The Biological Mechanism Of Symptoms Seasonal Variation Based On Hemolysis Of Erythrocyte And Anemia Of Patients With Thalassemia Disease

Background:Thalassemias encompass a group of hereditary hemolytic hemoglobin disorders characterized by the α-or β-globin gene mutations or deletions, the most common of which are α-and β-thalassemia.There is no effective treatment approach for thalssemia.Based on the theory of "nourishing the Kidney and replenishing marrow" and through 30 years of clinical practice,the group of professor of Wu Zhi-kui put forward a clearer understanding of the theory about using chinese medicine treating thalassemia from several aspects,such as theoretical basis,core pathogenesis, treatmental method rule,the action characteristics and possible mechanisms,which formed systemic theory and effective treatmental method and achieved a leading clinical achievement.lt is effective and scientific to apply the theory of "nourishing the Kidney and replenishing marrow"to guide clinical treatment by clinical studies.The research group of Wu Zhikui surveyed TCM syndromes of 112 cases of intermedia thalassemia in Guangxi and found that the most thalassemia patients manifest liver-kidney yin deficiency syndrome and essence-blood deficiency dyndrome.The disease severity of patients with thalassemia is related seasonal variation,ie,the hemolytic anemia in summer is more serious than that in winter.Its syndrome characterized by the unchanged basic type of syndrome,but its clinical symptoms have significantly seasonal changing (the severity of sydromes such as dizziness, hot flashes, night sweats, dysphoria fever and other symptoms aggravates in summer).The seasonal change of syndrome reflects the whole concept of yin-yang of four-season in TCM.The seasonal changes in patient’s condition may be related to seasonal changes in blood parameters.The clinical reports about seasonal variations in thalassemia and studies about its biological mechanisms are seldom found.Thalassemia patients in the high incidence of Guangxi acted as the objective of the study and the research was divided into three parts.The first part content is clinical studies of season change of thalassemia patients,which included two batches of clinical studies.The first batch was clincial observation for season changes of two groups patients with HbH disease and the second batch was clinical dynamic observation of seasonal variations about the same group patients with thalassemia disease.The second part content was carried out on the basis of clinical observation and explored the molecular mechanisms of seasonal change of syndromes of thalassemia from the aspect of erythrocyte hemolysis and anemia.The third part content was to further explore the molecular mechanism of YiSui Shengxue Granule (YSSXG) to treat thalassemia from the indicators related to erythrocyte hemolysis and anemia.Objective:Thalassemia patients were acted as the objective of the study.The study explored the objective laws of the seasonal changes of sydromes of patients with thalassemia disease from blood parameters and TCM symptom score.And the study also investigetded molecular mechanisms of seasonal changes of sydromes from the point of erythrocyte hemolysis and anemia.It explored inherent relationship between seasonal variations of syndromes and erythrocyte hemolysis or anemia,which can provide experimental evidence for the Chinese "seasons" theory to guide clinical timely treatment.Method:1 Clinical observation on syndromes of seasonal changes in patients with thalassemiaThalassemia patients considered as the objective of the study and the clinical observation was divided into two parts.The first batch of clinical observation:we selected two groups of patients with HbH disease in summer (in july) and winter(in december).The winter group had 35 cases with HbH disease(including 25 patients with non-deletion,10 patients with deletion) and the summer group had 31 cases with HbH disease(including 21 patients with non-deletion,10 patients with deletion).To compare differences of blood indicators and TCM symptom score between two groups and verify initially clinical rule of seasonal changes of syndromes in patients with thalassemia. The second batch of clinical observation:To select 11 cases with thalassemia disease(5 cases of a-thalassemia,6 cases of p-thalassemia), the researcher carried out continuous observation for the 11 cases of thalassemia patients and detected dynamically the level of blood parameters and TCM symptom score in winter(in december) and summer(in july),in order to further validate the objective law of patients with