毒热平注射液抗流感病毒作用及免疫调控机制的实验研究
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摘要
实验目的:
流感是流感病毒引起的急性呼吸道传染病,好发于冬、春季节。尤其是甲型流感病毒,由于其极易产生变异,制备有效的疫苗困难重重,而针对流感病毒感染的西药既有较强的毒副作用,又容易诱导病毒产生耐药性。这种现状下,在我国的中医药宝库中寻找有效药物、创造新型药剂来防治流感就成为一项很有意义的研究工作。
毒热平注射液是由黄芩、栀子、灯盏花、猪胆粉等四味药物组成的中药复方制剂,根据中医理论“毒损肺络”病机理论配伍研制而成,具有清热燥湿、凉血解毒、活血通络的功效。本实验采用流感病毒亚甲型鼠肺适应株(FM_1)感染小鼠作为模型,观察毒热平注射液对FM1不同时相感染小鼠的抗病毒作用及其对免疫功能的影响,以天然免疫中重要的模式识别受体Toll样受体为切入点,探讨其抗病毒作用及免疫调节作用的分子机制,为毒热平注射液的临床应用提供理论依据。
实验方法:
1.毒热平注射液对FM_1感染小鼠死亡保护率的影响
将ICR小鼠随机分为七组:正常组、模型组、利巴韦林组、双黄连组、毒热平注射液大、中、小剂量组,以4LD50的流感病毒液滴鼻感染小鼠造模,0.05mL/只,正常组以生理盐水滴鼻对照。造模后当日腹腔注射给药,每天1次,连续7天;正常组和模型组小鼠注射生理盐水对照。自造模当日起连续观察14天,计算各组死亡率及平均生活日。
2.毒热平注射液对FM1不同时相感染小鼠肺损伤及病毒增殖的影响
将ICR小鼠随机分为七组:正常组、模型组、利巴韦林组、痰热清组、毒热平注射液大、中、小剂量组,以4LD50的流感病毒液滴鼻感染小鼠造模,0.05mL/只,正常组以生理盐水滴鼻对照。造模后当日腹腔注射给药,每天1次;正常组和模型组小鼠注射生理盐水对照。按照要求在不同时间点处死小鼠,摘取肺脏,计算肺指数,同时各组取肺组织分别进行固定和匀浆,作病理形态学检测和肺组织中病毒滴度的检测。
3.毒热平注射液对FM1不同时相感染小鼠免疫功能的影响
动物分组、造模、给药方法同上。按照要求在不同时间点处死小鼠,无菌取脾脏做脾脏T、B淋巴细胞增殖活性的检测,采用MTT法;摘取小鼠肺脏进行匀浆,和小鼠血清一起用ELISA试剂盒检测炎症因子TNF-α、IFN-β、IFN-γ、IL-4、IL-12以及趋化因子IL-8、MIP-2、RANTES、IP-10等的含量。
4.毒热平注射液对FM1病毒感染小鼠免疫调控分子机制的研究
动物分组、造模、给药方法同上。按照要求在不同时间点处死小鼠,冰浴条件下无菌迅速摘取小鼠肺脏,置于无RNase EP管中,立即投入液氮中冷冻。每组取5个肺组织。用PCR法检测肺组织中TLR3及PKR mRNA的表达,用Western-blot法检测TLR3及PKR蛋白的表达。
实验结果:
1.毒热平注射液对FM_1感染小鼠死亡保护率的影响
模型组的死亡率为75%,平均生存时间为8.69d。毒热平注射液中剂量组死亡率为36.3%,与模型组比较,经x~2检验差异显著,P＜0.05;毒热平注射液大剂量组平均生存时间为10.8d,与模型组比较P＜0.05;毒热平中剂量组平均生存时间为11.81d,与模型组比较P＜0.01。
2.毒热平注射液对FM1不同时相感染小鼠肺损伤及病毒增殖的影响
2.1在不同时间点观察受试动物,可见正常组小鼠体重稳步上升,模型组小鼠的体重在三个时间点表现出先增加再下降的趋势,且均显著低于正常组。用药组中,利巴韦林组体重逐渐增加,痰热清组及毒热平各剂量组趋势同模型组。在每个时间点上,造模72h后,与模型组比较,发现毒热平中剂量组有明显差异,P＜0.05;造模120h后,毒热平小、中剂量组与模型组比较有明显差异,P＜0.05或P＜0.01。
2.2在不同时间点观察受试动物,可见模型组及各用药组的肺指数均呈现逐步升高的趋势。在每个时间点上,造模72h后,与模型组比较,利巴韦林组肺指数有显著性差异,P＜0.01;毒热平中、大剂量组有明显差异,P＜0.05;造模120h后,与模型组比较,发现利巴韦林组肺指数有极为显著性差异,P＜0.001;毒热平中剂量组有明显差异,P＜0.05。
2.3在不同时间点观察受试动物,可见模型组及各用药组肺组织中病毒滴度呈现逐步上升的趋势。感染120h后,与模型组相比,利巴韦林对肺脏中的病毒杀伤效果最明显,P＜0.001;毒热平注射液中剂量组对肺脏中的病毒亦有明显杀伤作用,P＜0.01,其余用药组与模型组比较无差异。
2.4 HE染色光镜观察正常组小鼠肺泡结构完整清晰,腔内未见渗出。模型组造模24h可见弥漫性肺泡壁增厚,肺泡壁血管扩张充血,有少量单个核细胞浸润,部分肺泡有少量出血。造模72h后可见弥漫性肺泡壁增厚,部分区域较前次显著增厚,有明显单个核细胞浸润。造模120h后可见支气管管腔内有出血及炎性渗出,支气管周围有多量淋巴细胞浸润;弥漫性肺泡壁增厚,局灶性肺泡实变。各用药组病变明显减轻,但随时间变化而病变均逐渐加重。利巴韦林120h组仍只见轻度病变;痰热清组、毒热平大、中、小剂量组病变均呈进行性加重,但均轻于同时期模型组。
3.毒热平注射液对FM1不同时相感染小鼠免疫功能的影响
3.1不同时间点观察受试动物,可见模型组脾脏T、B淋巴细胞增殖活性随时间变化呈现逐渐下降的趋势,造模后小鼠的T细胞增殖活性比B细胞增殖活性抑制更为明显。在三个时间点,模型组T细胞的增殖活性均明显受抑,P＜0.001,各用药组对其均有不同程度的恢复;造模24h后可见除利巴韦林组外的各中药组还表现出免疫促进的作用,与正常组相比显著增高,P＜0.001。造模72h后和120h,各用药组对小鼠的免疫抑制也有了不同程度的恢复,与模型组相比P＜0.001～P＜0.05,但均未恢复到正常组水平。各组B细胞增殖活性变化与T细胞近似,但变化程度较为缓和。
