地槐消银颗粒治疗银屑病血热证的临床及实验研究

【作者】 谢知音；

【导师】 段行武；

【作者基本信息】 北京中医药大学 ， 中医外科学（专业学位）， 2019， 博士

【摘要】 背景:银屑病是临床上常见的慢性皮肤病,严重影响患者身心健康。中医药对本病的认识有着悠久的历史,在历史演进中逐渐形成了从血论治的观点,并得到越来越多医家的认可,血热证是从血论治中最为常见的证型。地槐消银方在临床上应用多年,取得了较好的疗效,制作成颗粒剂后更加方便。目前关于中药治疗银屑病的机制探讨较多,磷脂酰肌醇-3-羟激酶/蛋白激酶B(phosphatidylinositol 3-hydroxy kinase/protein kinase B,PI3K/Akt)信号转导途径是当前的研究热点,不但在肿瘤的发病中有重要作用,在银屑病发病中也有重要作用。前期研究显示地槐消银颗粒可能通过本通路发挥作用。本研究主要验证地槐消银颗粒治疗银屑病血热证的临床疗效,并基于PI3K/Akt通路在动物和细胞层面进行机制研究。方法:临床研究:采用随机对照的观察方法,纳入符合银屑病血热证的患者72例,随机分为观察组和对照组。记录患者的一般资料、皮损PASI评分、中医证候评分。观察组给予地槐消银颗粒口服,对照组给予百癣夏塔热片口服,两组均外用医用凡士林。观察期为56天,治疗前后记录PASI评分及中医证候评分。观察地槐消银颗粒和百癣夏塔热片对于银屑病血热证PASI评分、中医证候评分、疗效等的影响,并计算各自的临床有效率。动物实验研究:通过外涂咪喹莫特建立诱导的银屑病样BALB/c小鼠模型,以地槐消银颗粒灌胃(分高、中、低剂量组),酶联免疫吸附法(ELISA)检测小鼠外周血清中细胞因子IL-2、IL-6、IL-8、IFN-γ、TNF-α的含量,Western blotting及RT-PCR检测皮损组织PI3K、Akt蛋白及mRNA的表达水平,探讨地槐消银颗粒对银屑病样小鼠皮损、细胞因子、通路蛋白及基因等的影响。细胞实验研究:应用表皮生长因子(EGF)刺激人角质形成细胞(human adult low calcium temperature,HaCaT)24小时,添加PI3K/Akt信号通路的特异性阻滞剂LY294002干预1小时,制备实验所需的细胞模型。再以地槐消银颗粒(分高、中、低剂量组)分别干预经EGF刺激和加入抑制剂的HaCaT细胞模型,观察地槐消银颗粒对经EGF刺激后的及加入抑制剂的HaCaT细胞增殖、凋亡、细胞周期和IL-2、IL-6、IL-8、IFN-γ、TNF-α含量的影响,Western blotting及RT-PCR检测细胞PI3K、p-Akt、Akt蛋白及PI3K、Akt mRNA的表达水平。结果:临床研究:给予观察组地槐消银颗粒治疗后,最后完成观察的共66例患者,各33例。观察组在治疗8周后有效率为87.88%,高于对照组的66.67%;其治疗后PASI评分明显低于对照组,有显著统计学差异(P<0.01);在治疗后其中医证候改善优于对照组,有显著统计学差异(P<0.01)。动物实验研究:1.地槐消银颗粒可以改善通过咪喹莫特造模的银屑病样小鼠的皮损状况,组织病理学表现主要是改善银屑病表皮增生及真皮血管增生、减轻炎症反应等;2.地槐消银颗粒可以抑制小鼠血清中IL-2、IL-6、IL-8、IFN-γ、TNF-α的分泌;3.通过对小鼠组织中PI3K mRNA及蛋白的测定,高剂量组效果最为明显,可以下调mRNA基因影响蛋白的生成,其发挥作用是通过PI3K/Akt途径实现,而对Akt mRNA及Akt蛋白均无影响。细胞实验研究:1.地槐消银颗粒可以抑制HaCaT细胞的增殖,加入通道抑制剂LY294002后其作用减弱,其抑制细胞增殖的作用通过PI3K/Akt通路实现;2地槐消银颗粒可以使细胞分裂停滞在G0-G1或者S期而使细胞无法增殖,同时可以增加细胞晚期凋亡率,这些影响在加入通道抑制剂LY294002后受到明显抑制;3.通过Western blotting及RT-PCR检测细胞PI3K、p-Akt、Akt蛋白及PI3K、Akt mRNA的表达水平,得出地槐消银颗粒可以通过下调PI3K mRNA水平进而降低PI3K蛋白的水平,加入通道抑制剂后这些效应消失。但对Akt蛋白和Akt mRNA均无影响。说明地槐消银颗粒通过下调PI3K mRNA水平进而影响PI3K蛋白的水平而发挥其作用;4.地槐消银颗粒可以抑制细胞因子IL-2、IL-6、IL-8、IFN-γ、TNF-α的分泌。PI3K/Akt通路在其中可能发挥了重要作用,这种效应明显体现在IL-2、IL-8、IFN-γ、TNF-α的分泌上,而在抑制IL-6的分泌上可能通过多种途径发挥作用;5.地槐消银颗粒通过PI3K/Akt通路发生作用,对PI3K基因及蛋白均有影响,并通过这个通路影响细胞因子IL-2、IL-6、IL-8、IFN-γ、TNF-α的分泌水平而发挥治疗银屑病的作用。结论:1.地槐消银颗粒治疗银屑病血热证疗效肯定,能改善患者的皮损严重程度并能降低中医证候评分,未出现不良反应。2.地槐消银颗粒可以改善通过咪喹莫特造模的银屑病样小鼠的皮损状况,其机制是通过下调PI3K mRNA合成抑制蛋白生成、抑制细胞因子生成等实现的。3.地槐消银颗粒可以通过PI3K/Akt通路发挥抑制HaCaT细胞增殖作用,并可以下调PI3K mRNA、抑制PI3K及pAkt蛋白合成,同时可以抑制多种细胞因子的分泌。综上所述,地槐消银颗粒治疗银屑病血热证临床疗效确切,其具体作用机制可能是通过下调PI3KmRNA及蛋白合成,同时抑制多种细胞因子的分泌,从而发挥促进细胞凋亡、抑制增殖的作用。更多还原

