Analysis Of Tongue Features In Elderly Patients With Fever From Acute Myeloid Leukemia And Effect Of Realgar On The Expression Of Mitochondrial Apoptosis Factors(Bcl-2、Bax、Cyt-C And AIF)in NB4 Leukemia Cells

Objective:In this study,acute myeloid leukemia in the elderly was studied,we hope to further explore the etiology,pathogenesis and clinical treatment of Acute Myeloid Leukemia（AML）in the elderly from the systematic perspective of principle-method-recipe-medicines in traditional Chinese medicine.1.The principle-method-recipe-medicines in traditional Chinese medicine,in the principle and method point of view,in this study,the TCM syndromes and tongue features of elderly patients with fever of acute myeloid leukemia were selected as the entry point,so as to achieve a more accurate description of the etiology,pathogenesis and syndrome type of TCM treatment for elderly patients with acute myeloid leukemia,and provide certain basis for the clinical dialectical treatment of TCM;2.The principle-method-recipe-medicines in traditional Chinese medicine,in the recipe and medicines point of view,In this study,the characteristic mineral realgar（As₄S₄）was selected as the starting point,through its effect on the expression of mitochondrial mediated apoptotic proteins Cyt-c,Bcl-2,AIF and Bax in NB4 leukemia cells,to explore the targeted regulatory effect of realgar on mitochondrial mediated apoptosis process,and to provide some experimental evidence for the targeted treatment of acute myeloid leukemia（AML）with realgar,so as to seek some theoretical basis for the development of new traditional Chinese medicine.Methods:1.Study and analysis of tongue characteristics of patients with acute myeloid leukemia fever:Selection of affiliated hospital of shandong university of TCM in January 2013-February 2018 were diagnosed with acute myeloid leukemia cells in elderly patients with fever,quasi observation cases 80 cases,including moderate and high fever group（40 cases temperature is greater than or equal to 38℃）,low thermal group（37℃temperature is greater than,less than 38℃）40 cases.Diagnostic criteria for acute myelogenous leukemia according to the blood disease diagnosis and curative effect of standard diagnostic criteria,TCM syndrome types reference in November 2001 for the fourth time physicians haematology professional committee of China association of Chinese medicine academic conference proposed leukemia syndrome diagnostic standard（trial scheme）and Shandong university of Chinese medicine affiliated hospital onset of the consumptive disease（acute leukemia）of traditional Chinese medicine diagnosis and treatment plan（trial edition）2012.Analyzes the TCM syndrome type and truthfully fill out the tongue like a watch list（see the appendix）,detailed observation records of the patient’s tongue like characteristics of tongue like content includes:the tongue color,tongue and the tongue coating（tongue coating color,the thickness of the coating on the tongue,tongue coating properties）three big aspects.2.The effects of Chinese herbal realgar（As₄S₄）on Bcl-2,Bax,Cyt-C and AIF expression of mitochondrial mediated apoptotic factors in NB4 cells:（1）By using ck-8 method,the increment effect of the NB4 cells in the logarithmic growth period under the gradient concentration and time of different effects was detected by using ck-8 method,and the corresponding IC50 value was detected.（2）Develop NB4 cell lines,realgar（As₄S₄）on NB4 cells（72h,0.075mg/ml As₄S₄）,immunofluorescence test to detect the mitochondria mediated apoptosis related factors of Cyt-C,AIF,Bcl-2,Bax expression level.