3.2不同时间点检测各组动物血清及肺匀浆中的细胞因子,可见模型组除IL-4水平随时间变化逐渐降低外,TNF-α、IFN-β、IFN-γ、IL-12以及趋化因子IL-8、MIP-2、RANTES、IP-10均呈现逐渐升高的趋势。各用药组对其有不同程度的干预作用。总体来说,相比而言,对各细胞因子的干预效果,痰热清组＞利巴韦林组＞毒热平大剂量组＞毒热平中剂量组,毒热平小剂量组一般来说效果较差。
4.毒热平注射液对FM1病毒感染小鼠免疫调控分子机制的研究
与正常组相比,模型组小鼠不同时相感染FM1后对肺组织中TLR3及PKR mRNA及蛋白的表达水平造成了不同程度的增高,药物亦发挥了不同程度的干预作用。总体而言,TLR3及PKR mRNA及蛋白的表达水平随时间变化呈现先升高(72h)、后下降(120h)的趋势,除毒热平小剂量组外,其他各用药组均可抑制其升高的水平,与模型组相比,P＜0.01或P＜0.05,但均不能恢复到正常组水平。
结论
1.毒热平注射液具有抗病毒作用
通过观察药物对肺脏中病毒滴度的影响发现,毒热平注射液中剂量组可明显抑制肺脏中病毒的增殖,具有一定的抗病毒作用。
2.毒热平注射液对FM_1感染小鼠的死亡和肺组织损伤具有保护和改善作用:
毒热平注射液中剂量组可明显降低FM1感染小鼠的死亡率,降低肺指数,改善肺脏病理变化,对FM_1感染小鼠的死亡和病毒引起的肺组织损伤具有保护作用。
3.毒热平注射液对FM_1感染小鼠免疫功能具有调节作用:
3.1流感病毒FM_1感染小鼠后,对脾脏T、B淋巴细胞的增殖活性具有明显的抑制作用,而毒热平注射液可对其有明显的恢复作用。
3.2毒热平注射液对FM_1感染小鼠血清及肺组织匀浆中细胞因子水平具有调节作用,可显著升高因病毒感染引起的IL-4下调以及抑制其他细胞因子和趋化因子的上调,减轻炎性病理损伤,进而恢复机体免疫功能的稳定和平衡,并帮助机体抵御病毒的侵害作用。
4.毒热平注射液可通过调节肺组织中TLR3和PKR的表达水平来发挥免疫调节作用。
OBJECTIVE
Influenza is a kind of acute respiratory infectious diseases caused by influenza virus, which usually occurs in the winter and spring.Preparing an effective vaccine for the influenza A virus is difficult because of its mutable feature.And the anti-influenza western medicines have strong side effects and easy induction of drug resistance.Under the status quo, to find effective anti-influenza drugs and new preparations has become a very meaningful research in Chinese medicine.
Dureping Injection is a Chinese medicine compound preparation,The sub-type of influenza virus strain,FM1,was used in this experiment as a pathogenic factor in mice.And the mice were used as a model to observe the antiviral activity and its effect on immune function of Dureping Injection.
METHODS
1.ICR mice were inoculated intranasally with FM_1,then treated with Dureping Injection for 7 days.The effect of Dureping Injection on the life protection and mean life day of FM1-infected mice after 14 days inoculation was observed.Normal group,Ribavirin Injection group and Shuanghuanglian Injection group were as control groups.
2.ICR mice were inoculated intranasally with FM_1,then treated with Dureping Injection for 1,3 and 5 days.At each timepoints,tested mice were sacrificed and the body weight and lung indexes were analysed;then lungs were fixed in formalin,routinely processed,and embedded in paraffin.Routine HE stained sections were examined.The lung homogenate was tested for virus titer in it.
