慢性乙肝肝胆湿热证Hsp70诱导缺陷的研究及清肝利湿汤干预
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摘要
目的：
1.通过小样本随机对照试验观察中医清肝利湿法治疗肝胆湿热型慢性乙型肝炎的疗效和安全性。
2.探索热应激诱导肝胆湿热型慢性乙型肝炎热休克蛋白70表达及细胞因子表达的水平,为了解慢性乙型肝炎肝胆湿热证的生物学本质提供资料。
方法：
1.纳入符合要求的受试者进行随机分组,试验组予中药清肝利湿汤加拉米夫定片,对照组予复方甘草酸苷片加拉米夫定片治疗,清肝利湿汤和复方甘草酸苷片疗程为4周,观察两组受试者的各单独症状指标和整体症状感受视觉模拟评分、ALT水平、病毒标志物及HBV DNA定量值的变化,并监测不良反应。
2.外周血液标本来自于肝胆湿热型、肝郁脾虚型慢性乙肝患者和正常人各9例。分离外周血单个核细胞(PBMC),进行或不进行热应激处理,使用westernblot方法检测热应激和免热应激处理的PBMC中Hsp70的表达,使用酶联免疫吸附法检测PBMC培养上清液中TNF-α、IFN-γ和IL-4的表达量。
结果：
1.共纳入75名受试者,其中试验组34例,对照组41例,采用意向性治疗原则。治疗后两组各单独症状和整体症状感受视觉模拟评分和ALT水平均出现下降,试验组在治疗第2,3,4周时的整体症状感受视觉模拟评分显著低于对照组(P<0.05),在治疗第3周时ALT水平显著低于对照组(P<0.05)。与治疗前比较,继续拉米夫定治疗12周时试验组HBV DNA定量改变值显著高于对照组(P<0.05)。两组患者均未出现严重不良反应。
2.免热处理的3组标本Hsp70及3种细胞因子在培养24h后表达水平相似,经热处理后,肝胆湿热组PBMC中Hsp70表达显著低于肝郁脾虚组和正常组。经热处理的肝胆湿热组PBMC培养上清液中TNF-α和IFN-γ表达水平显著高于同等条件下的肝郁脾虚组和正常组,而IL-4的表达则显著低于这2组。
结论：
1.中医清肝利湿法对肝胆湿热型慢性乙肝患者在肝炎症状、肝功能方面有改善作用,其抗病毒效应需进一步评估。
2.肝胆湿热型慢性乙型肝炎具有明显的Hsp70诱导缺陷,并存在Th1/Th2细胞因子表达失衡,这可能是慢性乙型肝炎肝胆湿热证具有特征性的免疫学和生物学本质。
Aim:
1. To evaluate the effectiveness and safety of eliminating-the-moist-heat-in-liver (Qinggan Lishi) therapy of traditional Chinese medicine (TCM) for the treatment of moist-heat-in-liver-and-gall-bladder-predominant (Gandan-Shire-predominant) chronic hepatitis B, a randomized controlled trial of small-size sample was designed and conducted.
2. The aim of the experiments in the 2nd part is to explore the heat-induced heat shock protein 70 expression and the alteration of cytokines level following heat treatment, thus to provide new information about the biological essence of moist-heat-in-liver-and-gall-bladder-predominant (Gandan-Shire-predominant) chronic hepatitis B(CHB).
Methods:
1. Patients who met the designed criteria were included and allocated randomly into 2 groups. Qinggan Lishi decoction, a traditional Chinese herb formula which was prescribed according to the principle of eliminating-the-moist-heat-in-liver therapy, was given to the experimental group combined with Lamivudine tablets. Compound Glycyrrhizin together with Lamivudine tablets were given to the controls. The administration duration of Qinggan Lishi decoction and Compound Glycyrrhizin tablets was 4 weeks. Visual analogue scores (VAS) of 7 independent symptoms and the general symptomatic VAS, ALT value, serum viral markers and HBV DNA quantitative value were recorded and compared between the 2 groups. Adverse events were monitored in the whole process of trial.
2. Blood samples were obtained from 9 patients with Gandan-Shire-predominant CHB,9 patients with stagnancy-of-liver-Qi-and-deficiency-of-spleen-Qi-predominant (Ganyu-Pixu-predominant) CHB, and 9 normal controls, respectively, and were allocated into 3 groups by the syndrome type of traditional Chinese medicine (TCM). Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples and were given heat treatment. Some were left untreated in order to compare with those treated. Western-blot assay were performed to test the level of heat shock protein 70 in PBMCs and ELISA assay to test the expression of TNF-α, IFN-γand IL-4 in the supernatant of PBMCs.