（3）Develop NB4cell lines,with different concentrations and different time points realgar（As₄S₄）acted on NB4 cells,and the expression levels of Bcl-2,Bax,Cyt-C and AIF of mitochondrial mediated apoptosis were detected by Western-blot method.Results:1.Study and analysis of the tongue characteristics of the elderly patients with acute myeloid leukemia fever:（1）In elderly patients with myelogenous leukemia(low grade fever is much on TCM syndrome type of qi and Yin deficiency type 23 cases（57.5%）,old myelogenous leukemia patients with high fever on the TCM syndrome type of poison type heat flourishing 28 cases（70%）;（2）In elderly patients with myelogenous leukemia feve low grade fever group,the maximum frequency of tongue color is the pale 16 cases（40.0%）,decreasing in turn:the reddish tongue 10 cases（25.0%）,the red tongue 7 cases（17.5%）,the dark purple tongue 7 cases（17.5%）,the crimson tongue 0 cases（0%）.The maximum frequency of tongue appearance is the normal tongue 20 cases（50%）,descending in turn:the swelling tongue 12 cases（30.0%）,the thin tongue 5 cases（12.5%）,and the shrink 3 cases（12.5%）.The maximum frequency of tongue coating is the less moss 20 cases（50%）,descending in turn:the thin moss 8 cases（20.0%）,the thick moss 7 cases（17.5%）,the no moss 5 cases（12.5%）.The maximum frequency of tongue coating color is the white 21 cases（52.5%）,descending in turn:the yellow 12 cases（30.0%）,the gray 4 cases（10.0%）,and the black3 cases（7.5%）.The maximum frequency of tongue coating is the dry moss 23case（57.5%）,descending in turn:the moisturizing moss 10 cases（25.0%）and the greasy moss 7 cases（17.5%）;（3）In elderly patients with myelogenous leukemia fever high fever group,the maximum frequency of tongue color is crimson tongue 19 cases（47.5%）,decreasing in turn:the red tongue 7 cases（17.5%）,the dark purple tongue 6 cases（15%）,the reddish tongue 5 cases（12.5%）,the pale tongue 3 case（7.5%）.The highest frequency of tongue appearance is the thin tongue 19 cases（47.5%）,descending in turn:the normal tongue 14 cases（35.0%）,the shrink tongue 4 cases（10.0%）,and the selling tongue 3 cases（7.5%）.The highest frequency of tongue coating is less moss 18 cases（45%）,decreasing in turn:thin moss 9 cases（22.5%）,no moss 7 cases（17.5%）,and thick moss 6 cases（15.0%）.The highest frequency of mossy color is the yellow 21（52.5%）,descending in turn:the white 9cases（22.5%）,the black 7cases（17.5%）,and the gray 3 cases（7.5%）.The highst frequency of tongue coating is dry moss 28 cases（70.0%）,descending in turn:the moisturizing moss 6 cases（15.0%）and the greasy moss 6 cases（15.0%）.2.Effects of Chinese traditional Chinese medicine realgar（As₄S₄）on Bcl-2,Bax,Cyt-C and AIF of mitochondrial mediated apoptotic factors in NB4 cells:（1）CCK-8 results:realgar inhibited the proliferation of acute myeloid promyeloid NB4 cell line in the logarithmic growth phase,and was positively correlated with the time and concentration of action.The results of the experimental group and the control group were different（P<0.05）,which was statistically significant.（2）immunofluorescence results:realgar can down-regulate the expression of apoptosis-related protein Bcl-2mediated by mitochondria in NB4 cell line,and up-regulate the expression of Cyt-C,AIF and Bax,showing a significant difference（P<0.01）compared with the control group.（3）Western blot method results:realgar can down-regulate the expression of apoptotic protein Bcl-2 mediated by mitochondria in NB4 cell lines,and up-regulate the expression of Cyt-C,AIF and Bax,which is different from the control group（P<0.05）,with statistical significance.Conclusion:1.（1）The patients with low fever of myeloid leukemia in the elderly are mostly Yin and Yin deficiency in TCM syndromes,and the patients with high fever in the elderly myeloid leukemia are mostly venomous in the TCM syndrome type;（2）The tongue image of senile myeloid leukemia patients with low fever is mainly manifested as pale white tongue,normal tongue body,little moss,white tongue,and dry moss.Its tongue image is more in line with qi-Yin deficiency type tongue image.（3）The tongue image of the elderly patients with myeloid leukemia is mainly characterized by tongue color,thin tongue,little moss,yellow tongue,and dry moss.Its tongue image is more in line with the toxic heat incandescent type of tongue image.2.Realgar（As₄S₄）single drug in the study showed a certain degree of antitumor activity,the early acute myeloid granulocyte can inhibit the proliferation of NB4 cell lines,make NB4 cells and mitochondria mediated apoptosis related factor to downgrade the Bcl-2 expression,Cyt-C,AIF,Bax expression.The results suggest that mitochondria may be an important target for realgar to promote the apoptosis of NB4 cells.