【作者】 张广丽；

【导师】 许华；

【作者基本信息】 广州中医药大学 ， 中医儿科学， 2011， 博士

【摘要】 目的：1.探讨儿童过敏性紫癜(HSP)与Toll样受体通路的相关性,进一步分析过敏性紫癜的发病机制及中药消癜汤可能作用靶点,为临床中医药防治HSP提供科学依据。2.分析儿童过敏性紫癜发病特征及消癜汤治疗过敏性紫癜的临床疗效。方法：1.采用前瞻性自身前后对照试验,观察过敏性紫癜患儿消癜汤治疗前后相关免疫指标的变化,并与健康对照组比较。选择2009年9月至2011年3月广州中医药大学第一附属医院儿科门诊及住院的符合入选标准的初诊过敏性紫癜病例20例,为治疗组,并设立健康对照组15例。其中治疗组男12例,女8例,平均年龄(7.1±2.0)岁；对照组男9例,女6例,平均年龄(6.9±1.1)岁。治疗组口服消癜汤(药物由广州中医药大学第一附属医院中药房及制剂室煎煮后提供),每日1剂,病情稳定后可予以隔日1剂,以4周为一疗程；健康对照组未给予药物治疗。采用荧光定量PCR检测HSP患儿治疗前后及健康对照组儿童外周血单核细胞TLR-2、TLR-4、NF-κB、IL-6mRNA表达水平及血沉、CPR、C3、C4值等相关指标的变化。2.采用回顾性队列研究的方法,根据360例过敏性紫癜患儿是否使用中药消癜汤分为消癜汤组(160例)和对照组(200例),其中,对照组根据主要治则治法分为对照1组(口服健脾益气、补肾等温补中药为主,共80例)和对照2组(口服疏风清热等祛邪中药为主,共120例),观察3组患儿一般情况分布(包括姓名、性别、年龄、发病季节、发病诱因、病程、证型分布等)、肾损害情况(血尿、蛋白尿分布及消退时间)、总疗效、临床症状消退时间、住院时间及复发率等,并进行统计分析。结果：(一)临床研究一1.HSP患儿治疗前后TLR2mRNA、NF-κBmRNA及IL-6 mRNA的变化HSP患儿治疗前外周血单核细胞TLR2mRNA、NF-κBmRNA及细胞因子IL-6mRNA表达明显高于与健康对照组(P<0.01或P<0.05)；口服消癜汤治疗一个疗程后,患儿外周血TLR2mRNA、NF-κBmRNA及细胞因子IL-6 mRNA表达明显下降,与治疗前比较有显著性差异(P<0.01或P<0.05)。2.HSP患儿治疗前后TLR2mRNA与NF-κBmRNA、IL-6 mRNA、血沉、CRP、C3、C4相关性分析HSP患儿治疗前单核细胞TLR2mRNA表达与NF-κBmRNA、IL-6 mRNA存在显著相关性(P<0.01)。口服消癜汤治疗后TLR2mRNA、NF-κBmRNA及细胞因子IL-6mRNA表达与健康对照组比较无显著性差异(P>0.05)。消癜汤治疗前后HSP患儿外周血单核细胞表面TLR2mRNA表达与血沉、C3、C4之间无相关性(P>0.05),与CRP显著相关(P<0.05或P<0.01)。3.HSP患儿治疗前后TLR4表达变化及相关性分析HSP患儿治疗前后外周血单核细胞TLR4 mRNA表达与健康对照组比较,无显著性差异(P>0.05)；口服消癜汤治疗一个疗程后,患儿TLR4 mRNA表达有所下降,但无统计学意义(P>0.05)。过敏性紫癜患儿治疗前后组单核细胞TLR4 mRNA表达与NF-κB、IL-6及血沉、CRP、C3、C4均无相关性。(二)临床研究二1.过敏性紫癜患儿发病情况360例过敏性紫癜患儿性别分布比较,男女比例为1.69：1,男性发病率高于女性。发病年龄主要为学龄期和学龄前期,以4～12岁学龄期儿童发病人数最多,占85.2%。秋冬季节发病患儿总数较春夏季节发病患儿总数多,春季和夏季发病率相近。患儿发病诱因由感染因素引起者占48.1%,以上呼吸道感染者居多,其次为消化道感染；由食物过敏诱发者占15.6%,其中以食用海鲜后发病者居多；由服用药物诱发者占13.1%；其他诱因引起者占23.3%,其他诱因包括接种疫苗、虫蚊叮咬、接触过敏物及外伤等。证型分布、病程方面三组相比无显著性差异(P>0.05)2.临床总疗效和症状改善情况临床总疗效方面,消癜汤组总有效率为85%,对照1组总有效率为73.8%,对照2组总有效率为75%。消癜汤明显优于对照组1组及对照2组(P<0.05),对照1组与对照2组比较无明显差异(P>0.05)症状改善方面,消癜汤组皮肤紫癜消退时间为(5.42±3.51)天,腹痛消退时间为(4.36±1.61)天,均明显低于对照1组和对照2组(对照1组分别为12.23±3.21天；8.13±1.05天,对照2组分别为10.16±3.21天；7.05±3.92天),具有显著性差异(P<0.05)。但在关节疼痛缓解时间方面与对照1组、对照2组比较,消癜汤组无显著性差异(P>0.05)；对照1组、对照2组之间相比无显著性差异(P>0.05)3.消癜汤对过敏性紫癜患儿肾损害的防治作用从治疗后出现肾损害情况分析,消癜汤组治疗4周、3个月、1年出现肾损害者比例分别为：5.62%、9.38%和5.63%。对照1组治疗4周、3个月、1年出现肾损害者比例分别为：20.00%、15.00%和18.75%。对照2组治疗4周、3个月、1年出现肾损害者比例分别为：15.83%、13.33%和12.50%。消癜汤组治疗后出现肾损害例数明显少于对照组1组和对照2组(P<0.05),而对照2组出现肾损害情况少于对照1组,二者有显著性差异(P<0.05)。从改善血尿、蛋白尿有效率比较,消癜汤组总有效率最高,其次为对照2组,对照1组最低。消癜汤组总有效率为81.7%,对照1组总有效率为60.7%,对照2组总有效率为77.5%。消癜汤组明显优于对照1组,有显著性差异(P<0.05)。对照2组优于对照1组(P<0.05)。消癜汤组总有效率高于对照2组,但无统计学意义(P>0.05)。从血尿、蛋白尿消退时间比较,消癜汤组血尿、蛋白尿消退时间较对照1组、对照2组明显缩短,差异有统计学意义(P<0.01)。对照2组血尿、蛋白尿消退时间较对照1组明显缩短(P<0.05或P<0.01)。从肾损害出现时间比较,消癜汤组肾损害出现的时间平均为(76±15)d,对照1组平均为(35±13)d,对照2组平均为(55±13)d,消癜汤组出现肾损害时间明显迟于对照组1组及对照2组,而对照2组出现肾损害时间迟于对照1组,差异均有显著性差异(P<0.05)4.住院时间和复发次率情况住院时间方面,消癜汤组、对照1组和对照2组住院<1周者分别占25.6%,20%和19.2%。住院1～2周者分别占41.3%,26.3%和26.7%。住院2～3周者分别占22%,36.2%和38.3%,住院>3周者分别占11.3%,17.5%和15.8%。消癜汤组住院时间明显少于对照组1及对对照2组,差异有统计学意义(P<0.01),但对照1组和对照2组住院时间比较,无显著性差异。复发率方面,消癜汤组占17%,对照1组占45%,对照2组占31.7%。消癜汤组复发率明显低于对照1组和对照2组(P<0.01)。对照2组复发率明显低于对照1组(P<0.01)。结论：1.本次研究结果提示消癜汤治疗前Toll样受体通路异常活化的有TLR2mRNA、NF-κBmRNA、IL-6mRNA。消癜汤治疗后TLR2mRNA, NF-κBmRNA、IL-6mRNA的高表达均恢复正常水平,且与健康对照组无显著性差异。而消癜汤治疗前后TLR4mRNA表达无明显变化,与健康对照组无明显差异。提示TLR2-NF-κB通路可能参与了过敏性紫癜的发生、发展,TLR2可能通过激活TLRs信号通路和一系列蛋白质级联反应激活转录因子NF-κB表达,释放炎症因子IL-6,参与了HSP的发病进程。消癜汤可能可以使TLR2表达下调,阻断或减弱NF-κB信号传导通路中的某些细胞因子表达,下调炎症因子IL-6,对炎症通路及炎症介质进行有效调控,起到治疗或减轻过敏性紫癜的效果。TLR4表达可能与过敏性紫癜发病机制无关。2.消癜汤治疗前后过敏性紫癜患儿血清TLR2与CRP水平呈显著正相关,推测TLR2反应了炎症的变化,可作为敏感的炎症指标。3.小儿过敏性紫癜男性发病率高于女性。发病年龄主要为学龄期和学龄前期,以4～12岁学龄期儿童发病人数最多。发病季节以秋冬季最为多见。发病诱因由感染因素引起者居多,其它诱因有食物过敏、服用药物、接种疫苗、虫蚊叮咬、接触过敏物及外伤等。4.消癜汤能有效减少过敏性紫癜患儿肾损害发生率,延缓肾损害出现的时间,治疗血尿、蛋白尿有较好的疗效,能缩短血尿、蛋白尿消退时间。同时,消癜汤能有效减少过敏性紫癜患儿住院时间及降低复发率,提高总疗效和改善皮肤紫癜及腹痛等症状。5.过敏性紫癜患儿急性期口服健脾益气、补肾等温补中药为主的患儿,其出现肾损害情况明显高于消癜汤组和口服疏风清热等祛邪中药为主的患儿,同时,其血尿、蛋白尿消退时间明显长于消癜汤组和口服疏风清热等祛邪中药为主的患儿；而且治疗后随访1年内复发率明显增高。因此,儿童过敏性紫癜急性期要慎用温补法。更多还原

