| Background:Psoriasis is a chronic inflammatory dermatosis caused by the synergistic effect of genetic, environmental and immune factors, which is characterized by excessive proliferation of keratinocytes and infiltration of immune cells. The incidence rate is 1% -3% worldwide, and demonstrated a continuous growth. Approximately 3% -10%of patients with psoriasis showed inflammatory lesions. In addition, other serious diseases as arthritis complicated by psoriasis, nonalcoholic fatty liver disease,osteoarthritis, cardiovascular disease and metabolic syndrome increased more physiological, psychological and economic burden of patients.Psoriasis vulgaris is the most common type of psoriasis. The cardinal characteristic pathological changes of the patients are Munro micro abscess formed by the accumulation of polymorphonuclear neutrophils (PMNs) in the epidermis. Munro micro abscess occurs only in the parakeratotic areas where keratinocyte (KC) is in active proliferation condition. The inflammatory feature of pustular psoriasis is more significant, it is worth mentioning that the infiltration of PMNs in the spongy micro abscess (Kogoj abscess) in the epidermis of patients with this type is more pronounced than psoriasis vulgaris, also the proliferation of KC is greater significantly.The above suggests that PMNs play an important role in the proliferation and inflammation of KC in psoriasis, as well as the occurrence and development of disease.Neutrophil elastase (NE) is a significant protease in the human body, its synthesis, storage and release mainly by PMNs, is a chymotrypsin superfamily member of serine protease. NE has always been the hot spots and focus of PMNs research. NE stimulates the proliferation of horn cells through the epidermal growth factor receptor (EGFR) signal pathway, possibly causing the accelerated proliferation of psoriatic lesions infiltrating by neutrophils. NE can also promote the expression and secretion of intercellular adhesion molecule (ICAM-1) and the inflammatory factors. These pathological changes may cause proliferation and differentiation of horn cells, anapetia and the changes of inflammatory cell infiltration to the epidermis and dermis. ICAM-1 is an important inflammatory factor that is expressed in large numbers when cells are stimulated by endotoxin LPS or inflammatory cytokines such as IL-1, IL-6 and TNF-?. ICAM-1 participates in PMNs adhesion and colonization,and the adhesion of PMNs to endothelial cells, KCs and T cells, which plays an important role in the response of sterile inflammatory tissue injury. The level of ICAM-1 in skin lesions in the psoriasis patients was significantly higher than that in the normal control group. The content of serum soluble ICAM-1 (sICAM-1) in psoriasis patients was significantly higher than that in the control group, so as the level of ICAM-1 in the skin lesions, and the elevated level was positively correlated with the lesion area. In addition, the excess release of proinflammatory cytokines including tumor necrosis factor-? (TNF-?), interleukin-6 (IL-6) and interleukin-8(IL-8), were detected in the lesions and circulation of psoriasis patients, and produced a cascade reaction.Trappin-2 is an inhibitor of endogenous NE mainly secreted by keratinocytes and epithelial cells, which can inhibit the activity of NE and promote its degradation. A great quantity of studies have suggested that Trappin-2 is highly expressed in inflammatory skin diseases and skin tumors. Trappin-2 inhibits the proliferation of keratinocytes and reduces the expression of inflammatory enhancers such as IL-6 and IL-8 and ICAM-1. Neutrophil infiltration is a significant feature of pustular psoriasis,Tanaka and other studies have indicated that NE expression in the skin lesions of patients with this type was significantly increased, and low concentrations of NE can induce the expression of Trappin-2, but when the degree of skin lesions increase and the concentration of NE is too high, the expression of Trappin-2 is inhibited, the proportion of NE / Trappin-2 promote, such as the level of Trappin-2 in the legions of patients with pustular psoriasis is significantly lower than that in patients with psoriasis v?lgaris, therefore you can infer the severity of psoriasis from the proportion of NE / Trappin-2. Previous studies have suggested that NE can significantly promote the proliferation and metabolism of HaCaT horn cells in vitro, whereas Trappin-2 slows the proliferation of HaCaT horn cells by inhibiting NE activity. Thus, the balance between NE and Trappin-2 plays an important role in the proliferation and inflammatory response of psoriasis.At present, early intervention in patients with psoriasis has