3.Animal grouping model,drug delivery methods Ibid.In accordance with the requirements,the mice were killed at different timepoints,the spleen T,B lymphocyte proliferation activity was detected with MTT method;ELISA kits were used with detection of cytokines,TNF-α,IFN-β,IFN-γ,IL-4,IL-12 and the chemokines IL-8,MIP-2,RANTES, IP-10 and so on,in the lung homogenate and serum of mice.
4.Animal grouping model,drug delivery methods Ibid.In accordance with the requirements,the mice were killed at different timepoints.TLR3 and PKR mRNA expression in lung tissues was Detected by PCR,and TLR3 and PKR protein expression in lung tissues was Detected by the Western-blot assay.
RESULTS
1.As Model group,the mortality rate was 75%,the average survival time was 8.69d. The mortality rate of the medium-dose Injection group of Dureping was 36.3%,compared with the model group,the significant difference x~2 test,P＜0.05;Dureping Injection of high-dose group the average survival time was 10.8d,compared with the model group P＜ 0.05;Dureping Injection of medium-dose group the average survival time was 11.81d, compared with the model group P＜0.01.
2.Observed in animal subjects at different timepoints,the normal mice can be seen a steady increase in body weight,body weight of mice in model group at three timepoints showed the first increase in a downward trend again,and were significantly lower than the normal group.As the drug groups,a gradual increase in body weight was observed in ribavirin group,while the same trend in every Dureping group and Tanreqing was seen compared with the model group.At each timepoint,72h after modeling,compared with the model group and found the body weight is significant differences in medium-dose group,P＜0.05;model 120h,the Dureping injection small,medium-dose group compared with the model group significant difference,P＜0.05 or P＜0.01.
Observed in animal subjects at different timepoints,we can see the model group and treatment group showed the lung index gradually increased trend.At each timepoint,72h after modeling,compared with the model group,the lung index ribavirin group were significantly different,P＜0.01;Dureping injection high,dose group,significant difference,P＜0.05;72h after modeling,compared with the model group,found that lung index ribavirin group extremely significant difference,P＜0.001;Dureping injection medium-dose group compared with the model group significant difference,P＜0.05.