【Abstract】 Objective:1.This research investigates the correlation between Henoch Schonlein purpura (HSP) in children and toll-like receptor pathway, further analyzes the pathogenesis of HSP and the possible targets of Xiao Dian Decoction, provides scientific basis for the clinical prevention and treatment of HSP by traditional Chinese medicine.2. To analvze the characteristics of HSP in children and observe the clinical effect of Xiao Dian Decoction in treating HSPMethods:1.A prospective before and after self control study is conducted,to observed the changes in immune parameters before and after Xiao Dian Decoction treatment, and compared with the healthy control group. Newly diagnosed 20 cases of HSP meeting the inclusion criteria in the outpatient and inpatient pediatric department of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine from september 2009 to March 2011, are as treatment group;and 15 cases of healthy kids are as healthy control group. There are 12 males and 8 females in treatment group, and their average age is (7.1±2.0) years old. There are 9 males and 6 females in control group, and their average age is (6.9±1.1) years old. Children in treatment group take Xiao Dian Decoction(Herbs are provided by pharmacy and preparation room of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine),is taken by the HSP children in the treatment group one dose per day, and one dose every two days after stable condition,4 weeks as a course of treatment. Children in healthy control group are not given any drug treatment. Fluorescent quantitative PCR is taken to detect the expression of TLR-2 mRNA, TLR-4 mRNA, NF-κB mRNA, IL-6mRNA in peripheral blood mononuclear cells and the value of ESR, C-reactive protein(CRP), C3, C4 in blood in treatment group before and after the treatment and those indicators are also detected from children in control group.2. A retrospective cohort study is conducted,360 cases of children with HSP are classified into Xiao Dian Decoction group(160 cases) and control group(200 cases) depending on whether taking Xiao Dian Decoction. Control group is classified into ontrol group 1 (80 cases) and control group 2(120 cases) depending on the main therapeutic principle. The warming and reinforcing herbs that can invigorating spleen and kidney are mainly taken in ontrol group 1, on the contrary, herbs eliminating pathogen are mainly taken in ontrol group2. The indicators such as the basic status (such as name, gender, incidence seasons, predisposing factors.), renal damage(such as hematuria, proteinuria, and their regression time),the total effect, the regression time of clinical symptoms, hospital stay and recurrent rate in one year, etc are observed in the three groups.Statistical analysis is performed after observation.Results:Clinical Research 11. Expression