| Objective1.To analysis the vitamin D receptor(VDR),nuclear factor-kappa B(NF-?B),inhibitor of nuclear factor-kappa B(I ?B)expression of vulgaris psoriasis,explore the correlation of protein expression and severity index(PASI),investigate the relation between protein expression and the type of traditional Chinese medicine(TCM).Those provide the rationales for the type of TCM and objective reference for clinical application.2.To investigate the influence of 25HVD3,interferon(INF)Y,interleukin(IL)-1,IL-4,IL-17,IL-22,VDR,NF-?B,I?B,VDRmRNA,NF-?BmRNA,I?BmRNA by YinXieIHao Decoction(YXIH)drug-containing serum(DCS),clarify the mechanism of YXIH in intervention of psoriasis vulgaris,and provide theoretical basis for the clinical promotion of compound.3.Adenovirus transfection method was included to construct stable VDR-/-Hacat cell.Investigate the influence of 25HVD3,INF-Y,IL-1,IL-4,IL-17,IL-22,VDR,NF-?B,I?B,VDRmRNA,NF-?BmRNA,I?BmRNA by different concentrations of YXIH.MethodsPart 1:Clinical research102 cases,which conformed for the diagnostic criteria of psoriasis vulgaris,were included in the reaserch.All cases were from the dermatology outpatient and inpatient of the first affiliated hospital of GuangZhou university of TCM.PASI score,histopathological staining and immunohistochemi cal examination were conducted after patients signed the informed consents.We detected the integral optical density(IOD)of target protein VDR,NF-?B,I?B with image-proplus 6.0(IPP6.0).Comparing the protein expression level of normal group and psoriasis group,analysising the correlation of protein expression and the PASI score.Comparing the protein expression level of blood heat syndrome(BHS),blood deficiency syndrome(BDS),blood stasis syndrome(BSS)group.Different expression level of target protein in psoriasis vulgaris patient would lay a foundation of the subsequent experimental research.Part 2 Experimental research1.The impact of YXIH decoction on the psoriatic cell modelGrouping:blank group(BG),model group(MG),low dose group(LD),middle dose group(MD),high dose group(HD).BG was cultured with normal serum,MG,LD,MD,HD were induced with TNF-a and cultured with corresponding drug serum.25HVD3,The concentration of INF-?,IL-1,IL-4,IL-17,IL-22 in the culture supernatants were measured by enzyme-linked immunosorbent assays(ELISA).The protein expression of VDR?NF-?B?I?B of each group were determined with western blotting(WB).Gene expression of VDRmRNA,NF-?BmRNA,I?BmRNA of each group were determined with reverse transcription-polymerase chain reaction(RT-PCR)?2.Conduction and verification of VDR-/-cell modelGrouping:VDR-NC group,VDR-433 group,VDR-544 group,VDR-775 group.Four plasmids were provided by GenePharma,were transfected into Hacat cell,transfection efficiency of plasmids were detected by fluorescence microscope.The protein and gene expression of VDR of each group were determined with WB,RT-PCR to selecte high quality and stable VDR-/-cell.3.Controlled plasmid and VDR775 plasmid effects on Hacat cellsGrouping:Blank group(BG),model group(MG),controlled plasmid transfected model group(NC-MG),VDR-775 plasmid transfected model group(si-MG).According to the general steps to culture cell.The concentration of 25HVD3,INF-?,IL-1,IL-4,IL-17,IL-22 levels in the culture supernatants were measured by ELISA.The protein expression of VDR,NF-?B,I?B of each group were measured with WB.Gene expression of VDRmRNA,NF-? BmRNA,I?BmRNA of each group were tested with RT-PCR.4.The influence of YXIH for Hacat before and after VDR was silencedGrouping:controlled plasmid transfected blank group(NC-BG),controlled plasmid transfected model group(NC-MG),controlled plasmid transfected low dose group(NC-LD),controlled plasmid transfected middle dose group(NC-MD),controlled plasmid transfected high dose group(NC-HD),VDR-775 plasmid transfected blank group(si-BG),VDR-775 plasmid transfected model group(si-MG),VDR-775 plasmid transfected low dose group(si-LD),VDR-775 plasmid transfected middle dose group(si-MD),VDR-775 plasmid transfected high dose group(si-HD).According to the general steps to