脾虚与皮肤机械屏障功能关系的部分实验研究

【作者】 何丹；

【导师】 苗维纳； 林青；

【作者基本信息】 成都中医药大学 ， 中西医结合基础， 2008， 硕士

【摘要】 目的建立脾虚复合皮肤机械屏障功能障碍瘙痒小鼠模型,观察小鼠瘙痒行为、表皮神经酰胺含量和表皮厚度的变化,以及中药复方黄连解毒汤、参苓白术散对其的影响作用,为进一步从中医临床的证治规律出发,探讨健脾方药与特应性皮炎之间的相关性、脾虚与皮肤机械屏障功能障碍之间的关系提供实验依据,并为研究脾虚本质提供新的研究思路和方法。方法1.小鼠表皮脂质的抽提及表皮中神经酰胺含量测定方法研究抽提动物表皮脂质,采用薄层层析、BIO-RAD GelDoc凝胶成像系统采集图像、Quantity One软件分析测定神经酰胺含量,并对含量测定方法进行了方法学考察。2.皮肤机械屏障功能障碍瘙痒小鼠模型的考察用有机溶剂造成小鼠瘙痒模型,考察造模0天、3天、5天、7天小鼠瘙痒行为及表皮神经酰胺含量,以确定造模天数。3.黄连解毒汤对脾虚小鼠皮肤机械屏障功能障碍瘙痒的作用分别复制大黄、利血平所致小鼠脾虚模型,并在此基础上复制小鼠皮肤机械屏障功能障碍瘙痒模型,给予黄连解毒汤水煎液,记录动物两小时内瘙痒次数,测定神经酰胺含量,测量表皮厚度。4.黄连解毒汤对小鼠皮肤机械屏障功能的影响及参苓白术散对其的干预作用采用黄连解毒汤水煎液给予小鼠灌胃,并在此基础上复制小鼠皮肤机械屏障功能障碍瘙痒模型,再给予参苓白术散水煎液治疗,记录动物两小时内瘙痒次数,测定神经酰胺含量,测量表皮厚度。结果1.小鼠表皮脂质的抽提及表皮中神经酰胺含量测定方法研究方法学考察,得出:神经酰胺在0.416～2.08μg范围内,含量与密度积分值有良好线性关系,相关系数R=0.9953;同板精密度良好,RSD=1.09%;异板精密度良好,RSD=2.10%;对照品溶液与供试品溶液在2小时内显色稳定;重现性良好,RSD=3.03%;平均加样回收率为99.58%,RSD=1.46%;样品测定结果RSD=3.10%。2.皮肤机械屏障功能障碍瘙痒小鼠模型的考察造模5天动物与空白组动物比较,瘙痒行为最为明显,P＜0.001;表皮神经酰胺含量降低最为明显,P＜0.001。3.黄连解毒汤对脾虚小鼠皮肤机械屏障功能障碍瘙痒的影响作用(1)瘙痒行为与正常组动物比较,单纯瘙痒组动物瘙痒次数明显增加(P＜0.01);两种脾虚瘙痒组动物瘙痒次数少于单纯瘙痒组动物,利血平模型组P＜0.05;各给药组(黄连解毒汤)动物瘙痒次数均值小于相应脾虚组动物瘙痒次数均值。(2)神经酰胺含量①大黄所致脾虚,各组均值:空白组＞瘙痒组＞大黄组＞给药组,瘙痒组与空白组比较,P＜0.001,大黄组与瘙痒组、给药组与大黄组比较,P＜0.05。②利血平所致脾虚,各组均值:空白组＞瘙痒组＞利血平组＞给药组,瘙痒组与空白组、利血平组与瘙痒组比较,P＜0.001;给药组与利血平组比较,神经酰胺含量没有提高。(3)表皮厚度复制皮肤机械屏障功能障碍瘙痒模型后,与空白组比较,瘙痒组表皮明显增厚(P＜0.001)。大黄和利血平所致的脾虚也能够使表皮增厚,与空白组比较P＜0.05;与瘙痒组比较,大黄脾虚模型组轻度增厚。给药组动物表皮厚度略小于相应脾虚模型组,两组间比较,没有统计学意义。4.黄连解毒汤对小鼠皮肤机械屏障功能的影响及参苓白术散对其的干预作用(1)瘙痒行为单纯瘙痒组动物瘙痒次数明显增加,与正常组动物比较,P＜0.01;采用黄连解毒汤复制脾虚模型后,黄连解毒汤模型组动物瘙痒次数少于单纯瘙痒组动物(P＜0.05),给药组(参苓白术散)与黄连解毒汤模型组动物比较瘙痒次数减少(P＜0..05)。(2)神经酰胺含量各组均值:空白组＞瘙痒组＞给药组(参苓白术散)＞黄连解毒汤组,瘙痒组与空白组比较,P＜0.01,给药组(参苓白术散)与黄连解毒汤组比较,P＜0.05。(3)表皮厚度复制皮肤机械屏障功能障碍瘙痒模型后,单纯瘙痒组动物表皮增厚,与空白组比较,P＜0.001。黄连解毒汤能够加剧此种表皮增厚,黄连解毒汤模型组与瘙痒组比较,P＜0.05;参苓白术散可明显抑制此种表皮增厚,给药组动物表皮厚度明显低于黄连解毒汤模型组,P＜0.001。结论本次实验采用表皮神经酰胺含量测定、瘙痒行为观察、表皮厚度测量等作为检测指标,复制了皮肤机械屏障功能障碍瘙痒模型,与空白组比较,动物瘙痒行为明显、表皮神经酰胺含量下降,表皮增厚,说明皮肤机械屏障功能被破坏时表皮神经酰胺含量降低,而脾虚又会进一步降低表皮的神经酰胺含量,导致皮肤机械屏障功能障碍程度加重。在单纯瘙痒模型动物的基础上,利血平、大黄、黄连解毒汤三种方法所致脾虚模型小鼠的表皮神经酰胺含量降低,皮肤机械屏障功能进一步下降。健脾方药参苓白术散能够在一定程度上改善之。脾虚与皮肤机械屏障功能障碍之间有一定关联,在特应性皮炎的治疗中,健脾方药可能对于异位性皮炎患者皮肤屏障功能的修复发挥了重要的作用。更多还原