of TLR2mRNA, NF-κBmRNA and IL-6mRNA before and after HSP treatmentExpression of TLR2mRNA, NF-κBmRNA and IL-6mRNA in peripheral blood mononuclear of children with HSP are higher than healthy control group before treatment(P<0.01 or P<0.05). But the expression of TLR2mRNA, NF-κBmRNA and IL-6mRNA in peripheral blood monocytes decrease obviously after taking Xiao Dian Decoction for a course of treatment, there is significant difference compared with pre-treatment (P<0.01 or P<0.05).2. Correlation analysis between TLR2mRNA and NF-κBmRNA、IL-6 mRNA、ESR、CRP、C3、C4 before and after HSP treatmentSignificant correlation is found between expression of TLR2mRNA and NF-κBmRNA、IL-6 mRNA、ESR、CRP、C3、C4 before treatment in the monocytes of children with HSP (P<0.01).Compared with healthy control group, there is not significant difference in the expression of TLR2mRNA、NF-κBmRNA and IL-6 mRNA after taking Xiao Dian Decoction for a course of treatment (P>0. 05). There is not significant correlation between expression of TLR2mRNA in monocytes and ESR、C3、C4 before and after treatment (P>0.05). But There is significant correlation between expression of TLR2mRNA in monocytes and CRP before and after treatment(P<0.05 or P<0.01).3. Expression of TLR4mRNA and correlation analysis before and after HSP treatmentThere is not significant difference for TLR4mRNA expression in peripheral blood monocytes before and after treatment in children with HSP compared with healthy control group(P>0.05). Expression of TLR4mRNA decrease after taking Xiao Dian Decoction for a course of treatment in children with HSP, but there is not statistically significant (P>0.05). There is not significant correlation between expression of TLR4mRNA in monocytes and NF-κB, IL-6, ESR, CRP, C3, C4 before and after HSP treatment.Clinical Research 21. The clinical features of HSP in childrenComparison of gender distribution of 360 cases of HSP in children is that male to female ratio is 1.69. Incidence of male is higher than female.The age of disease occurrence is mainly distributed in school age and preschool. The largest incidence is occurred during school-age 4 to 12 years old, accounting for 85.2%.Total incidence of HSP in fall and winter seasons is more than that in spring and summer, Incidence of HSP in spring is close in summer. Infection factors account for about 48.1%in all incentives of disease, in which upper respiratory tract infection is most common, and gastrointestinal infection is second. Food allergy accounts for 15.6%, in which seafood is the most common. Oral drugs account for 13.1%. Other incentives account for 23.3%, including vaccination, mosquito insect bites, exposure to allergens and trauma etc.In terms of card type distribution and duration of HSP in children, Xiao Dian Decoction has no significant difference comparing with control group 1 and control group 2 (P>0.05)2. Overall clinical efficacy and symptoms improvement comparisonIn terms of overall clinical efficacy, the total effective rate of Xiao Dian Decoction is 85%, control group 1 is 73.8%, control group 2 is 75%. The total effective rate of Xiao Dian Decoction is better than that of control group 1 and control group 2(P<0.05), There is not significant different between control group 1 and control group 2 in