| Objective:To explore the role of Th17 cells in the pathogenesis of psoriasis and mechanism of compound glycyrrhizin in the treatment of animal model of psoriasis,through detection of levels of Th17 cell and related cytokines and HaCaT cell proliferation and related factor expression, provide reliable experimental basis and theoretical support for the treatment of psoriasis with compound glycyrrhizin.Methods:1, In vivo test:Take 24 New Zealand white rabbits were randomly divided into normal group, model group, the treatment group and the control group. Conduct modeling process using rabbit model of psoriasis based on HULTH the modeling method, after successful modeling of one week in accordance with predetermined protocols on rabbits administered by continuous administration after six weeks of rabbit skin pathology form of psoriasis and related inflammatory cytokines were examined to learn about the changes. (1) modeling and skin tissue after treatment were collected, sliced prepare specimens through a microscope to observe changes in their skin tissue and cell morphology, structure pathology was observed. (2) by collecting skin tissue, skin tissue to observe Appearance, (3) preclude the detection of skin tissue by ELISA Th-17, IL-6, TNF-a and PGE2 content. (4) using Western blot, the skin protein MMP-1 and MMP-3’s.2. In vitro experiments:Experimental study HaCaT cell growth (1) SNMC promote: Take keratinocytes in vitro cell culture model (HaCaT) cell experiments conducted in vitro, experiments with cells cultured in vitro by HaCaT third generation as, HaCaT cells were observed for groups, cells were randomly divided into control group, low dose group, middle dose group, high dose group (containing compound glycyrrhizin final concentration of Oug/L,25ug/L,50ug/L, 100ug/L of 10% FBS DMEM). Using flow cytometry 72h after intervention HaCaT cell cycle distribution analysis GO/G1, S, and the number of G2/M phase of the cell. Expressing cells and the use of cyclinD1, CDK4, p21 protein in western-blot assay. Experimental study on apoptosis of HaCaT (2) SNMC inhibition:the 3rd generation HaCaT cells were used as the experimental cells were randomly divided into high-dose and low-dose, medium-dose and control groups, and no dosing of the model group, which plus normal tissue culture medium containing 10% fetal bovine serum and cultured, other groups were added 10% FBS DMEM containing intervention Immol/L of 24h after adding the corresponding concentrations of the drug are also intervention 24h, and then through an electron microscope Morphological changes of the organization and HaCaT cells were collected, and then analyzed by flow cytometry for apoptosis HaCaT cells, test protein was detected by Western-Blot method, the indicators include Bcl-2, Bax, NF-κB, and then HaCaT cells through the expression of colorimetric detection of apoptotic factors, indicators Caspase-3, Caspase-9, an inflammatory skin tissue factor expression of inflammatory reactions, indicators include the Th-17, IL-6, TNF-a and PGE2.Results:1, In vivo results:observe rabbit skin tissue sections, blank group can be seen in vivo tissue surface of its skin is smooth, neat arrangement of their HaCaT cells their uniform. The organizational structure of the model group subject to certain destruction, thinning phenomenon, HaCaT cells arranged disorderly, multiple tissue surface visible phenomenon disappeared; while the treatment group and the control group of skin tissue damage has been reduced, less skin surface cracks. EIISA detect skin tissue Th-17, IL-6, TNF-α and PGE2 assay results showed that:the ratio, Th-17, IL-6, TNF-α and PGE2 expression in skin treatment group model group significantly lower (P<0.05); compared with the control group, the treatment group Th-17, there was no significant difference (P TNF-a and PGE2 content> 0.05), but slightly higher than IL-6 (P<0.05). Western-blot results showed that skin control group of MMP-1, MMP-3 protein is low. Model group skin MMP-1, MMP-3 protein was significantly higher in the treatment group and the control group MMP-1, MMP-3 protein was significantly lower than the model group, and there was no significant difference in the two groups.(2) In vitro results:Research HaCaT cell proliferation SNMC promote:Study on Morphological Structure:visible drug intervention groups after 72h the cells showed a normal cell state, the cells are mainly circular, oval and polygonal nuclei mainly in the main round, and shows irregular lobulated, nuclear chromatin distribution, in which high-dose group were seen more nuclear division phenomenon, its cytoplasm rich rough endoplasmic reticulum. Cell cycle distribution:The study found significantly lower in the high dose group of GO/G1 phase compared with control group (P<0.01), while the G2/M phase was significantly higher than the control and low dose group (P< 0.05). HaCaT cells express CyclinDl, CDK4 and p21 protein:intervention after 72h, CyclinDl high dose group expression,, CDK4 protein expression was significantly higher than the other two groups of low-dose group and model group (P<0.05); at the same time P21 protein, low-dose group and the control group also was significantly higher than in the high-dose group (P<0.05).SNMC HaCaT cell apoptosis inhibition studies:HaCaT cell morphology observed:Compared with the control group, after the emergence of the model by modeling group to produce a large number of cell necrosis, HaCaT cell apoptosis in a serious, high dose and medium-dose group after treatment through drug intervention withered death and necrosis are less frequent. HaCaT cells Bax, Bcl-2, NF-κB protein expression:HaCaT cells after induction of apoptosis after drug intervention, Bax protein expression of its HaCaT cells, middle dose group, high dose group was significantly lower than the model group, high dose group was significantly lower than the low-dose group; HaCaT cells express Bcl-2 through the study found, and there is dependency, high dose and dose dose than the low dose and control group, and NF-κB expression have the same characteristics, high dose and the dose should be lower than the low dose and control group, Western blot detection of Caspase-3, Caspase-9 protein expression:middle dose group, high dose group was significantly lower than the model, situation apoptotic response, high-dose and mid-dose to below the low-dose and control groups; protein activity also shows the high dose and dose-apoptotic proteins to be lower than the low-dose and control groups; middle dose group and high dose was significantly lower than the low dose group. Th-17, IL-6, TNF-α and PGE2 assay results:compared with model group, high, medium and low dose group Th-17, IL-6, TNF-α and PGE2 expression was significantly lower (P<0.05); and as the dose increased, L-1 (3, IL-6, TNF-α and PGE2 expression was significantly lower (P<0.05)Conclusion:1, compound glycyrrhizin can delay degeneration psoriatic skin tissue model, which can inhibit skin tissue related inflammatory cytokines Th-17, IL-6 expression, TNF-α and PGE2, and inhibit MMPs in expression, there is a certain amount of their inhibitory effect relationship.2, compound glycyrrhizin in vitro can promote the proliferation of HaCaT cells, which may be due to compound glycyrrhizin can promote CyclinDl, CDK4 increased expression of cytokines related to cell cycle, and apoptosis related factor has been the proliferation of expression.3, compound glycyrrhizin in vitro modeling can be suppressed after HaCaT cell apoptosis and necrosis, the main mechanism is due to the drug compound glycyrrhizin inhibits the expression of inflammatory cytokine expression and apoptosis-related protein, thereby inhibiting HaCaT cell apoptosis. |
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