Observed in animal subjects at different timepoints,we can see the model group and treatment group in the virus titer of lung tissue showed a gradual upward trend.120h after infection,compared with the model group,the virus titer in the lungs of ribavirin has extremely significant difference,P＜0.001;Dureping injection medium-dose group has also shown clear signs of antiviral effect,P＜0.01,and the remaining treatment group compared with the model group is no difference.
HE staining under light microscope to observe the normal mice clear alveolar structural integrity,no exudation cavity.After 24h,Model group can be seen diffuse alveolar wall thickening,dilatation of alveolar wall congestion,a small amount of infiltration of mononuclear cells,some alveolar bleeding.Model can be seen after 72h diffuse alveolar wall thickening,and some regions significant thickening than previous states,marked infiltration of mononuclear cells also can be seen.120h after modeling we can see that there is bleeding bronchial lumen and inflammatory exudate,the volume of lymphocytic infiltration around the bronchia;diffuse alveolar wall thickening,focal alveolar consolidation.The treatment group lesions decreased,but changes gradually increase over time.120h after infection,ribavirin group is still only mild disease;Tanreqing group,Dureping large,medium and small dose group showed a progressive increase lesion,but are lighter than the same period of the model group.
3.Observation of subjects at different timepoints,animals,shows that the model group of spleen T,B lymphocyte proliferation activity gradually showed a downward trend over time,after modeling,T cell proliferation activity than the B cell proliferation inhibition activity becomes more apparent.In the three timepoints,Tcell proliferation activity of model group was significantly inhibited,P＜0.001,the treatment groups recover in varying degrees; after 24h,the various traditional Chinese medicine preparations were shown as immunization accelerate,compared with the normal group,significantly higher,P＜0.001.72h and 120h after modeling,the treatment group of mice with immune suppression are also different levels of the restoration,120h each group is stronger than 72h.Compared with model group,P＜0.001～P＜0.05,but not the resumption of the level of the normal group.B cell proliferation of each group is similar to T cell,but to some extent,rather unconspicuous.
At different timepoints the serum and lung homogenate cytokines in each group of animals were detected.It can be seen in addition to IL-4 level decreased over time,TNF-α, IFN-β,IFN-γ,IL-12 and more factor IL-8,MIP-2,RANTES,IP-10 showed a gradual rise in model group.The treatment group may have different levels of intervention effect.Generally speaking,the comparison of the effect of cytokine intervention,Tanreqing Group＞ribavirin group＞Dureping high-dose drug group＞medium-dose group,in general to small-dose group, less effective.
4.Compared with normal group,TLR3 and PKR mRNA and protein expression in the lung tissue of model group mice displayed varying degrees of increase at different time after infection.Drugs played a role in different levels of intervention.Overall,TLR3 and PKR mRNA and protein expression levels change over time increased at first(72h),and then decreased(120h) of the trend.With the exception of Dureping small-dose group,the other groups display a higher level compared with the model group,P＜0.01 or P＜0.05,but can not be recovered to normal level.
CONCLUSION
1.Through the observation of the effects on the virus titer of the lungs,we find that of Dureping Injection medium-dose group could inhibit the proliferation of virus in the lungs, which displayed a certain degree of antiviral activity.
2.Dureping Injection medium-dose group can significantly reduce mortality in mice infected with FM1,reduce lung index,improve the pathological changes in the lungs of infected mice.It is proved that Dureping Injection can protect the death and injury of the lung tissue caused by FM1 virus infection.
3.Dureping Injection group can significantly recovered the inhibited proliferation of spleen T/B lymphocyte caused by FM1 virus infection.It can also recover the changes of cytokines level in the serum and lung tissue homogenate caused by FM1 virus infection.It can increase the reduction of IL-4 and down-regulate other cytokines and chemokines due to virus infection.It can alleviate pathology of inflammatory injury,recover immune function and thus the stability and balance,and to help the body to resist against the virus.
4.Dureping Injection play a role in immune regulation by regulating the expression levels of TLR3 and PKR mRNA and their protein in lung tissues.
