The Expression Of TLR4、TLR9mRNA And Dwonstream MyD88、TRIF MRNA In Peripheral Blood Mononuclear Cells Of Patients With Pityriasis Rosea

Objective: Our topic was to investigate the expression of TLR4mRNA、TLR9mRNA、MyD88mRNA and TRIF mRNA in the peripheral bloodmononuclear cells (PBMC) in patients with Pityriasis rosea(PR) using SYBRgreen I Real-time fluorescence quantitative PCR, and study the role of TLRSin the pathogenesis of PR. PR is a common and self-limited inflammatory skindisease in clinical,the couse of which is6-8weeks, Its Characteristic lesion isoval or circular rosiness、cardinal or yellow-red macules or papules withbran-like scales,which long axis are consistented with striae. The pathogenyand pathogenesis of PR are unknown, Up to now it is thought to be relatedwith Virus infection and cellular immunity.There are two sets of immune defense mechanisms to resist the invasionof pathogens: innate immunity and adaptive immunity[1]. Innate immunity isthe first line of the host defense.The immune responses utilize the skin andmucous membrane epithelium physical barriers to prevent infection andrespond quickly. Adaptive immune responses are slower process which ismediated by T and B cells and their antigen receptors are highly diversifiedbecause of the complexity of recombinant DNA, so they can identifyabnormal antigens as well as conservative antigens. Innate immunity identifypathogens through pathogen associated molecular patterns (PAMPs). Inmammals, the receptors which are sponsible for the identification of PAMPsare called pattern recognition receptors (PRRs). Toll-like receptors (TLRS) arekinds of main PRRs in innate immune system, which are germline-encodedand expressed in the immune and non-immune cells in an established form[1].TLRS are hot research points in recent years that widely distributed. TLRSexpresse in immune cells as well as non-immune cells and are thought to be the bridge between innate immunity and adaptive immunity. After TLRSrecognize pathogens, they can mediate the transmembrane signal transductionof the innate immunity, and activate a variety of proinflammatory factor suchas IL-12, TNF-α, which are involved in the anti-infection of inflammatoryprocess. TLRS play a regulatory role in the natural immune and adaptiveimmune responsealso by producing cytokines such as TNF-α and IL-1,chemokines such as IL-8and MIP2, costimulatory molecules such as CD40andCD80and adhesion molecules such as ICAM-1[2-4]and by up-regulating ofcostimulatory molecules in the surface of antigen presenting cells such asCD80/CD80[5,6]. TLR4is mainly distributed in antigen presenting cells, theligand of which is gran negative bacteria cell wall with lipopolysaccharide.TLR9is expressed in many human cells whose ligand is low methylatedcytosine phosphate guanosine (CpG) DNA. This motif are prevalented in theDNA of bacteria and viruses but rare in mammals. In recent years, TLRS havesignificant progresses in skin inflammation, skin malignant tumor and defensemechanisms.Method：In cases group,12males and14famales were involved in the study. Theywere outpatients with PR from department of dermatology in the secondhospital of Hebei Medical University, the average age was22.5years old andthe course was3days to1month in cases group.The inclusion criteria:(1)Typical clinical manifestations: oval or circular rosiness、 cardinal oryellow-red macules or papules with bran-like scales,whose long axis wereconsistented with striae in chest and back;(2) All the cases were withoutimmune diseases, allergic disease, cancer and other serious systemic diseases;(3) All the cases had not taken medicine which could affect immunologicalfunction for one month such as corticosteroids and immunosuppressive agentand antihistamines.20healthy persons without allergic disease, autoimmunedisease and family history, were involved in control group. Their gender, agehad no statistical differences compared with the case group. The expression ofTLR4mRNA, TLR9mRNA, MyD88mRNA and TRIF mRNA were detected in PBMC of26PR Patients and20healthy volunteers by SYBR Green IReal-time fluorescence quantitative PCR technique. The Experimental datawas analysed by SPSS13.0statistical software and when P<0.05it hadstatistical significance.We selected the normality test and the Mann-Whitneyas statistical method and the measured data was described by median orquartile.Results:1The RQ values of gene TLR4mRNA: After the test of normality, P＜0.1, so the data disobeyed Gaussian distribution. The results of case group andcontrol group were expressed by median or quartile, and respectively were8.102/11.87；7.275/4.96. Statistical results of the expression of TLR4mRNA in patients with PR did not show statistical significance compared withthe normal controls (P=0.09＞0.05).2The RQ values of gene TLR9mRNA: After the test of normality, P＜0.1, so the data disobeyed Gaussian distribution. The results of case group andcontrol group were expressed by median or quartile, and respectively were1.592/3.86；3.13/8.93. Statistical results of the expression of TLR9mRNAin patients with PR had statistical significance compared with the normalcontrols (P=0.03<0.05).3The RQ values of gene MyD88mRNA: After the test of normality, P＜0.1, so the data disobeyed Gaussian distribution. The results of case groupand control group were expressed by median or quartile, and respectively were1.5005/1.445；1.7445/1.297. Statistical results of the expression of MyD88mRNA in patients with PR did not show statistical significance compared withthe normal controls (P=0.36＞0.05).4The RQ values of gene TRIF mRNA: After the test of normality, P＜0.1, so the data disobeyed Gaussian distribution. The results of case group andcontrol group were expressed by median or quartile, and respectively were3.0115/4.431；3.443/5.970. Statistical results of the expression of TRIFmRNA in patients with PR did not show statistical significance compared withthe normal controls (P=0.23＞0.05). Conclusisons:1The results of the expression of TLR4mRNA and TRIF mRNA inperipheral blood mononuclear cell of patients with PR both did not showstatistical significance compared with the normal controls (P＞0.05),whichindicate that the expression of TLR4mRNA andTRIF mRNA in the casegroup had no difference compared with the normal control.So we speculatethe pathogenesis of PR may not related with the infection of gram-negativebacteria.2The results of the expression of TLR9mRNA in peripheral bloodmononuclear cell of patients with PR had statistical significance comparedwith the normal controls (P<0.05), the expression of TLR9mRNA in the casegroup is lower than that in the normal control, the cause may be that thecertain DNA sequences in organism inhibits the expression of TLR9.Wespeculate TLR9may take part in the pathogenesis of PR.3The results of the expression of MyD88mRNA in peripheral bloodmononuclear cell of patients with PR did not show statistical significancecompared with the normal controls(P＞0.05), which indicate that theexpression of MyD88in the case group had no difference compared with thenormal control.The possible reason is that TLR9is not the only TLR in theupstream of the MyD88-dependent pathway and multiple TLRS utilize theMyD88-dependent pathway to iniate signaling.