been shown to significantly reduce systemic inflammatory response and relieve symptoms. But the pathogenesis of psoriasis is complicated, so its target drug is still limited. The major methods of treating psoriasis include conventional systemic treatments (such as cyclosporine, methotrexate, acitretin and photochemical therapy) and biological agents (such as infliximab, etanercept, efalizumab and adalimumab). However, there are many potential side effects of these treatments, including fever, fatigue, skin itching and other symptoms. At present, only 25% of patients with psoriasis are satisfied with the treatment of the disease. In addition to the above problems, the single target sites and high prices of these agents also limit their clinical development and application. Therefore, it is an urgent demand to develop effective therapies for the disease.Tripterygium wilfordii polycoride (TWP) is a water chloroform extract extracted from traditional Chinese medicine Tripterygium wilfordii, with immunosuppressive,antiinflammatory, anti-tumor and other biological activity, thereby improving the body's inflammatory status and immune function. TWP has been widely used in the clinical treatment of autoimmune diseases and inflammation-related diseases, and has achieved a more recognized efficacy. Meta analysis confirmed that Tripterygium on the progress of the various types of psoriasis are effective, but the possible role of the molecular mechanism still remains unknown.Therefore, in this article, we decided to thoroughly study the mechanism of Tripterygium wilfordii polyglycoside in the treatment of psoriasis and whether the balance between NE and Trappin-2 is also involved in its potential molecular mechanism to provide a more favorable scientific basis for the therapeutic targets of Tripterygium wilfordii polyglycoside treating psoriasis.Objective:Via constructing two models of in vitro injury: HaCaT cell which is human immortalized epidermal cell proliferation model induced by NE (neutrophil elastase)and the HaCaT cell inflammation model induced by TNF-a associated with NE are used to study the effects of tripterygium wilfordii polyglycoside on HaCaT cell proliferation, the secretion of inflammatory factors as well as the expression of ICAM-1, and to explore the mechanism of inhibiting HaCaT cell proliferation and inflammation.Method:1. HaCaT cells were stimplated with different concentrations of NE at different time.The optimal concentration and time of NE were determined by comparing the activityof cell proliferation and the level of inflammatory factors. HaCaT cells were treated with different concentrations of Trappin-2 and TWP at different time conducting the cytotoxicity test to determine the optimal concentration and time of Trappin-2 and TWP.2. The intervention effect of TWP and Trappin-2 on HaCaT cell proliferation was observed by NE-induced HaCaT cell proliferation model.3. To investigate the effect of TNF-? and NE on the proliferation of HaCaT cells and the expression of inflammatory factors in the HaCaT cell inflammation model induced by TNF-a associated with NE. TWP and Trappin-2 were intervened to observe the effect to the model by the intervention effect of TWP and Trappin-2.4. The changes of HaCaT cells proliferation was detected by CCK-8 kit.5. The levels of interleukin-6 (IL-6), interleukin-8 (IL-8), NE and Trappin-2 in the cultured supernatant were measured by enzyme-linked immunosorbent assay (ELISA)kit.6. The expression of intracellular adhesion molecule-1 (ICAM-1) was detected by Western Blot.Result:1. The effect of NE on the proliferation of HaCaT cells and the level of inflammatory factors(1) At the same time, the proliferation activity of HaCaT cells was significantly increased with the increase of NE concentration when NE concentration was ? 10 IU/L (P <0.05), but the proliferation activity of HaCaT cells was significantly decreased when NE concentration was ? 50IU/L (P<0.05).(2) The expression level of IL-6, IL-8 and ICAM-1 in HaCaT cells was slightly changed at the NE concentration of 0.1-100 IU/L, but there was no significant difference with the time and concentration (P> 0.05).