Pathogenetic Exploration Of The Hormone-dependent Dermatitis And The Intervention Effect Of Tripterygium H.h. By The Approach Of GC/GR Mediated NF-κB/IκB Signaling Pathway

IntroductionHormone-dependent dermatitis (HDD) is a inflammatory dermatosis caused by incorrect use of topical glucocorticoid (GC). the pathogenesis is unclear, and there is no satisfactory clinical treatment.Topical GC is the original cause of hormone-dependent dermatitis, and GC need combined with intracellular glucocorticoid receptor (GR) to do its work, therefore the pathogenesis of hormone-dependent dermatitis is obviously related to the interaction between GC-GR.Study has showed that there are a large number of the release of inflammatory cytokines occurs in the process of hormone-dependent dermatitis, and aggregation and expression of inflammatory cytokines may be associated with multiple signaling pathways activated,which NF-κB/IkB signaling pathway have attracted the most attention. GR and NF-κB are a pair of mutually antagonistic transcriptional regulation factors in immune and inflammation responses. NF-κB can mediate a variety of immune and inflammatory related cytokines and activation process of medium, while GR inhibits NF-κB activation gene expression.Accordingly, we speculate that GR mediated NF-κB/IκB signaling pathway may play a central role in regulating the development of hormone-dependent dermatitis.Tripterygium hypoglaucum hutch (THH) is one kind of traditional chinese medicine which has the effect such as anti-inflammatory, anti-tumor, immune suppression, is widely used in a variety of allergic diseases, autoimmune diseases. In recent years, clinical studies showed that THH have achieved a better curative effect on treating hormone-dependent dermatitis, it can significantly reduce the recurrence rate after the drug stopped. However, the specific mechanism of action is unclear, we hypothesize that:THH may inhibit the expression of inflammatory cell factors, have the effect of anti-inflammation and immune regulation through the signal pathways above.In this paper, we will use the hormone-dependent dermatitis guinea pig model, making the process of inflammatory reaction in the model as the main line of the study, with GR, NF-κB/IκB as the breakthrough point, from the perspective of signaling pathways to research GR, NF-κB/IκB regulation and control of the process of hormone-dependent dermatitis and its upstream and downstream of cytokines (IL1, IL-4, TNF-a) expression changes, to help elucidating the mechanism of hormone-dependent dermatitis.Taking THH for intervention method, its action mechanism will discussed in order to optimize the experimental foundation for clinical treatment.ObjectiveThis stuty will preliminary explore the pathogenesis of hormone-dependent dermatitis and the effects of Tripterygium hypoglaucum hutch(THH) on nf-kappa B/I kappa B(NF-κB/IκB) signal pathways by determinating the key molecules of NF-κB/IκB signal pathway with the animal model of hormone-dependent dermatitis, and provide xperimental basis for the clinical application of THH.Methods1. Establish hormone-dependent dermatitis animal model by using topical0.02%clobetasol propionate ointment.2.36male guinea pigs were randomly divided into blank control group, model group (7d group,15d group,21d groups and28d group) and GR blocking group, each group of6.4model groups were drawed blood on the7th day,15th day (date of drug withdrawal),21st day (6days after the drug withdrawal)28th day (13days after the drug withdrawal) by heart puncture, respectively,by using ELISA method for determination of serum IL-1, IL-4levels. Take skin tissue samples, to determine the nf-kappa Bp65, I kappa B protein expression levels using immunohistochemical method, to detect GRmRNA, TNF alpha mRNA transcription level using the reverse transcription polymerase chain reaction (RT-PCR) method. Observe the change of the indicators in different phases of the model building.3. In15days of continuous use, topical steroid was stopped for GR blocking group.20mg/kg body weight by subcutaneous injection at a concentration of lOg/L of RU486(mifepristone) oil,1times/day for6days was used. The21st day, the animals were sacrificed by cervical dislocation. Cutting the experimental area of skin tissue, and detected the expression level of NF-κB and IκB with SABC immunohistochemical method, and compareed with the21days group which sacrificed the same day. Observed the effect on the expression of NF-κB and IκB after GR blocked.4.40guinea pigs randomly selected from48guinea pigs, were used to reproduce hormone-dependent dermatitis model. After successful modeling, they were randomly divided into five groups(model group, low-dose THH group, middle dose THH group, large-dose THH group and topical steroid diminishing group).The remaining eight guinea pigs served as blank control. Control group and model group were treated with