| 1 BackgroundPrimary immune thrombocytopenia derived fatigue is one of the most suffering complaints of patients with ITP.Almost 90%of the patients with chronic ITP are suffering from fatigue to different extents.When undertaking the project of ’Mechanism study of treatment of ITP by theory of Spleen’ supported by the national key basic research development plan,we found that JIANPIYIQISHEXUEFANG based on the theory of spleen can significantly improve the symptoms of fatigue,balance the immune system and improve the peripheral platelet count of patients with ITP,which suggested that ITP is the disease that JIANPIYIQISHEXUEFANG could show the best effect on.Related mechanism and basic studies have also shown that JIANPIYIQISHEXUEFANG has the effects of prolonging the weight-bearing swimming time of mice and increasing the speed of movement of zebrafish,which suggested that JIANPIYIQISHEXUEFANG has the anti-fatigue effect.Therefore,we proposed the theoretical hypothesis that mitochondrial dysfunction as well as energy metabolism imbalance may be the key mechanism of ITP fatigue.This thesis was carried out in accordance with the content of the theoretical hypothesis,focusing on the correlation of ITP-derived fatigue and mitochondrial function.2 ObjectiveTo clarify the mechanism of JIANPIYIQISHEXUEFANG treating ITP-derived fatigue,we established the mice models of ITP by passive immune modeling method,chose JIANPIYIQISHEXUEFANG as the experimental medicine,and tested indicators of fatigue and mitochondrial function.3 Methods3.1 Establishment of ITP mice model and tests of fatigueWe established the ITP mice model with 90 BALB/c mice(M/F=1/1)through immunological way.90 mice were randomly divided into five groups according to their PLT level:normal group,model group,Prednisone group,medium JIANPIYIQISEXUE group and high JIANPIYIQISEXUE group.All mice in treatment groups was given with the corresponding drug by 0.1 mL/10 g daily for 8 days.3.2 Indicators3.2.1 Indicators of effectivenessHere are the indicators of effectiveness:①The outlooking and activity condition of mice.②The duration of weighted swimming of mice on the day before the experiment,1st day of treatment and the last day of treatment.③The body weight and organ index of ITP mice.④The peripheral blood tests(PLT,PDW,HGB,WBC)of mice on the day before injecting APS,the 4th and the 8th day of injecting APS,the 1st,the 4th and the 8th treatment day.3.2.2 Effect of prescription JIANPIYIQISHEXUE on mitochondrial functionWe tested ROS level in mice spleen tissue from each group with frozen section ROS testing kit.We purified the DNA and mtDNA of mice spleen tissue,testing the copy number of mtDNA in mice spleen tissue by qPCR technique.We also tested the ATP level of mice colon tissue from each group with ATP testing kit.3.2.3 Molecular mechanism of prescription JIANPIYIQISHEXUE on regulating mitochondrial functionSDHA is the crucial part of mitochondrial complexⅡ,and ClpP as well as LonP1 play important roles in keeping protein balance of mitochondria.We tested the expression level of SDHA,ClpP,and LonP1 in spleen,liver,heart,lung,brain,colon and skeletal muscle of mice,both on transcriptional and translational levels with qPCR and Western blot technique.4 Results4.1 Indicators of ITP derived fatigue4.1.1 The duration of weighted swimming of mice.There was no significant difference among each group before the APS injection(p>0.05).On the 8th day after APS injection,duration of each experimental group were significantly shorter than that of normal group(p<0.01).No significant difference was detected among each treatment group(p>0.05).On the last day of treatment,duration of model group and Prednisone group consistently got shorter,whereas duration of medium JIANPIYIQISHEXUE group and high JIANPIYIQISHEXUE group were significantly longer than that of model group(p<0.01).No significant difference was detected between medium JIANPIYIQISHEXUE group and high JIANPIYIQISHEXUE group.4.1.2 The body weight and organ index of ITP mice.No significant difference of body weight was found between each group on the last day of treatment(p>0.05).Spleen index of each experimental group were significantly higher than that of normal group(p<0.01).Spleen index of each treatment group were significantly lower than that of model group(p<0.05).Liver index of model group and Prednisone group were both significantly higher than that of normal group(p<0.01).Liver index of medium JIANPIYIQISHEXUE group(p<0.05)and high JIANPIYIQISHEXUE group(p<0.01)were both significantly lower than that of model group.Heart index of model group,Prednisone group and medium JIANPIYIQISHEXUE group were significantly higher than that of normal group(p<0.05).Lung index of Prednisone group was significantly higher than that of normal group(p<0.05).No significant difference of Kidney index was found among each group(p>0.05).Colon index of medium JIANPIYIQISHEXUE group was significantly higher than that of normal group(p<0.05).Brain index of model group was significantly higher than that of normal group(p<0.05).4.1.3 The peripheral blood tests of mice.No significant difference of PLT was detected