【Abstract】 BackgroundPsoriasis,one common chronic skin disease,which seriously impacts the physical and mental health of patients.Traditional Chinese medicine(TCM),which has a long history,has great understanding of the disease,and in the evolution of history,traditional Chinese hemo-modulating therapy rise in response to the proper time and conditions,which has got acceptance by increasing doctors.Blood heat syndrome is the most common syndrome type in hemo-modulating therapy.Dihuai Xiaoyin prescription has been used in clinic for many years and good curative effect has been achieved,besides,it would be more convenient if we make the prescription into granules.Recently,there are numerous discussion about the mechanism of traditional Chinese medicine,and the pathway of phosphatidylinositol 3-hydroxykinase/protein kinase B(PI3K/Akt)signal transduction is the current research hotspot,which do not only plays an essential role in the pathogenesis of tumor,but also play a significant role in the pathogenesis of psoriasis.Previous studies have confirmed that Dihuai Xiaoyin granules might play a role through this pathway.The purpose of this study was to verify the clinical efficacy of Dihuai Xiaoyin granules in the treatment of psoriasis with blood heat syndrome,and to explore the mechanism at the animal and cellular level based on the PI3K/Akt pathway.MethodClinical study:We adopted the randomized controlled observation that 72 patients who suffer from psoriasis with blood heat syndrome were randomly divided into observation group and control group.General information,skin lesion PASI score and TCM syndrome score were recorded.The patients in the observation group were given Dihuai Xiaoyin granules orally,and the patients in the control group were given psoriasis xataret tablets orally,besides,both groups would be treated with topical medical vaseline.The observation period was 56 days.PASI score and TCM syndrome score were recorded before and after treatment.To observe the effects of Dihuai Xiaoyin granule and psoriasis xatahot tablets on psoriasis with blood heat syndrome,which means that,the change of PASI score,TCM syndrome score and efficacy,besides,calculating their respective clinical effective rates.Animal experiment research:To build psoriasis BALB/c mouse model by externally using imiquimod,after which,we treated the mouse model with Dihuai Xiaoyin granular((high,medium and low dose group)by intragastric administration.And then,the enzyme linked immunosorbent assay(ELISA)would be used to detect the content of peripheral blood cytokines,which contain IL-2,IL-6,IL-8,IFN-γ and TNF-α,besides,the method of Western blotting and RT-PCR would be used to detected the expression level of PI3K,Akt protein and mRNA in the lesion tissue.All we talked above were to discuss the influence of Dihuai Xiaoyin granule to the psoriatic mouse,the observation targets mainly contain the lesion tissue,cytokines,proteins related to pathways and gene,etc.Cell experimental study:Human adult low calcium temperature(HaCaT)was stimulated by epidermal growth factor(EGF)for 24 hours,and LY294002,specific blocker of PI3K/Akt signaling pathway,was added to intervene for 1 hour to prepare the cell model required for the experiment.And then,the HaCaT cell model,which had been stimulated by EGF and added with inhibitor,would be intervened by Dihuai Xiaoyin particles(high,medium and low dose group)respectively.After which,we observed the proliferation,apoptosis,cell cycle of the HaCaT cell model and the influence of the content of IL-2,IL-6,IL-8,IFN-γ,TNF-α,besides,Western blotting and RT-PCR would be used to detect the expression level of PI3K,p-Akt,Akt protein and PI3K,Akt mRNA.ResultClinical study:After 8 weeks of treatment,a total of 66 patients were observed