Results:
1. Seventy-five patients,34 for the experimental and 41 for the controlled, were included. One patient of experimental group was ruled out by the intention-to-treat analysis. The VAS on each single independent symptom and general symptomatic VAS and ALT value of both groups were all decreased after the initiation of treatment. At the 2nd,3rd, and 4th week, the experimental had significantly lower VAS on each of the 7 independent symptoms and general symptomatic VAS than the controlled had (P<0.05). Moreover, the ALT value of the experimental group was significantly lower than that of the controlled (P<0.05). At the 12th week of continuous administration of Lamivudine tablets, the differential value of HBV DNA quantity of experimental group was significantly higher than that of control (P<0.05). No severe adverse events occurred during the whole trial.
2. The untreated PBMCs from 3 groups had similar expression capability of Hsp70 and 3 cytokines following 24h of normal incubation. However, the situation was different on the PBMCs treated by incubation at 42.5℃for 16h. Gandan Shire group showed significantly less expression of Hsp70 than the other 2 groups whereas had significantly higher level of TNF-αand IFN-y in the supernatant of PBMCs with heat treatment. Gandan Shire group also was tested significantly lower expression of IL-4 in the supernatant compared with that of the other 2 groups.
Conclusions:
1. The eliminating-the-moist-heat-in-liver (Qinggan Lishi) therapy of traditional Chinese medicine may have beneficial effect on improving the symptoms due to hepatitis and liver function for a particular group of patients with chronic hepatitis B who are supposed to be discriminated and identified in the TCM manner. However, the anti-viral effect remains to be verified by further clinical trials.
2. The Gandan-Shire-predominant CHB shows evident deficiency of heat-inducibility of Hsp70 and imbalance of Thl/Th2 cytokine expression, which may convey specific characteristics of CHB with traditional Chinese medical Gandan Shire syndrome in terms of immunological and biological essence.
1.通过小样本随机对照试验观察中医清肝利湿法治疗肝胆湿热型慢性乙型肝炎的疗效和安全性。
2.探索热应激诱导肝胆湿热型慢性乙型肝炎热休克蛋白70表达及细胞因子表达的水平,为了解慢性乙型肝炎肝胆湿热证的生物学本质提供资料。
方法：
1.纳入符合要求的受试者进行随机分组,试验组予中药清肝利湿汤加拉米夫定片,对照组予复方甘草酸苷片加拉米夫定片治疗,清肝利湿汤和复方甘草酸苷片疗程为4周,观察两组受试者的各单独症状指标和整体症状感受视觉模拟评分、ALT水平、病毒标志物及HBV DNA定量值的变化,并监测不良反应。
2.外周血液标本来自于肝胆湿热型、肝郁脾虚型慢性乙肝患者和正常人各9例。分离外周血单个核细胞(PBMC),进行或不进行热应激处理,使用westernblot方法检测热应激和免热应激处理的PBMC中Hsp70的表达,使用酶联免疫吸附法检测PBMC培养上清液中TNF-α、IFN-γ和IL-4的表达量。
结果：
1.共纳入75名受试者,其中试验组34例,对照组41例,采用意向性治疗原则。治疗后两组各单独症状和整体症状感受视觉模拟评分和ALT水平均出现下降,试验组在治疗第2,3,4周时的整体症状感受视觉模拟评分显著低于对照组(P<0.05),在治疗第3周时ALT水平显著低于对照组(P<0.05)。与治疗前比较,继续拉米夫定治疗12周时试验组HBV DNA定量改变值显著高于对照组(P<0.05)。两组患者均未出现严重不良反应。
2.免热处理的3组标本Hsp70及3种细胞因子在培养24h后表达水平相似,经热处理后,肝胆湿热组PBMC中Hsp70表达显著低于肝郁脾虚组和正常组。经热处理的肝胆湿热组PBMC培养上清液中TNF-α和IFN-γ表达水平显著高于同等条件下的肝郁脾虚组和正常组,而IL-4的表达则显著低于这2组。
结论：
1.中医清肝利湿法对肝胆湿热型慢性乙肝患者在肝炎症状、肝功能方面有改善作用,其抗病毒效应需进一步评估。
2.肝胆湿热型慢性乙型肝炎具有明显的Hsp70诱导缺陷,并存在Th1/Th2细胞因子表达失衡,这可能是慢性乙型肝炎肝胆湿热证具有特征性的免疫学和生物学本质。
Aim:
1. To evaluate the effectiveness and safety of eliminating-the-moist-heat-in-liver (Qinggan Lishi) therapy of traditional Chinese medicine (TCM) for the treatment of moist-heat-in-liver-and-gall-bladder-predominant (Gandan-Shire-predominant) chronic hepatitis B, a randomized controlled trial of small-size sample was designed and conducted.