among each group before APS injection(p>0.05).On the 4th day of injecting APS,PLT of each experimental group were significantly lower than that of normal group(p<0.01).No significant difference of PLT was found between each treatment group and model group(p>0.05).On the 1st treatment day,PLT of each experimental group were still significantly lower than that of normal group(p<0.01).On the 4th treatment day,only PLT of high JIANPIYIQISHEXUE group was significantly higher than that of model group(p<0.01).On the 8th treatment day,PLT of each treatment group was significantly higher than that of model group(p<0.01).No significant of PLT was detected among each treatment group(p>0.05).No significant difference of PDW,HGB or WBC was detected among each group on each testing timing point(p>0.05).4.2 Indicators of mitochondrial function4.2.1 ROS of each experimental group was significantly higher than that of normal group(p<0.05).ROS of each treatment group was significantly lower than that of model group(p<0.05).ROS of Prednisone group was significantly lower than that of high JIANPIYIQISHEXUE group(p<0.01),and ROS of high JIANPIYIQISHEXUE group was significantly lower than that of medium JIANPIYIQISHEXUE group(p<0.05).4.2.2 The mtDNA copy number of each experimental group was significantly lower than that of normal group(p<0.01).No significant difference of mtDNA copy number was detected among each treatment group(p>0.05).4.2.3 ATP level in model group was significantly lower than that in normal group(p<0.01).ATP level in each treatment group was significantly higher than that in normal group and model group(p<0.01).ATP level in high JIANPIYIQISHEXUE group was significantly higher than that of Prednisone group and medium JIANPIYIQISHEXUE group(p<0.01).4.3 Molecular indicator regulating mitochondrial function4.3.1 SDHA in spleen,heart and brain of ITP mice in model groups were significantly higher than that of normal groups(p<0.05).SDHA in spleen,heart and brain of ITP mice in Prednisone group and high JIANPIYIQISHEXUE group were significantly lower than that of model groups(p<0.05).No significant difference of SDHA in brain was found between Prednisone group and normal group(p>0.05).These trends were consistent with those of transcriptional level testing.Western blot showed no significant trend in SDHA of liver,lung and skeletal muscle,however,qPCR showed that mRNA of SDHA in liver and skeletal muscle of model groups were higher than those in normal groups(p<0.05),and each treatment could downregulate mRNA of SDHA to different extent(p<0.05).4.3.2 ClpP in spleen,heart and skeletal muscle of ITP mice in model groups were significantly higher than that of normal groups(p<0.05).SDHA in spleen of ITP mice in each treatment group were significantly lower than that of model group(p<0.05).No significant difference of ClpP in skeletal muscle was found between Prednisone group and normal group(p>0.05),high JIANPIYIQISHEXUE group and normal group(p>0.05).No significant difference of ClpP in spleen was found between medium JIANPIYIQISHEXUE group and normal group(p>0.05),high JIANPIYIQISHEXUE group and normal group(p>0.05).ClpP in heart of Prednisone group and high JIANPIYIQISHEXUE group were significantly lower than that of model group(p<0.05).These trends were consistent with those of transcriptional level testing.4.3.3 LonP1 in spleen of ITP mice in model group was significantly lower than that of normal group(p<0.05).LonP1 in spleen of ITP mice in Prednisone group and medium JIANPIYIQISHEXUE group were significantly higher than that of model group(p<0.05).No significant difference of LonP1 in spleen was found between medium JIANPIYIQISHEXUE group and normal group(p>0.05).LonP1 in heart and skeletal muscle in model group were significantly higher than that of normal group(p<0.05).No significant difference of LonP1 in heart was found between medium JIANPIYIQISHEXUE group and normal group(p>0.05).LonP1 in skeletal muscle of Prednisone group and medium JIANPIYIQISHEXUE group were significantly lower than that of model group(p<0.05).These different translational leveltrends were consistent with those of corresponding transcriptional level testing results.5 ConclusionJIANPIYIQISHEXUEFANG could significantly relieve ITP derived fatigue,and regulate immune system.JIANPIYIQISHEXUEFANG could significantly reduce the ROS level in spleen of ITP mice and increase the ATP level in colon of ITP mice to improve the mitochondrial function of mice.The expressions of SDHA,ClpP and LonP1 in most organs and skeletal muscle could reactively increase under stress conditions caused by passive immune modeling.JIANPIYIQISHEXUEFANG could down regulate the expressions of SDHA,ClpP and LonP1 in corresponding organs,suggesting that SDHA,ClpP and LonP1 could be the molecular targets that JIANPIYIQISHEXUEFANG helps to keep the balance of mitochondrial proteins and improve the mitochondrial function.We found that spleen,liver,heart and skeletal muscle of mice were more sensitive to our experiments and treatments,which makes them preferred organs and tissues for further research. |
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