernatant. Compound Q-l could significantly down-regulate the protein expression levels of p65, p-p65, P-IκBα, p38, p-p38 induced by TNF-α, but had no marked effects on IKBCX, ERK, p-ERK, JNK and p-JNK proteins. Conclusion Compound Q-l can significantly inhibit the proliferation of MH7A cells, reduce the expression of inflammatory cytokines TNF-α and IL-6 in cell supernatant, and down-regulate the protein expressions of p65, p-p65, IKBCX, p-LKBA, p38, p-p38 induced by TNF-α. The possible mechanism of action is related to NF-κB and p38MAPK sig-naling pathways.
ABSTRACT
BACKGROUND: The relationship between nuclear factor (NF)-kB signaling pathway and intervertebral disc degeneration is a hotspot in the field of orthopedics. In-depth investigation on various signaling pathways in the intervertebral disc contributes to understanding the mechanism of intervertebral disc degeneration. OBJECTIVE: To investigate the regulation of the serum containing Yishen Huoxue Tongluo Recipe on the NF-kB signal pathway of human intervertebral disc nucleus pulposus cells under different hydrostatic pressures, attempting to explore the possible therapeutic mechanism and target of Yishen Huoxue Tongluo Recipe in the treatment of degenerative diseases of the intervertebral disc from the perspective of molecular biology. METHODS: Passage 3 Human intervertebral disc nucleus pulposus cells were divided into eight groups and were cultured in the drug-containing serum of Yishen Huoxue Tongluo Recipe. The cells were intervened for 2, 4, and 6 hours under different hydrostatic loading conditions (0.3,1, and 3 MPa). The morphology and growth of nucleus pulposus cells were observed by an inverted phase contrast microscope. Ultrastructural changes of intervertebral disc nucleus pulposus cells were observed by a transmission electron microscopy. Cell counting kit-8 method was used to detect the proliferation activity of nucleus pulposus cells. Annexin V-FITC/Propidium Iodide apoptotic kit was used to double-stain nucleus pulposus cells to detect the cell apoptotic rate. Western blot method was used to detect the changes of NF-kB p65, Collagen II, ADAMTS-4, MMP-13 and Caspase-3 in nucleus pulposus cells. RESULTS AND CONCLUSION: (1) Under the same pressure and time, the morphology and growth of nucleus pulposus cells in the pressure+drug-containing serum groups were better than those in the normal pressure group and the simple pressure groups. Among them, the 0.3 and 1 MPa pressure+drug-containing serum groups had more intact cell morphology and better cell growth than the 3 MPa pressure+ drug-containing serum group. (2) The proliferative activity of nucleus pulposus cells was higher in the pressure+drug-containing serum group, and there was a significant difference between the 0.3 MPa pressure+drug-containing serum group and the 0.3 MPa simple pressure group (P < 0.05). (3) The apoptotic rate of nucleus pulposus cells in the pressure+drug-containing serum group was lower than that in the normal pressure group and simple pressure intervention group (P < 0.05). (4) The expression of Collagen II and Caspase-3 increased in the pressure+drug-containing serum group, while the expression of NF-kappa B p65, ADAMTS-4 and MMP-13 decreased in the pressure+drug-containing serum group (P < 0.05). To conclude, Yishen Huoxue Tongluo Recipe can increase cell activity, reduce cell apoptosis and effectively delay the degeneration of nucleus pulposus cells. Its mechanism is likely to promote the expression of Collagen II and Caspase-3 through the NF-kB signaling pathway of nucleus pulposus cells of intervertebral disc, and inhibit the expression of NF-kB p65, ADAMTS-4 and MMP-13.