(3) Low concentrations of NE significantly increased Trappin-2 levels in HaCaT cells, however high concentrations of NE reduced Trappin-2 levels.Based on the above data analysis, the concentration of 10 IU/L and the time of 24h to stim?late were the optimal stim?lation time and the optimum time of NE. At this time and concentration, the difference in the results of subsequent experiments is minimal.2. The effect of Trappin-2 on HaCaT proliferationThe viability of HaCaT cells changed slightly after treating with Trappin-2 at 10-100 ng / ml for 12h, and there was no significant difference (P>0.05). However,pretreatment with Trappin-2 at 25-100 ng / ml for 24 or 48h significantly reduced HaCaT cell viability and the change was in a dose-dependent manner(P<0.05).Based on the above data analysis, the selected concentration of 25 ng / ml and time of 24h was the optimal stim?lating concentration and the optimal time of Trappin-2. At this concentration and time, the difference in the results of subsequent experiments is minimal.3. The effect of TWP on HaCaT proliferationCompared with the control group (TWP =0?g/ml), TWP (1-100?g/ml) had a slight change in HaCaT cell viability,and there was no significant difference (P>0.05), suggested that there is no obvious cytotoxic effect to HaCaT cell by TWP(<100 ?g/ml). In addition, compared with NE group, TWP at the dose of 50 ?g/ml and the 24h-treatment has the best inhibition on HaCaT cell. Therefore, the optimal concentration and optimal time of TWP were determined by the combination of 50?g/ml and 24h.4. The effect of TWP/Trappin-2 on proliferation model of HaCaT cells induced by low concentration of NE(1) The effect of TWP/Trappin-2 on viability of HaCaT cells induced by low concentration of NEThe viability of HaCaT cells was significantly increased after pretreatment with NE (10 IU/L) for 24h compared with the control group (P <0.01). In addition, TWP and Trappin-2 did not significantly alter HaCaT cell viability (P> 0.05). Compared with NE group, pretreatment with TWP (50?g/ml) or Trappin-2 (25ng /ml) for 24h co?ld significantly decrease the activity of HaCaT cells after 10 IU/L NE treatment for 24h (P<0.05).(2) The effect of TWP/Trappin-2 on the balance of NE and Trappin-2 in Hacat cells induced by low concentration of NEThe levels of NE and Trappin-2 in Hacat cells were significantly increased after NE(10 IU/L) pretreatment for 24h (P<0.01). In addition, TWP and Trappin-2 did not significantly alter NE and Trappin-2 levels. However, pretreatment with TWP (50?g/ml) or Trappin-2 (25ng/ml) for 24h could significantly down-regulate NE levels in Hacat cells and significantly up-regulate Trappin-2 levels compared with NE group(P<0.05). Thus, the ratio of NE and Trappin-2 in Hacat cells was significantly higher than that in the control group; and pretreatment with 50?g/ml TWP or 25ng/ml Trappin-2 for 24h co?ld significantly reduce this ratio(P<0.05).5. The effect of TWP/Trappin-2 on TNF-? associated with NE inducing HaCaT cell inflammation model(1) IL-6, IL-8, NE and Trappin-2 levels as well as NE/Trappin-2 ratio were significantly increased after TNF-? (10ng/ml) and TNF-?+NE treatment of HaCaT cells compared with the control group (P<0.05).(2) Compared with TNF-? group and TNF-?+NE group, IL-6, IL-8 and NE levels were significantly decreased meanwhile Trappin-2 level was significantly increased after TWP stim?lated the HaCaT cell model induced by TNF-?+NE (P<0.05). The expression of IL-6, IL-8, NE and Trappin-2 in the HaCaT cell model induced by TNF-?+NE stim?lated by Trappin-2 was similar to that of TWP (P<0.05).(3) Compared with the control group, after TNF-a+NE pretreatment,HaCaT cells expressing the level of ICAM-1 protein increased significantly. The expression of ICAM-1 was significantly decreased after TWP / Trappin-2 stim?lated the HaCaT cell model induced by TNF-a+NE (P <0.05).Conclusion:1. Low concentration of NE (?10IU/L) co?ld significantly increase HaCaT cell viability and Trappin-2 level, while high concentration of NE (?50IU/L) inhibited HaCaT cell viability and decreased Trappin-2 expression.2. When the concentration of Trappin-2 was ?25ng/ml and the time was ?24h, the proliferation of HaCaT cells was significantly decreased meanwhile did not change significantly with the increase of concentration.3. TWP had no significant cytotoxic effect on HaCaT cells and did not significantly alter the expression of NE and Trappin-2 under the condition of TWP concentration?100?g/ml, the time less than 48h, and the change of HaCaT cell viability was not statistically significant.4. Pretreatment with TWP (50?g/ml) and Trappin-2 (25ng/ml) for 24h could effectively inhibit NE-induced HaCaT cell proliferation and significantly decrease NE level, up-regulate Trappin-2 level and decrease NE/Trappin -2 ratio.5. TWP or Trappin-2 co?ld significantly inhibit the up-reg?lation of IL-6 and IL-8 induced by NE+TNF-a in HaCaT cells as well as significantly decrease NE content and NE/Trappin-2 ratio, up-regulate the level of Trappin-2. In addition, TWP or Trappin-2 can down-regulate ICAM-1 protein expression.6. There is a similar effect on HaCaT betweeent TWP and Trappin-2, which suggests that TWP may be a potent activator of Trappin-2. |
PDF Full Text Request