流感是流感病毒引起的急性呼吸道传染病,好发于冬、春季节。尤其是甲型流感病毒,由于其极易产生变异,制备有效的疫苗困难重重,而针对流感病毒感染的西药既有较强的毒副作用,又容易诱导病毒产生耐药性。这种现状下,在我国的中医药宝库中寻找有效药物、创造新型药剂来防治流感就成为一项很有意义的研究工作。
毒热平注射液是由黄芩、栀子、灯盏花、猪胆粉等四味药物组成的中药复方制剂,根据中医理论“毒损肺络”病机理论配伍研制而成,具有清热燥湿、凉血解毒、活血通络的功效。本实验采用流感病毒亚甲型鼠肺适应株(FM_1)感染小鼠作为模型,观察毒热平注射液对FM1不同时相感染小鼠的抗病毒作用及其对免疫功能的影响,以天然免疫中重要的模式识别受体Toll样受体为切入点,探讨其抗病毒作用及免疫调节作用的分子机制,为毒热平注射液的临床应用提供理论依据。
实验方法:
1.毒热平注射液对FM_1感染小鼠死亡保护率的影响
将ICR小鼠随机分为七组:正常组、模型组、利巴韦林组、双黄连组、毒热平注射液大、中、小剂量组,以4LD50的流感病毒液滴鼻感染小鼠造模,0.05mL/只,正常组以生理盐水滴鼻对照。造模后当日腹腔注射给药,每天1次,连续7天;正常组和模型组小鼠注射生理盐水对照。自造模当日起连续观察14天,计算各组死亡率及平均生活日。
2.毒热平注射液对FM1不同时相感染小鼠肺损伤及病毒增殖的影响
将ICR小鼠随机分为七组:正常组、模型组、利巴韦林组、痰热清组、毒热平注射液大、中、小剂量组,以4LD50的流感病毒液滴鼻感染小鼠造模,0.05mL/只,正常组以生理盐水滴鼻对照。造模后当日腹腔注射给药,每天1次;正常组和模型组小鼠注射生理盐水对照。按照要求在不同时间点处死小鼠,摘取肺脏,计算肺指数,同时各组取肺组织分别进行固定和匀浆,作病理形态学检测和肺组织中病毒滴度的检测。
3.毒热平注射液对FM1不同时相感染小鼠免疫功能的影响
动物分组、造模、给药方法同上。按照要求在不同时间点处死小鼠,无菌取脾脏做脾脏T、B淋巴细胞增殖活性的检测,采用MTT法;摘取小鼠肺脏进行匀浆,和小鼠血清一起用ELISA试剂盒检测炎症因子TNF-α、IFN-β、IFN-γ、IL-4、IL-12以及趋化因子IL-8、MIP-2、RANTES、IP-10等的含量。
4.毒热平注射液对FM1病毒感染小鼠免疫调控分子机制的研究
动物分组、造模、给药方法同上。按照要求在不同时间点处死小鼠,冰浴条件下无菌迅速摘取小鼠肺脏,置于无RNase EP管中,立即投入液氮中冷冻。每组取5个肺组织。用PCR法检测肺组织中TLR3及PKR mRNA的表达,用Western-blot法检测TLR3及PKR蛋白的表达。
实验结果:
1.毒热平注射液对FM_1感染小鼠死亡保护率的影响
模型组的死亡率为75%,平均生存时间为8.69d。毒热平注射液中剂量组死亡率为36.3%,与模型组比较,经x~2检验差异显著,P＜0.05;毒热平注射液大剂量组平均生存时间为10.8d,与模型组比较P＜0.05;毒热平中剂量组平均生存时间为11.81d,与模型组比较P＜0.01。
2.毒热平注射液对FM1不同时相感染小鼠肺损伤及病毒增殖的影响
2.1在不同时间点观察受试动物,可见正常组小鼠体重稳步上升,模型组小鼠的体重在三个时间点表现出先增加再下降的趋势,且均显著低于正常组。用药组中,利巴韦林组体重逐渐增加,痰热清组及毒热平各剂量组趋势同模型组。在每个时间点上,造模72h后,与模型组比较,发现毒热平中剂量组有明显差异,P＜0.05;造模120h后,毒热平小、中剂量组与模型组比较有明显差异,P＜0.05或P＜0.01。
2.2在不同时间点观察受试动物,可见模型组及各用药组的肺指数均呈现逐步升高的趋势。在每个时间点上,造模72h后,与模型组比较,利巴韦林组肺指数有显著性差异,P＜0.01;毒热平中、大剂量组有明显差异,P＜0.05;造模120h后,与模型组比较,发现利巴韦林组肺指数有极为显著性差异,P＜0.001;毒热平中剂量组有明显差异,P＜0.05。
2.3在不同时间点观察受试动物,可见模型组及各用药组肺组织中病毒滴度呈现逐步上升的趋势。感染120h后,与模型组相比,利巴韦林对肺脏中的病毒杀伤效果最明显,P＜0.001;毒热平注射液中剂量组对肺脏中的病毒亦有明显杀伤作用,P＜0.01,其余用药组与模型组比较无差异。
2.4 HE染色光镜观察正常组小鼠肺泡结构完整清晰,腔内未见渗出。模型组造模24h可见弥漫性肺泡壁增厚,肺泡壁血管扩张充血,有少量单个核细胞浸润,部分肺泡有少量出血。造模72h后可见弥漫性肺泡壁增厚,部分区域较前次显著增厚,有明显单个核细胞浸润。造模120h后可见支气管管腔内有出血及炎性渗出,支气管周围有多量淋巴细胞浸润;弥漫性肺泡壁增厚,局灶性肺泡实变。各用药组病变明显减轻,但随时间变化而病变均逐渐加重。利巴韦林120h组仍只见轻度病变;痰热清组、毒热平大、中、小剂量组病变均呈进行性加重,但均轻于同时期模型组。
3.毒热平注射液对FM1不同时相感染小鼠免疫功能的影响
3.1不同时间点观察受试动物,可见模型组脾脏T、B淋巴细胞增殖活性随时间变化呈现逐渐下降的趋势,造模后小鼠的T细胞增殖活性比B细胞增殖活性抑制更为明显。在三个时间点,模型组T细胞的增殖活性均明显受抑,P＜0.001,各用药组对其均有不同程度的恢复;造模24h后可见除利巴韦林组外的各中药组还表现出免疫促进的作用,与正常组相比显著增高,P＜0.001。造模72h后和120h,各用药组对小鼠的免疫抑制也有了不同程度的恢复,与模型组相比P＜0.001～P＜0.05,但均未恢复到正常组水平。各组B细胞增殖活性变化与T细胞近似,但变化程度较为缓和。