at last,the effective rate of Dihuai Xiaoyin granule was87.88%,higher than66.67%of the control group.The PASI score after treatment was significantly lower than that of the control group,and the difference was statistically significant(P<0.01).The improvement of TCM syndrome was better than that in the control group,and the difference was statistically significant(P<0.01).Animal experiment research:1.Dihuai Xiaoyin granules could ameliorate the skin lesions of mouse with psoriasis induced by imiquimod.The main effect on pathology is to improve epidermal hyperplasia and dermal vascular hyperplasia in psoriasis and reduce inflammatory reaction;2.Dihuaixiao silver granule can inhibit the secretion of i1-2,il-6,il-8,IFN-,TNF-in serum of mice;3.We detected the PI3K mRNA and protein in mouse tissues,which drew a conclusion that the high-dose group was the most effective one.It could down-regulate the expression of mRNA gene and affect the production of protein,and this function was realized through the PI3K/Akt pathway,besides,it had no effect on Akt mRNA and Akt protein.Cell experimental study:1.Dihuai xiaoyin granule can inhibit the proliferation of HaCaT cells,and its effect could be weakened when LY294002 pathway was inhibited,and its inhibitory effect on cell proliferation is realized through the PI3K/Akt pathway;2.Dihuai xiaoyin granules can make cell division stagnating in G0-G1 or S period,which prevent cell proliferation,and can also increase the rate of late stage cell apoptosis.These effects were significantly inhibited after the addition of LY294002 the pathway inhibition;3.We detected the expression level of PI3K,p-Akt,Akt protein and PI3K,Akt mRNA through the method of Western blotting and RT-PCR,which drew a conclusion that Dihuai Xiaoyin granules could reduce the level of PI3K protein by down-regulating the level of PI3K mRNA,and these effects would disappear after the addition of pathway inhibitors,however,the level of Akt protein and Akt mRNA were stable.All we talked above demonstrate that Dihuai Xiaoyin granules could exert its effect by down-regulating the level of PI3K mRNA and thereby affecting the level of PI3K protein;4.Dihuai Xiaoyin granules could inhibit the secretion of IL-2,IL-6,IL-8,IFN-γ and TNF-α,in which,the PI3K/Akt pathway may play an essential role,and this effect was obviously reflected in the secretion of IL-2,IL-8,IFN-γ and TNF-α,while it may play a role in inhibiting the secretion of il-6through a variety of ways;5.Dihuai Xiaoyin granules act through the PI3K/Akt pathway,and have an effect on the PI3K gene and protein,through which,it affects the secretion level of a variety of cytokines such asIL-2,IL-6,IL-8,IFN-γ,TNF-α and play a role in the treatment of psoriasis.ConclusionThe efficacy of Dihuai Xiaoyin granule in the therapy of psoriasis with blood-heat syndrome is certain with no adverse reactions occurred,which could ameliorate the severity of skin lesions of patients and reduce the TCM syndrome score;2.Dihuai Xiaoyin granule could ameliorate the skin lesions of mouse with psoriasis that induced by imiquimod,and the mechanism is achieved by down-regulating PI3K mRNA synthesis to inhibit protein production and cytokine production.3.Dihuai Xiaoyin granule could inhibit the proliferation of HaCaT cells through the PI3K/Akt pathway,it could also down-regulate the expression of PI3K mRNA,inhibit the synthesis of PI3K and pAkt proteins,and inhibit the secretion of a variety of cytokines.To sum up,Dihuai Xiaoyin granule has a definite clinical effect in the treatment of psoriasis with blood heat syndrome,and its specific mechanism may be to promote apoptosis and inhibit proliferation by down-regulating PI3KmRNA and protein synthesis and inhibiting the secretion of a variety of cytokines.更多还原