culture cell.The concentration of 25HVD3?INF-?,IL-1,IL-4,IL-17,IL-22 levels in the culture supernatants were measured by ELISA.The protein expression of VDR?NF-?B?I?B of each group were measured with WB.Gene expression of VDRmRNA,NF-? BmRNA,I?BmRNA of each group were determined with RT-PCR.ResultsPart 1:Clinical research1.The expression of VDR and I?B protein was significantly lower than normal group in psoriasis skin tissue(P<0.01),while NF-?B protein was significantly higher(P<0.01).2.VDR expression was negatively correlated with PASI score(r=-0.475,P=0.000),while NF-?B?I? B epression was positively correlated(r=0.093,P=0.352;r=0.037,P=0.713).The expression of NF-?B in BHS was higher than the other two groups,the expression of NF-? B,I ?B was lower and VDR expression was higher than other groups in BSS,the expression of VDR was lower than other groups in BDS.Part 2 Experimental research1.The impact of YXIH decoction on the psoriatic cell modelCompared with the BG group,25HVD3 was significantly reduced(P<0.01)in MG group,INF-?,IL-1,IL-4,IL-17,IL-22 were significantly increased(P<0.01);VDR,I?B protein and gene expression levels were significantly decreased(P<0.01),NF-?B protein and gene expression levels were increased significantly(P<0.01).Compared with the MG group,the concentration of 25HVD3 were significantly increased(P<0.01),while INF-?,IL-1,IL-4,IL-22 of LD,MD,HD were significantly reduced(P<0.01),IL-17 of LD,MD,HD were significantly reduced(P<0.05).The expression of I?B,VDR protein in LD,MD,HD was significantly incressedC(P<0.05),while NF-?B was obviously decreased(P<0.01).The expression of VDRmRNA in MD,HD was significantly incressed(P<0.05),the expression of I?BmRNA in LD,MD,HD was apparently incressed(P<0.05).2.Conduction and verification of VDR-/-cell modelThe expression of VDR gene and protein was minimum when VDR775 plasmid was transfected.The transfection expression of VDR775 plasmid was the highest when MOI = 100,and the transfection expression of VDR NC plasmid was the highest when MOI=300.3.Controlled plasmid and VDR775 plasmid effects on Hacat cells Compared with the BG group,25HVD3 was significantly reduced(P<0.01)in MG group,INF-?,IL-1,IL-4,IL-17,IL-22 were significantly increased(P<0.01);VDR,I?B protein and gene expression levels were significantly decreased(P<0.01),NF-?B protein and gene expression levels were increased significantly(P<0.01).Compared with the MG group,the expression of protein,gene and cytokines expression wasn' t changed much in NC-MG group(P>0.05).Compared with the NC-MG group,the concentration of 25HVD3 were significantly decreased(P<0.01),INF-y,IL-1,IL-17,IL-22 was apparently increased(P<0.01);the expression of VDR gene and protein was decreased obviously(P<0.01),the expression of NF-?B and I?B protein was increased apparently(P<0.01);NF-? BmRNA was significantly increased(P<0.01);I?B was' t changed much(P<0.01).4.The influence of YXIH for VDR Hacat before and after the silence? The effects of YXIH drug-containing serum on reference plasmid transfected HacatCompared with the NC-BG group,the concentration of 25HVD3 was significantly decreased in NC-MG group(P<0.01),INF-?,IL-1,IL-4,IL-17,IL-22 was increased significantly(P<0.01);the expression of VDR,I?B gene and protein was decrease obvioursly(P<0.01),NF-?B gene and protein was increased apparently(P<0.01).Compared with the NC-MG group,the concentration of 25HVD3 was obviously increased in NC-LD group,INF-?,IL-1,IL-4 was significantly decreased(P<0.05),the expression of NF-?B protein was decreased significantly(P<0.01),I ?B,protein was increased obvioursly(P<0.01).The concentration of 25HVD3 was obviously increased in NC-MD group(P<0.01),INF-?,IL-1,IL-4,IL-17 were apparently reduced(P<0.01),the expression of VDR,I?B protein and gene was significantly increased(P<0.05),NF-?B protein and gene expression were significantly reduced(P<0.01).The concentration of 25HVD3 was obviously increased in NC-HG group(P<0.01),INF-?,IL-1,IL-4,IL-17 was apparently decreased(P<0.01);the expression of VDR,I?B protein expression was significantly