vaseline, applied once a day, topical steroid diminishing group:the1st week start with0.1%hydrocortisone butyrate cream2times/d, the2nd weekl times/d, the3rd week once every other day, the4th week, once every3days.Low-dose THH group, middle-dose THH group and large-dose THH group were given THH solution lml/kg,2ml/kg,4ml/kg by gavage once daily, respectively. Each group were treated4weeks before withdrawal.5days later, the serum IL-1, IL-4levels, the NF-κBp65, IκB protein expression levels and the GR mRNA, TNF-a mRNA transcript levels from skin tissue were measured, the changes of these indicators in different intervention conditions were Observed. Results1. Compared with the blank control group, the serum IL-1, IL-4in the group of7day and the15day showed a downward trend, while the21d group and the28d group increased. Serum IL-1of the28days group were significantly higher than normal controls, the difference was statistically significant (P<0.05).2. Compared with the blank control group, the expression of NF-κB in the skin tissue in7d group, and15d group showed a downward trend, after the withdrawal, the21d group was significantly higher and28d group declined slightly, the difference was statistically significant (P<0.05). Compared with the blank control group, the skin tissue IκB in the7d group,15d group showed an upward trend, the differences were statistically significant (P<0.05). In the21d group,28d group gradually decreased, but the expression is still higher than the control group.3. GRa mRNA expression in the7d group has increased, then decreased until28d group increased slightly. GRa mRNA expression in15d group,21d group and28d group was significantly lower than the control group, the difference was statistically significant (P<0.05). Compared with the control group, the expression of TNF-a mRNA of skin tissue in the7d group and15d group showed a downward trend, while that of the21d group was significantly increased, the difference was statistically significant (P<0.05). that of the28d group gradually decreased, but the expression is still higher than the control group.4. Compared with the blank control group and the21d group, the expression of NF-κBp65of the GR blocking group were significantly increased, while IκB were significantly lower, the differences was statistically significant (P<0.01).5. After four weeks treatment, compared with the model group, the serum IL-1, IL-4, skin tissue NF-κB and TNF-a mRNA of the hormone diminishing group and the THH groups were decreased to a varying degrees.The inhibition effects of THH was more obvious with doses increasing. Serum IL-1, IL-4, NF-κB, TNF-α mRNA expressions of high-dose THH group were significantly lower than that of the model group, the difference was statistically significance (P<0.05). Compared with the model group, the IκB expression of THH groups and the hormone diminishing group increased in varying degrees.The IκB expressions of the hormone group and the high-dose THH group significantly increased, the difference was statistically significant (P<0.05).6. After four weeks treatment, compared with the model group, the GRa mRNA levels of hormone diminishing group significantly reduced, and the difference was statistically significant (P<0.05), while the GRa mRNA levels of all THH groups did not significantly decreased (P>0.05).Conclusions1. In hormone-dependent dermatitis guinea pig model, long term using potent topical GC can reduce the GR levels of the skin tissues, and can keep GR maintaining a low level expression for a long time after drug withdrawal. GC can activate IκB expression, restrain NF-κB expression simultaneously, inhibit proinflammatory cytokines such as IL1, TNF-α, thereby inhibiting inflammation.2. After blocking GR, compared with the same time model group, the NF-κB activity of GR blocked group has significantly increased, and while the IκB expression decreased, which show the reduced GR expression inhibits IκB and enhance the expression of NF-κB activity in the case of hormone-dependent dermatitis, and thereby increase the inflammatory conditions. In other word, GR would inhibit immune and inflammation-related genes NF-KB-induced. so it can be inferred:the low level GR inhibiting IκB expression, thereby activating NF-κB/IκB pathway may be the cause of the occurrence of hormone-dependent dermatitis.3. THH can inhibit serum IL-1, IL-4levels and levels of TNF-α mRNA of skin tissue, increased IκB expression and inhibit the expression of NF-κB in the skin of HDDguinea pig in a concentration-dependent manner, but no significant effect on GRa mRNA levels. It suggest that:the anti-inflammatory mechanism of THH on the condition of hormone-dependent dermatitis is not mediated by GR, but probably by inhibiting TNF-a levels, activating IκB expression, thereby preventing nuclear translocation of NF-κB, lowered its transcriptional regulation of inflammation factors expression.