the overall clinical efficacy comparison (P>0.05)In terms of symptoms improvement, The regression time of skin purpura in Xiao Dian Decoction is (5.42±3.51) days, and the regression time of abdominal pain in Xiao Dian Decoction is (4.36±1.61) days, which are significantly lower than control group 1 and control group 2 (P<0.05). But In terms of joint pain, Xiao Dian Decoction has no significant difference comparing with control group 1 and control group 2 (P>0.05)3. The function of prevention and treatment of Xiao Dian Decoction for renal damage caused by HSPOn analysis of renal damage appearing after treatment, the respective rate of occurrence of renal damage in 4 weeks,3 months and 1 year in Xiao Dian Decoction group respectively is 5.62%、9.38% and 5.63%, those in control group 1 respectively is 20.00%、15.00% and 18.75%, those in control group 2 respectively is 15.83%、13.33% and 12.50%. The number of renal damage after treatment in Xiao Dian Decoction group is significantly less than that in control group 1 and control group 2 (P<0.05). The number of renal damage in control group 2 is significantly less than that in control group 1, there is a significant difference between control group 1 and control group 2 (P<0.05)On comparison of effective rate of hematuria and proteinuria improvement, the totally effective rate of Xiao Dian Decoction group is highest, that is 81.7%, and the second is control group 2, the rate is 77.5%, control group 1 is lowest, that is 60.7%. The totallv effective rate of Xiao Dian Decoction is better than control group 1, there is a significant difference (P<0.05) The totally effective rate of control group 2 is better than control group 1 (P<0.05). The totally effective rate of Xiao Dian Decoction is higher than control group 1, but there is not significant different (P>0.05)On comparison of the regression time of hematuria and proteinuria, Xiao Dian Decoction group is shorter than control group 1 and control group 2, there are significant differences (P<0.01).The regression time of hematuria and proteinuria in control group 2 is shorter than control group 1 (P<0.05 or P<0.01)On comparison of the time renal damage occurs, The average time of renal damage occurrence in Xiao Dian Decoction group is (76±15) days, (35±13) days in control group 1, and (55±13) days in control group 2. The average time of renal damage occurrence in Xiao Dian Decoction group is significant later than control group 1 and control group 2 (P<0.05), and the time in control group 2 is later than control group 1, there is significant different (P<0.05)4. Hospital stay and recurrence rateThe proportion of hospital stay less than one week in Xiao Dian Decoction group account for 25.6%,1 to 2 weeks account for 41.3%,2 to 3 weeks account for 22%, more than 3 weeks account for 11.3%. The proportion of hospital stay less than one week in control group 1 account for 20%,1 to 2 weeks account for 26.3%,2 to 3 weeks account for 36.2%, more than 3 weeks account for 17.5%. The proportion of hospital stay less than one week in control group 2 account for 19.2%,1 to 2 weeks account for 26.7%,2 to 3 weeks account for 38.3%, more than 3 weeks account for 15.8%. The hospital stay in Xiao Dian Decoction group is less than control group 1 and control group 