thalassemia existting seasonal variations of syndromes.2 Experimental study about the molecular mechanisms of seasonal variation of syndrome based on erythrocyte hemolysis or anemia in thalassemia patientsThe experimental study was carried out on the base of the clinical studies, and we reasearched differences in the level of indicators related to erythrocyte hemolysis and anemia between winter and summer,such as the level of SI and SF,the erythrocytic membrane oxidative damage,the erythrocytic inclusion bodies,the erythrocytic membrane related enzymes activity,the hematopoietic cytokine activity,the erythrocytic membrane protein mRNA expression by using by using real-time PCR,Elisa, transmission electron microscopy(TEM),chemical color and other molecular biology techniques.We explored inherent relationship between seasonal variations of syndromes and erythrocyte hemolysis or anemia.3 Experimental study about the molecular mechanisms of YSSXG treating thalssemia based on erythrocyte hemolysis or anemiaSixteen patients with thalassemia (8 cases of α-thalassemia and 8 cases of β-thalassemia) disease were collected and treated with YSSXG for 3 months.From the point of erythrocyte hemolysis or anemia,we researched changes of globin protein mRNA expression,the erythrocytic membrane anti-oxidative damage,the erythrocytic membrane protein mRNA expression,the erythrocytic membrane related enzymes activity,the hematopoietic cytokine activity,DNA methyltransferase mRNA and protein expression,BCL11A mRNA expression before and after the treatment,in order to further explore the molecular mechanisms of YSSXG treating thalssemia.Results:1 Clinical observation on the seasonal variation of syndromes in patients with thalassemia1) Clinical observation of the seasonal variation influence on the severity of syndromes in patients with HbH diseaseThe Hb level of the winter group in patients with HbH disease is significantly higher than that of the summer group(P<0.05),and levels of Retand HbH are significantly lower than those of the summer group (P<0.05). The difference between two groups in RBC has no statistical significance.Whether the non-deletion type or deletion type of the winter group,the main blood index Hb is higher than that of the summer group.TCM syndrome quantitative score of sallow complexion,onyx color light, tinnitus, oropharyngeal drying of the winter group are significantly lower than those of the summer group(P<0.01 or P<0.05).There are no differences in TCM syndrome quantitative score of soreness and weakness of waist or knees,night sweats between two groups.TCM syndrome quantitative score of pale complexion, tinnitus, oropharyngeal drying of the non-deletion type in winter group are significantly lower than those of the summer group.TCM syndrome quantitative scores of sallow complexion,onyx color light,soreness and weakness of waist or knees,oropharyngeal drying of the deletion type in winter group are significantly lower than those of the summer group.Whether the non-deletion type or deletion type of the winter group,the majority of TCM Syndrome quantitative scores are lower than the summer group,indicating that the symptoms of HbH patients in summer group were more serious than those of the winter group which is consistent the level of Hb of two groups.2) Clinical observation of the seasonal variation influence on the severity of syndromes in 11 cases patients with thalssemia diseaseThe the winter Hb level of 11 cases patients with thalassemia is significantly higher than that in summer(P<0.05).There are no differences in blood indexes RBC、 Ret between two seasons.TCM syndrome quantitative scores of onyx color light,eat less,night sweats of 11 case of patients with thalssemia in winter are significantly lower than those in summer(P<0.05),and there are no differences in TCM syndrome quantitative scores of dizziness,languid,soreness and weakness of waist or knees between two seasons.Clinical studies showed that:the clinical symptoms of erythrocytic hemolysis and anemia in patients with thalassemia surely exits seasonal variations,and the severity of the clinical symptoms is consistent with the level of the main blood index Hb level.2 Experimental study about the molecular mechanisms of seasonal variation of syndrome based on erythrocyte hemolysis or anemia in thalassemia patients1) The levels of serum iron (SI) and ferritin (SF) in summer and winter were showed that:The levels of SI,SF of the summer group of 31 cases patients with HbH disease is significantly higher than those of the winter group of 35 cases patients with HbH disease(P<0.05).The summer levels of SI,SF