2. The aim of the experiments in the 2nd part is to explore the heat-induced heat shock protein 70 expression and the alteration of cytokines level following heat treatment, thus to provide new information about the biological essence of moist-heat-in-liver-and-gall-bladder-predominant (Gandan-Shire-predominant) chronic hepatitis B(CHB).
Methods:
1. Patients who met the designed criteria were included and allocated randomly into 2 groups. Qinggan Lishi decoction, a traditional Chinese herb formula which was prescribed according to the principle of eliminating-the-moist-heat-in-liver therapy, was given to the experimental group combined with Lamivudine tablets. Compound Glycyrrhizin together with Lamivudine tablets were given to the controls. The administration duration of Qinggan Lishi decoction and Compound Glycyrrhizin tablets was 4 weeks. Visual analogue scores (VAS) of 7 independent symptoms and the general symptomatic VAS, ALT value, serum viral markers and HBV DNA quantitative value were recorded and compared between the 2 groups. Adverse events were monitored in the whole process of trial.
2. Blood samples were obtained from 9 patients with Gandan-Shire-predominant CHB,9 patients with stagnancy-of-liver-Qi-and-deficiency-of-spleen-Qi-predominant (Ganyu-Pixu-predominant) CHB, and 9 normal controls, respectively, and were allocated into 3 groups by the syndrome type of traditional Chinese medicine (TCM). Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples and were given heat treatment. Some were left untreated in order to compare with those treated. Western-blot assay were performed to test the level of heat shock protein 70 in PBMCs and ELISA assay to test the expression of TNF-α, IFN-γand IL-4 in the supernatant of PBMCs.
Results:
1. Seventy-five patients,34 for the experimental and 41 for the controlled, were included. One patient of experimental group was ruled out by the intention-to-treat analysis. The VAS on each single independent symptom and general symptomatic VAS and ALT value of both groups were all decreased after the initiation of treatment. At the 2nd,3rd, and 4th week, the experimental had significantly lower VAS on each of the 7 independent symptoms and general symptomatic VAS than the controlled had (P<0.05). Moreover, the ALT value of the experimental group was significantly lower than that of the controlled (P<0.05). At the 12th week of continuous administration of Lamivudine tablets, the differential value of HBV DNA quantity of experimental group was significantly higher than that of control (P<0.05). No severe adverse events occurred during the whole trial.
2. The untreated PBMCs from 3 groups had similar expression capability of Hsp70 and 3 cytokines following 24h of normal incubation. However, the situation was different on the PBMCs treated by incubation at 42.5℃for 16h. Gandan Shire group showed significantly less expression of Hsp70 than the other 2 groups whereas had significantly higher level of TNF-αand IFN-y in the supernatant of PBMCs with heat treatment. Gandan Shire group also was tested significantly lower expression of IL-4 in the supernatant compared with that of the other 2 groups.
Conclusions:
1. The eliminating-the-moist-heat-in-liver (Qinggan Lishi) therapy of traditional Chinese medicine may have beneficial effect on improving the symptoms due to hepatitis and liver function for a particular group of patients with chronic hepatitis B who are supposed to be discriminated and identified in the TCM manner. However, the anti-viral effect remains to be verified by further clinical trials.
2. The Gandan-Shire-predominant CHB shows evident deficiency of heat-inducibility of Hsp70 and imbalance of Thl/Th2 cytokine expression, which may convey specific characteristics of CHB with traditional Chinese medical Gandan Shire syndrome in terms of immunological and biological essence.
引文
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[42]Wu YC, Yen WY, Lee TC, Yih LH. Heat shock protein inhibitors,17-DMAG and KNK437, enhance arsenic trioxide-induced mitotic apoptosis[J]. Toxicol Appl Pharmacol.2009; 236(2):231-238.
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[44]El Golli Bennour E, Rodriguez-Enfedaque A, Bouaziz C, et al. Toxicities induced in cultured human hepatocarcinoma cells exposed to ochratoxin A:oxidative stress and apoptosis status[J]. J Biochem Mol Toxicol.2009; 23(2):87-96
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[49]Giraldo PC, Ribeiro AD,Simoes JA, et al. Circulating heat shock proteins in women with a history of recurrence vulvovaginitis[J]. Infect Dis Obstet Gynecol, 1999; 7(3):128-132.
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