3.2不同时间点检测各组动物血清及肺匀浆中的细胞因子,可见模型组除IL-4水平随时间变化逐渐降低外,TNF-α、IFN-β、IFN-γ、IL-12以及趋化因子IL-8、MIP-2、RANTES、IP-10均呈现逐渐升高的趋势。各用药组对其有不同程度的干预作用。总体来说,相比而言,对各细胞因子的干预效果,痰热清组＞利巴韦林组＞毒热平大剂量组＞毒热平中剂量组,毒热平小剂量组一般来说效果较差。
4.毒热平注射液对FM1病毒感染小鼠免疫调控分子机制的研究
与正常组相比,模型组小鼠不同时相感染FM1后对肺组织中TLR3及PKR mRNA及蛋白的表达水平造成了不同程度的增高,药物亦发挥了不同程度的干预作用。总体而言,TLR3及PKR mRNA及蛋白的表达水平随时间变化呈现先升高(72h)、后下降(120h)的趋势,除毒热平小剂量组外,其他各用药组均可抑制其升高的水平,与模型组相比,P＜0.01或P＜0.05,但均不能恢复到正常组水平。
结论
1.毒热平注射液具有抗病毒作用
通过观察药物对肺脏中病毒滴度的影响发现,毒热平注射液中剂量组可明显抑制肺脏中病毒的增殖,具有一定的抗病毒作用。
2.毒热平注射液对FM_1感染小鼠的死亡和肺组织损伤具有保护和改善作用:
毒热平注射液中剂量组可明显降低FM1感染小鼠的死亡率,降低肺指数,改善肺脏病理变化,对FM_1感染小鼠的死亡和病毒引起的肺组织损伤具有保护作用。
3.毒热平注射液对FM_1感染小鼠免疫功能具有调节作用:
3.1流感病毒FM_1感染小鼠后,对脾脏T、B淋巴细胞的增殖活性具有明显的抑制作用,而毒热平注射液可对其有明显的恢复作用。
3.2毒热平注射液对FM_1感染小鼠血清及肺组织匀浆中细胞因子水平具有调节作用,可显著升高因病毒感染引起的IL-4下调以及抑制其他细胞因子和趋化因子的上调,减轻炎性病理损伤,进而恢复机体免疫功能的稳定和平衡,并帮助机体抵御病毒的侵害作用。
4.毒热平注射液可通过调节肺组织中TLR3和PKR的表达水平来发挥免疫调节作用。
OBJECTIVE
Influenza is a kind of acute respiratory infectious diseases caused by influenza virus, which usually occurs in the winter and spring.Preparing an effective vaccine for the influenza A virus is difficult because of its mutable feature.And the anti-influenza western medicines have strong side effects and easy induction of drug resistance.Under the status quo, to find effective anti-influenza drugs and new preparations has become a very meaningful research in Chinese medicine.
Dureping Injection is a Chinese medicine compound preparation,The sub-type of influenza virus strain,FM1,was used in this experiment as a pathogenic factor in mice.And the mice were used as a model to observe the antiviral activity and its effect on immune function of Dureping Injection.
METHODS
1.ICR mice were inoculated intranasally with FM_1,then treated with Dureping Injection for 7 days.The effect of Dureping Injection on the life protection and mean life day of FM1-infected mice after 14 days inoculation was observed.Normal group,Ribavirin Injection group and Shuanghuanglian Injection group were as control groups.
2.ICR mice were inoculated intranasally with FM_1,then treated with Dureping Injection for 1,3 and 5 days.At each timepoints,tested mice were sacrificed and the body weight and lung indexes were analysed;then lungs were fixed in formalin,routinely processed,and embedded in paraffin.Routine HE stained sections were examined.The lung homogenate was tested for virus titer in it.