in 11 cases patients with thalassemia have a tendency of higher than those of the winter.2) The levels of the MDA contents and antioxidative enzyme acivities in summer and winter were showed that:The SOD acivity of the winter group of 35 cases patients with HbH disease is significantly higher than that of the summer group of 31 cases patients with HbH disease.The activity of GSH-Px and the MDA content have no significant differnce between the winter and the summer group.The winter activities of SOD,GSH-Px in 11 cases patients with thalassemia are significantly higher than those of the winter,and the MDA content is markedly lower than that of the winter.3) The level of erythrocytic inclusion body content in summer and winter were showed that:We can discover that there are plenty of erythrocytic inclusion bodies in patients with thalassemia in summer,and we also can find that in the red cells there are several erythrocytic inclusion bodies in the winter,but the amount of the winter is less than that of the summer.4) The level of erythrocytic membrane protein mRNA expression in summer and winter were showed that:The mRNA expression levels of SPTA1,SPTB,EPB4.1 of the winter group of 35 cases patients with HbH disease are significantly higher than those of the summer group of 31 cases patients with HbH disease(P<0.01).The mRNA expression level of SPTA1 in 11 cases patients with thalassemia is significantly higher than that of the winter(P<0.05),and the winter levels of SPTB,EPB4.1 in 11 cases patients with thalassemia have a tendency of higher than those of the summer.5) The level of hematopoietic cytokine activities in summer and winter were showed that:The SCF acivity of the winter group of 35 cases patients with HbH disease is significantly higher than that of the summer group of 31 cases patients with HbH disease(P<0.05).The activity of IL-3 has no significant differnce between the winter and the summer group.The activities of SCF,EPO in 11 cases patients with thalassemia in the winter are significantly higher than those of the summer(P<0.05 or P<0.01),and the IL-3 content is markedly lower than that of the summer(P<0.05).6) The level of erythrocytic membrane related enzymes activity in summer and winter were showed that:The acivities of Na+-K+-ATPtase,T-ATPase of the winter group of 35 cases patients with HbH disease are significantly higher than those of the summer group of 31 cases patients with HbH disease(P<0.05).The activity of Ca2+-Mg2+-ATPtase has no significant differnce between the winter and the summer group.The activities of Na+-K+-ATPtase,T-ATPase in 11 cases patients with thalassemia in the winter are markly higher than those of the summer(P<0.05 or P<0.01),and the activity of Ca2+-Mg2+-ATPtase has no significant differnce in the winter and summer.The results of the experimental study showed that:The influence of seasonal variation on levels of indicators about erythrocyte hemolysis or anemia may be the material basis of syndromes seasonal variation in patients with thalassemia.3 Study about the molecular mechanisms of YSSXG treating thalssemia based on erythrocyte hemolysis or anemia3.1 Clinical observation about YSSXG treating 16 cases of patients with thalssemia(8 cases of α-thalassemia and 8 cases of β-thalassemia)1) Comparison of blood parameters before and after treatment:Compared with pre-treatment measurements for 8 cases with α-thalassemia,levels of Hb concentrations after 2 to 3 months treatment and RBC counts after 3 months treatment were significantly increased (P<0.01 or P<0.05),and the Ret count markedly decreased (P<0.05).Compared with pre-treatment measurements for 8 cases with β-thalassemia,levels of HbF were significantly increased after lto 3 months treatment; Hb concentrations and RBC counts have kept increasing after treament; and the Ret count has an obvious increase after 2 months of post-treatment.2) Comparison of TCM symptoms scores before and after treatment:Scores of TCM symptoms,such as Pale complexion,tidal fever and night sweating, dizziness,Palpitation,dry throat decreased significantly compared with those of pre-treatment(P<0.01 or P<0.05) and the syndrome score of soreness and weakness of waist or knees has a decrease tendency compared with that pre-treatment.3.2 Study on molecular mechanisms of YSSXG treating thalssemia1) Comparison of globin chain ratio for thalassemia:Fora-thalassemia patients,the relative level of α-globin to β-globin expression is markedly increased(P<0.01);Aγ or Gγ-globin toβ-globin have no statistical change.For β-thalassemia patients,the relative level of Aγ-globin to α-globin expression has an increasing