【Abstract】 PurposeThe splenic asthenia mouse models and pruritus mouse model induced by impairment of cutaneous permeability barrier function are copied in company with, The scratching behavior are observed,the content of ceramides is determined and the thickness of epidermis is determined.Observe the effect of traditional Chinese drug Huanglianjiedu Decoction,Senling Baishu Powder to the mouse models,Because of traditional Chinese medicine clinical regularity,we’d like to study the relationship between traditional Chinese drug for invigorating the spleen and atopic dermatitis, splenic asthenia and cutaneous permeability barrier function by the experimental study,and provide new research ideas and methods for the study about the hypostasis of splenic asthenia.Methods1.Epidermal lipids extraction of mouse and evaluating the method for determining the content of epidermal ceramidesEpidermal lipids of mouse are extracted by routine method,separated by thin-layer chromatography(TLC),shot by BIO-RAD GelDoc system.Epidermal ceramides is determined by Quantity One.And then evaluate this methodology.2.The study of pruritus mouse model induced by impairment of cutaneous permeability barrier function.To select the pruritus model for using,the rostral skin of mouse is defatted by organic solvent to make the pruritus model,the scratching behavior are observed and the content of ceramides is determined after making the pruritus model for 0day, 3days,5 days and 7 days.3.The effect of Huanglianjiedu Decoction to splenic asthenia models and pruritus mouse model induced by impairment of cutaneous permeability barrier function.The two splenic asthenia mouse models are induced by Rhubarb,Reserpine.And then pruritus model is copied on the two splenic asthenia mouse models. Huanglianjiedu Decoction is administrated.The scratching frequency is recorded, the content of ceramides is determined and the thickness of epidermis is determined.4.The effect of Huanglianjiedu Decoction to the mouse model of cutaneous permeability barrier function and the interference effect of ShenLing BaiZhu Decoction.The splenic asthenia mouse models are induced by Huanglianjiedu Decoction, And then pruritus model is copied on the splenic asthenia mouse models. ShenLingBaiZhu Decoction is administrated as treatment.The scratching frequency is recorded,the content of ceramides is determined and the thickness of epidermis is determined.Results1.Epidermal lipids extraction of mouse and evaluating the method for determining the content of epidermal ceramidesThe ceramides content and density score have good linear relationship when the ceramides content is between 0.416μg and 2.08μg,R=0.9953;The precision of the same plate is good,RSD=1.09%;The precision of different plates is good,RSD= 2.10%;The stability of control article solution and sample solution is good within 2h. The reproducibility is good,RSD=3.03%;The average recovery is 99.58%, RSD=1.46%.The RSD of simple determination is 3.10%.2.The study of pruritus mouse model induced by impairment of cutaneous permeability barrier functionThe scratching behavior is most significant after making the pruritus model for 5 days,P＜0.001,vs control group.The average contents of ceramides are lower after making the pruritus model for 5 days,P＜0.001,vs model for 0day.3.The effect of Hnanglianjiedu Decoction to splenic asthenia models and pruritus mouse model induced by impairment of cutaneous permeability barrier function. Scratching behavior Compare to the control group,The average scratching frequency of pruritus model mice increase in evidence(P＜0.01).The average scratching frequency of two splenic asthenia pruritus model mice is less than the scratching frequency of pruritus model mice.Reserpine group P＜0.05.The average scratching frequency of medication administration team is less than the average scratching frequency of two splenic asthenia pruritus model mice.Ceramide content①Splenic asthenia induced by Rhubarb,average:control group＞pruritus group＞Rhubarb group＞medication administration team.P＜0.001, pruritus group vs control group.P＜0.05,Rhubarb group vs pruritus group, medication administration team vs Rhubarb.