3.Animal grouping model,drug delivery methods Ibid.In accordance with the requirements,the mice were killed at different timepoints,the spleen T,B lymphocyte proliferation activity was detected with MTT method;ELISA kits were used with detection of cytokines,TNF-α,IFN-β,IFN-γ,IL-4,IL-12 and the chemokines IL-8,MIP-2,RANTES, IP-10 and so on,in the lung homogenate and serum of mice.
4.Animal grouping model,drug delivery methods Ibid.In accordance with the requirements,the mice were killed at different timepoints.TLR3 and PKR mRNA expression in lung tissues was Detected by PCR,and TLR3 and PKR protein expression in lung tissues was Detected by the Western-blot assay.
RESULTS
1.As Model group,the mortality rate was 75%,the average survival time was 8.69d. The mortality rate of the medium-dose Injection group of Dureping was 36.3%,compared with the model group,the significant difference x~2 test,P＜0.05;Dureping Injection of high-dose group the average survival time was 10.8d,compared with the model group P＜ 0.05;Dureping Injection of medium-dose group the average survival time was 11.81d, compared with the model group P＜0.01.
2.Observed in animal subjects at different timepoints,the normal mice can be seen a steady increase in body weight,body weight of mice in model group at three timepoints showed the first increase in a downward trend again,and were significantly lower than the normal group.As the drug groups,a gradual increase in body weight was observed in ribavirin group,while the same trend in every Dureping group and Tanreqing was seen compared with the model group.At each timepoint,72h after modeling,compared with the model group and found the body weight is significant differences in medium-dose group,P＜0.05;model 120h,the Dureping injection small,medium-dose group compared with the model group significant difference,P＜0.05 or P＜0.01.
Observed in animal subjects at different timepoints,we can see the model group and treatment group showed the lung index gradually increased trend.At each timepoint,72h after modeling,compared with the model group,the lung index ribavirin group were significantly different,P＜0.01;Dureping injection high,dose group,significant difference,P＜0.05;72h after modeling,compared with the model group,found that lung index ribavirin group extremely significant difference,P＜0.001;Dureping injection medium-dose group compared with the model group significant difference,P＜0.05.