trend.2) Comparisons of the MDA content and anti-oxidant demage enzymes activity:activities of erythrocytic SOD and GSH-Px increase significantly (P<0.01),and the erythrocytic MDA content obviously decrease(P<0.01).3) Comparison the level of erythrocyte membrane skeletal proteins mRNA expressions:The level of SPTA1,SPTB mRNA expression are markedly increased (P<0.01),and EPB4.1 mRNA has no statistical change.4) Comparison levels of activities of Na+-K+-ATPtase,Ca2+-Mg2+-ATPtase and T-ATPase:The levels of activities ofNa+-K+-ATPtase,T-ATPtase increase significantly when compared with those of pre-treatment(P<0.05),and Ca2+-Mg2+-ATPtase activity had no significant chang.5) Comparison levels of hematopoietic cytokine activities:Activities of SCF,GM-CSF increase significantly and the activity of IL-3 decrease markly when compared with those of pre-treatment(P<0.01).6) Comparison the level of DNA methyltransferase expressionsrFor 8 cases of a-thalassemia patients,levels of mRNA expression of DNMT1, DNMT3A, and DNMT3B and protein expression of DNMT1 and DNMT3A significantly decreased after treatment with YSSXG(P<0.05 or P<0.01).The protein expression of DNMT3B had a decreasing trend compared with that of pre-treatment.7) Comparison the level of BCL11A mRNA expressions:For 8 cases of β-thalassemia patients,the level of BCL11A mRNA expression which negatively regulates HbF expression markedly decreased(P<0.05).The positive clinical efficacy and changes of indicators related to erythrocyte hemolysis and anemia suggest that YSSXG can promote erythro--poiesis,extend the life of RBC,and reduce erythrocyte hemolysis.Conclusions:1 Clinical observation on seasonal changes of syndromes in patient with thalassemiaSeasonal changes influence on the main blood parameter Hb and syndrome quantitative score in patients with thalassemia showed that:The Hb level of patients with thalassemia in winter is significantly higher than that of the summer. The majority of TCM Syndrome quantitative scores in winter are lower than those of the summer,indicating that the symptoms of HbH patients in summer are more serious than those of the winter which is consistent the level of Hb of two groups,which verify that syndrome severity exists the objectivity of seasonal variation. Clinical observations suggest that the seasonal variation influences hemolytic and anemia symptoms and the severity of the clinical symptoms is consistent with the level of Hb,which indicates the hemolysis in high temperature season is more serious than that of in lower temperature season.2 Experimental study on the correlation between seasonal variation of syndromes in patients with thalassemia and measurements of indicators related to hemolytic and anemia1) Measurements of SI,SF showed that:The levels of SI,SF in summer is higher than those of the winter, which indicates that the possible mechanism of the hemolysis and anemia in summer more serious than of the winter is related to patients with thalassemia exites serious iron overload.2) Measurements of the MDA content and anti-oxidative damage enzyme activities showed that:Activities of erythrocytic SOD, GSH-Px in winter are higher than those of the summer,and the MDA content in winter is lower than that of the summer. The more lipid oxidative MDA aggravates oxidative damage,and stimulates the body’s compensatory mechanisms,which make levels of SOD,GSH-Px to compensatory increase.3) Measurements of erythrocytic inclusion bodies content showed that:The content of inclusion bodies in summer is more than that of the winter.Inclusion bodies in the erythrocytic membrane can lead to erythrocytic hemolysis and anemia by oxidation and mechanical damage,which may be one of reasons why patients with thalassemia exists the more serevity of hemolysis and anemia in summer.4) Measurements of erythrocytic membrane skeletal protein mRNA showed that:Expression levels in patients with thalassemia in winter are higner than in summer.Erythrocyte membrane skeletal proteins are essential to maintain the stability of the structure and morphology of RBC,suggesting that patients with thalassemia existing syndromes seasonal changes is related to the mRNA expression difference in winter and summer.5) Measurements of hematopoietic cytokine activities showed that:Activities of SCF,EPO in patients with thalassemia in summer are lower than those of the winter.SCF and Epo can cooperate to promote the proliferation and differentiation of erythroid, and improve symptoms of anemia.6) Measurements of erythrocyte membrane related enzyme activities showed that:Activities of Na+-K+-ATPase,T-ATPase in summer are significantly lower than those in winter.Erythrocyte membrane ion permease can affect the structure and stability of RBC.3 Study on the correlation between clinical efficacy and indicators of erythrocytic hemolysis and anemia by using YSSXG in treating thalassemia3.1 Clinical boservation on treating 16 cases of thalassemia with YSSXGYSSXG can effectively improve the clinical symptoms and increase the level of the main blood parameter Hb of patients with thalassemia. We can discover that the degree of improvement in clinical symptoms is consistent with the level of Hb.The clinical study shows that YSSXG is effective in treating thalassemia disease. 