2 (P<0.01).There was no significant difference between control group 1 and control group 2 on hospital stay comparison.On comparison of recurrence rate, the recurrence rate in Xiao Dian Decoction group is 17%, the recurrence rate in control 1 is 45%, the recurrence rate in control 2 is 31.7%. The recurrence rate in Xiao Dian Decoction group is less than control group 1 and control group 2 (P<0.01). The recurrence rate in control group 2 is less than control group 1 (P<0.01)Conclusion1. The results of study indicate that the receptor pathways of TLR2mRNA, NF-κBmRNA and IL-6 mRNA are abnormally activated before the treatment of Xiao Dian Decoction, and the high expression of TLR2mRNA, NF-κBmRNA and IL-6 mRNA decrease to nornal level after the treatment of Xiao Dian Decoction, which is not significant different compared with healthy control group.The result indicates that the pathway of TLR2 to NF-κB may participate in the pathogenesis of HSP. TLR2 may participate HSP process by activating TLRs signal pathway and series of cascade of protein reactions to activate transcription factor NF-κB expression and release inflammatory factor IL-6.Xiao Dian Decoction may block or weaken some cytokine expression in NF-κB signal transduction pathway, downregulate inflammatory factor IL-6, effectively control inflammatory pathway and inflammatory mediators, to reach the effect of treating HSP. TLR4 expression may be unrelated to the pathogenesis of HSP. 2. TLR2 expression is significantly and positively correlated with CRP levels in the serum of children with HSP before and after treatment of Xiao Dian Decoction. So it is guessed that TLR2 expression reflects the changes of inflammatorv reaction and is taken as sensitive indicators of inflammation3. The incidence of HSP in boys was higher than that in girls. Onset age is mainly focused on school age and preschool. Autumn and winter are the most common incidence seasons.Infection factors account for main incentives of disease and other incentives are food allergy,oral drugs, vaccination, mosquito insect bites, exposure to allergens and trauma etc.4. Xiao Dian Decoction can effectively reduce the incidence of renal damage in children with HSP and delay the time of renal damage occurence. It has a better therapeutic effect in treating hematuria and proteinuria, Which can shorten the regression time of hematuria and proteinuria. Xiao Dian Decoction can also effectively reduce the hospital stay and the recurrence rate.It can improve the total effect and improve symptoms such as skin purpura and abdominal pain.5. In the acute stage of HSP, the incidence of renal damage in the children with HSP who mainly take warning and reinforcing herbs such as strengthening spleen and replenishing qi and reinforcing kidney herbs, is higher than that in the children with HSP who mainlv take Xiao Dian Decoction or eliminating-pathogen herbs such as dispelling wind and clearing heat herbs, at the same time, the regression time of hematuria and proteinuria in the former is significantly longer than that in the latter. The recurrence rate in the former is higher than that in the latter after treatment in one year.So.it is considered that warning and reinforcing method is cautious in the treatment of acute HSP in children.更多还原