Observed in animal subjects at different timepoints,we can see the model group and treatment group in the virus titer of lung tissue showed a gradual upward trend.120h after infection,compared with the model group,the virus titer in the lungs of ribavirin has extremely significant difference,P＜0.001;Dureping injection medium-dose group has also shown clear signs of antiviral effect,P＜0.01,and the remaining treatment group compared with the model group is no difference.
HE staining under light microscope to observe the normal mice clear alveolar structural integrity,no exudation cavity.After 24h,Model group can be seen diffuse alveolar wall thickening,dilatation of alveolar wall congestion,a small amount of infiltration of mononuclear cells,some alveolar bleeding.Model can be seen after 72h diffuse alveolar wall thickening,and some regions significant thickening than previous states,marked infiltration of mononuclear cells also can be seen.120h after modeling we can see that there is bleeding bronchial lumen and inflammatory exudate,the volume of lymphocytic infiltration around the bronchia;diffuse alveolar wall thickening,focal alveolar consolidation.The treatment group lesions decreased,but changes gradually increase over time.120h after infection,ribavirin group is still only mild disease;Tanreqing group,Dureping large,medium and small dose group showed a progressive increase lesion,but are lighter than the same period of the model group.
3.Observation of subjects at different timepoints,animals,shows that the model group of spleen T,B lymphocyte proliferation activity gradually showed a downward trend over time,after modeling,T cell proliferation activity than the B cell proliferation inhibition activity becomes more apparent.In the three timepoints,Tcell proliferation activity of model group was significantly inhibited,P＜0.001,the treatment groups recover in varying degrees; after 24h,the various traditional Chinese medicine preparations were shown as immunization accelerate,compared with the normal group,significantly higher,P＜0.001.72h and 120h after modeling,the treatment group of mice with immune suppression are also different levels of the restoration,120h each group is stronger than 72h.Compared with model group,P＜0.001～P＜0.05,but not the resumption of the level of the normal group.B cell proliferation of each group is similar to T cell,but to some extent,rather unconspicuous.
At different timepoints the serum and lung homogenate cytokines in each group of animals were detected.It can be seen in addition to IL-4 level decreased over time,TNF-α, IFN-β,IFN-γ,IL-12 and more factor IL-8,MIP-2,RANTES,IP-10 showed a gradual rise in model group.The treatment group may have different levels of intervention effect.Generally speaking,the comparison of the effect of cytokine intervention,Tanreqing Group＞ribavirin group＞Dureping high-dose drug group＞medium-dose group,in general to small-dose group, less effective.
4.Compared with normal group,TLR3 and PKR mRNA and protein expression in the lung tissue of model group mice displayed varying degrees of increase at different time after infection.Drugs played a role in different levels of intervention.Overall,TLR3 and PKR mRNA and protein expression levels change over time increased at first(72h),and then decreased(120h) of the trend.With the exception of Dureping small-dose group,the other groups display a higher level compared with the model group,P＜0.01 or P＜0.05,but can not be recovered to normal level.
CONCLUSION
1.Through the observation of the effects on the virus titer of the lungs,we find that of Dureping Injection medium-dose group could inhibit the proliferation of virus in the lungs, which displayed a certain degree of antiviral activity.
2.Dureping Injection medium-dose group can significantly reduce mortality in mice infected with FM1,reduce lung index,improve the pathological changes in the lungs of infected mice.It is proved that Dureping Injection can protect the death and injury of the lung tissue caused by FM1 virus infection.
3.Dureping Injection group can significantly recovered the inhibited proliferation of spleen T/B lymphocyte caused by FM1 virus infection.It can also recover the changes of cytokines level in the serum and lung tissue homogenate caused by FM1 virus infection.It can increase the reduction of IL-4 and down-regulate other cytokines and chemokines due to virus infection.It can alleviate pathology of inflammatory injury,recover immune function and thus the stability and balance,and to help the body to resist against the virus.
4.Dureping Injection play a role in immune regulation by regulating the expression levels of TLR3 and PKR mRNA and their protein in lung tissues.
引文
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