increased(P<0.01),NF-?B protein and gene was significantly decreased P<0.05).?The effects of YXIH drug-containing serum on VDR775 plasmid transfected HacatCompared with the si-BG group,the concentration of 25HVD3 was signify cantly decreased in si-MG group(P<0.01),INF-?,IL-1,IL-4,IL-17,IL-22 was increased significantly(P<0.01);the expression of VDR,I?B protein and gene was obviously decreased(P<0.01),NF-?B protein and gene was increased apparently(P<0.01).Compared with the si-MG group,the concentration of 25HVD3 was increased significantly in si-LD(P<0.01),INF-?,IL-1,IL-4,IL-17 was obvioustly decreased(P<0.05);the expression of VDR protein and gene was significantly increased(P<0.05),NF-?B was decreased apparently(P<0.05).The concentration of 25HVD3 was obviously increased in si-MD group(P<0.01),INF-Y,IL-4,IL-17,IL-22 was significantly reduced(P<0.05);the expression of VDR protein was significantly increased(P<0.05),NF-?B protein and gene was apparently decreased(P<0.01),I ?B protein and gene expression was obvioustly incresed(P<0.01).The concentration of 25HVD3 was obviously increased in si-HD(P<0.01),INF-?,IL-1,IL-4,IL-17,IL-22 were significantly reduced(P<0.05),the expression of VDR protein and gene was significantly increased(P<0.01),NF-?B protein and gene expression was decreased apparently(P<0.01),I?BmRNA were increase significantly(P<0.05).?The influence of YXIH for Hacat before and after VDR was silencedCompared with the corresponding controls,The concentration of IL-I?1 IL-4?IL-17?IL-22 was obviously increased in si-MG group(P<0.01);the expression of VDR protein and gene was significantly decreased(P<0.01)in si-MG group.The concentration of 25HVD3 was obviously decreased in si-MG(P<0.01),INF-??IL-1?IL-17?IL-22 was obviously increased(P<0.01);the expression of VDR protein and gene was significantly decreased(P<0.05),NF-?B protein and gene was increased apparently(P<0.1).The concentration of 25HVD3 was obviously decreased in si-LD(P<0.01),INF-??IL-1?IL-17?IL-22 was obviously increased(P<0.05);the expression of VDR protein and gene was significantly decreased(P<0.01),NF-?B protein and gene was increased apparently(P<0.05),I?B protein was increased apparently(P<0.01).The concentration of 25HVD3 was obviously decreased in si-MD(P<0.01),INF-??IL-1?IL-17?IL-22 was obviously increased(P<0.05),the expression of VDR protein and gene was significantly decreased(P<0.05),NF-?B protein and gene was increased apparently(P<0.01).The concentration of 25HVD3 was obviously decreased in si-HD(P<0.01),INF-??IL-1?IL-4?IL-17?IL-22 was obviously increased(P<0.05);the expression of VDR protein and gene was significantly decreased(P<0.01),NF-?B protein and gene was increased apparently(P<0.01);the expression of I?BmRNA was decreased apparently(P<0.01).Conclusion1.There exists different expression level of VDR,NF-?B,I?B in psoriasis vulgris,the suppression of VDR signal may be associated with the occurrence and development of psoriasis.Differential expression of protein may provide reference for syndrome differentiation and clinical medication2.YXIH decoction could promote 25HVD3 expression,and inhibit inflammation cytokines,such as INF-?,IL-1,IL-4,IL-17,IL-22,its mechanism might be related to the promotion of VDR and I?B,and suppression of NF-?B inflammatory signal.3.VDR775 migh be a perfect plasmid to conduct VDR-/-cell model.The biochemical function of psoriatic cell model wasn't when VDR NC plasmid was transfected.4.VDR-/-psoriatic cell model was conducted in this research,we found that NF-?B inflammatory signal pathway was excessively activated in psoriatic cell model when VDR was knockdown.We also deduced that there exit "cross-talking"between VDR signal and NF-?B sinal pathway.Moreover,VDR may inhibit inflammation signal by regulating the NF-?B pathway.5.YXIH decoction could also inhibit inflammatory response after target gene VDR was knockdown,but the suppression was lower than before.VDR may be one of the important targets of YXIH that regulating NF-?B pathway to inhibit inflammation reaction. |
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