②Splenic asthenia induced by Reserpine average:controlgroup＞pruritus group＞Reserpine group＞medication administration team.P＜0.001,pruritus group vs control group,Reserpine group vs pruritus.Ceramide content of medication administration team comparing with it of pruritus group is not improve.Thickness of epidermis After copying the pruritus mouse model,the epidermis thickens.P＜0.001,vs control group.Splenic asthenia induced by Rhubarb and Reserpine can aggravate the thickening of epidermal.P＜0.05,vs control group.The average epidermal thickness of Rhubarb model mouse is less than the one of pruritus model mouse.The average epidermal thickness of medication administration team mouse is less than the one of corresponding splenic asthenia model mouse, comparing with two group is not statistically significant.4.The effect of Huanglianjiedu Decoction to the mouse model of cutaneous permeability barrier function and the interference effect of ShenLing BaiZhu Decoction.Scratching behavior Compare to the control group,The average scratching frequency of pruritus model mice increase in evidence(P＜0.01).Splenic asthenia induced by Huanglianjiedu Decoction is copied,The average scratching frequency of the splenic asthenia pruritus model mice is less than the scratching frequency of pruritus model mice(P＜0.05).The average scratching frequency of medication administration team is less than the average scratching frequency of the splenic asthenia pruritus model(P＜0.05)Ceramide content average:control group＞pruritus group＞medication administration team＞Huanglianjiedu Decoction group.P＜0.001,pruritus group vs control group.P=0.052,Huanglianjiedu Decoction group vs pruritus group,P＜0.05,medication administration team vs Huanglianjiedu Decoction group.Thickness of epidermis After copying the pruritus mouse model,the epidermis thickens.P＜0.001,vs control group.Splenic asthenia induced by Huanglianjiedu Decoction can aggravate the thickening of epidermal.P＜0.05,vs pruritus group. ShenLingBaiZhu decoction can inhibit the thickening of epidermis significantly.The average epidermal thickness of medication administration team mouse is less than the one of Huanglianjiedu Decoction model mouse,P＜0.001.ConclusionsThe experimental use of epidermal ceramides content,scratching behavior, Thickness of epidermis as a testing target,The pruritus mouse model induced by impairment of cutaneous permeability barrier function is copied,compared with the control group,The scratching behavior is most significant,the content of epidermal ceramides is reducible,the epidermis thickens.According to the results,The epidermal ceramides content will decrease when the cutaneous permeability barrier function is destroyed.Splenic asthenia will aggravate the decrease of ceramides and the cutaneous permeability barrier disorders.The epidermal ceramides content of the three splenic asthenia models mice induced by Rhubarb,Reserpine and Huanglianjiedu Decoction is decreased.The three splenic asthenia models can make the poor cutaneous permeability barrier function poorer.ShenLingBaiZhu Decoction can improve it.更多还原