3.2 Experimental study about the influence of YSSXG on indicators related to the erythrocytic hemolysis and anemia in patients with thalassemia1) The detecting measurements of globin chains ratio showed that:Forα-thalassemia patients,the relative expression level of α-globin to β-globin was markedly increased.Forβ-thalassemia patients,the relative expression level of Aγ-globin to α-globin had an increasing trend.YSSXG may improve symptoms of erythrocytic hemolysis and anemia by promoting globin chain ratio balance of patients with thalassemia.2) The detecting measurements of DNA transferase mRNA and protein expression showed that:YSSXG can downregulate DNA methyltransferase mRNA and protein expression for patients with α-thalassemia,which can ease DNA methylation inhibiting the α- globin transcription promoter region,and thus can increase the synthesis of α- globin chains and improve erythrocyte hemolysis and anemia.3) The detecting measurements of BCL11A mRNA expression showed that:YSSXG can downregulate BCL11A mRNA expression for patients with P-thalassemia, which can indicates that YSSXG may promote Ay-globin mRNA expression to increase the synthesis of HbF,and thus relive symptoms of erythrocytic hemolysis and anemia by downregulating the BCL11A mRNA expression.4) The detecting measurements of the MDA content and anti-oxidative enmyze showed that:The activities of SOD,GSH-Px are markly increased and the content MDA is obviously decreased after YSSXG treatment.YSSXG can inhibit free radical damaging erythrocytic membrane,maintain the structure and function integrity of erythrocytic membrane,prolong the life of RBC,and alleviate the erythrocytic hemolysis and anemia by increasing the anti-oxidant damage enmyze activity.5) The detecting measurements of erythrocytic membrane skeleton mRNA expression showed that:The epxression levels of SPTA1,SPTB are significantly increased after YSSXG treatment,whcih indicates that YSSXG can maintain the structure integrity of erythrocytic membrane by upregulating erythrocytic membrane skeleton mRNA expression.6) The detecting measurements of erythrocytic membrane related enmyzes showed that:The activity levels of Na+-K+-ATPase,T-ATPase are significantly increased after treatment,which indicates that YSSXG may increase activities of erythrocytic membrane related enmyzes by reducing the injury by peroxidation products to erythrocyte membrane,and thus can be better to maintain the erythrocytic membrane fluidity, deformation,integrity in order to reduce hemolysis anemia.7) The detecting measurements of hematopoietic cytokine activity showed that: The hematopoietic positive regulator cytokines activities of SCF,GM-CSF are significantly increased and the activity of IL-3 is markly decreased after treatment. YSSXG may promote the proliferation and differentiation of erythroid progenitor cells and a variety of hematopoietic cell,enhance effective,and relieve symptoms of patients with thalassemia by increasing the activities of SCF,GM-CSF.The main innovation:As the breakthrough point to seasonal variations influence to disease for the the first time to explore the impact of seasonal variations on the clinical manifestations of thalassemia patient from two aspects of the main blood parameters and TCM quantitative score,we find that the level of Hb in summer is lower than that of the winter,and the severity of TCM in summer are serious than those of the winter, which indicates that the impact of seasonal variations on the clinical manifestations is consistent with the level of Hb level and verify the fact that the disease severity of patients with thalassemia is related seasonal variation, ie,the hemolytic anemia in summer is more serious than that in winter.We also explore the possible molecular mechanism of that change.The results rich Chinese "four seasons" theory and provide experimental evidence for clinical timely treatment of thalassemia,which has an important theoretical and practical significance.