| Fuzi is the processed goods of Aconitum carmichaeli Debx, which belongs to ranunculaceae plants. Fuzi is pungent in flavor, hot in nature, and toxic. It acts on the heart, spleen and kidney channels. The effects of Fuzi are reviving yang for resuscitation, supplementing fire and Yang, dispersing cold and alleviating pain. It can be used in clinic for depletion of yang, collapse and yang deficiency. Modern researches have proved that Fuzi has the effects of strengthening the heart, resisting arrhythmia, anti-inflammation, analgesia, etc. It can be used for shock caused by acute myocardial infarction, hypotension, CHD and angina pectoris in clinic etc, now. Fuzi was recorded in Shen Nong Materia Medica firstly, and it was used as low-grade because of its toxicity. So processing technology of Fuzi is being emphasized, and in Chinese medicine Fuzi is one example preserving effects and decreasing toxicity through processing. Modern researches have found that AC,MA and HA are the main components of Fuzi. And these DDAs of Fuzi are easy hydrolysis during processing which will reduce toxicity of Fuzi.There are many kinds of methods for Fuzi processing. Now the limit- examines methods of aconitine, is used as the main quality control method of Fuzi. But the influence of processing, TCM theory on quality, effects, toxicity and safety of Fuzi is ignored. So the effects of Fuzi decreased just because of excessive processing, and the current standards of processing do not pay attention to the effects of Fuzi.As one part of the 10th Five Years Key Programs for Science and Technology Development of China, the former study results indicated that the concentration of AC, MA and HA would decrease with the processing time prolonging. But HA and MA would still exist during the long processing time. It was also found that DDAs had toxicity and the effect of strengthening heart, and HA was the safest one. All of these show that HA perhaps can be used as the new standard for evaluation of Fuzi.In view of the results above, the safest processed Fuzi, which has the special effect of reviving yang for resuscitation (RYFR), will be selected through the safety evaluation to Fuzi processed during different time. The correlation between content changes of AC,HA & MA and TD50,ED50 of Fuzi processed during different time will be analyzed with multiple correlation analysis. And then, the toxic effect of HA on the cultured cardiac cells will be observed. At last, I will study the possible mechanism of HA-induced cardiotoxicity. 1 Research on the correlation between changes of DDAs content and safety of the processed FuziClinical effect is influenced by the quality of processed Fuzi. It is important for Fuzi to establish the quality assessment. Based on the RYFR of Fuzi, the acute toxicity on heart and cardiotonic effect of the processed Fuzi was observed in my research. The safety of 7 kinds of Fuzi processed during different time was evaluated according to TD50, ED50 and TI. The key component related to RYFR and acute toxicity on heart of Fuzi would be found through the correlation analysis between the changes of AC, MA, HA and TD50, ED50 of the processed Fuzi.1.1 Study the acute toxicity of Fuzi processed during different time on heart in ratsAccording to the changes of rats'ECG after duodenal administration during 30 min, the cardiac toxicity of 7 kinds of Fuzi processed during different time was evaluated. The result showed that the cardiac toxicity of Fuzi decreased gradually with the increasing of the processing time. The TD50 of these 7 kinds of Fuzi processing during different time are 0.4500g/kg, 0.2185g/kg, 0.4925g/kg, 0.6190g/kg, 2.399g/kg, 2.009g/kg and 4.4060g/kg respectively.1.2 Effect of Fuzi processed during different time on improving cardiac function of ratsThe changes of rats'cardiac function in 120min, such as +dp/dtmax, LVSP, t-dp/dtmax, HR, were observed with MedLab-U after duodenal administration. According to the changes of these indexes in 30, 60, 90,105 and 120min, it is found that the 7 kinds of processed Fuzi can improve cardiac function of rats to some extent.1.3 Safety evaluation of Fuzi processed during different timeThe ED50 (+dp/dtmax & LVSP)of 7 kinds of Fuzi processed during different time were calculated with Bliss methods. According to the formula that TI=TD50/ED50,the safety ranges of these processed Fuzi were calculated. The results show that the cardiotonic action of the processed Fuzi is weakened gradually with the increasing of the processing time. And Fuzi with high effect and low toxicity must be processed 100min.1.4 Study on correlation analysis of content changes and safety of Fuzi processed during different timeTo study the relationship between ED50,TD50,TI and content changes of DDAs of 7 kinds of the processed Fuzi,and analyze the correlation between the content changes and effect, toxicity of these processed Fuzi with multiple linear regression analysis, it is found that the decreasing of toxicity and effect of these processed Fuzi is related with the decreasing of DDAs. In the processed Fuzi with high effect and low toxicity (No.5), which has been processed 100min, AC has been not detected, proper MA and HA still existed. In addition, AC is positive correlation with TD50; HA is positive or no correlation with TD50; MA is positive correlation with TD50.HA is positive correlation with ED50 (LVSP or +dp/dtmax).But AC and MA correlation results are different because of analysis methods.Summary: The processed Fuzi with high effect and low toxicity (No.5), which has been processed 100min, is better than others in RYFR and safety. HA can be used as the new standard of processed Fuzi reviving yang for resuscitation.2 Toxicity research on the primary cultured cardial cells of HA According to the results above, there was significant correlation between HA and the special effect of RYFR, toxicity of the processed Fuzi. So it is very important for standardized processing of Fuzi to study the toxic effect of HA and the possible mechanism of HA-induced cardiotoxicity.2.1 Establishment of cultured cardiac cellsThe survival rate of cardiac cells was over 90% by Trypan blue dying. Under the invert microscope, cardiac cells growed and attached wall, like spindle, diamond or polygon, and extended pseudopodia, aggregate and had spontaneous beating. After culturing 48 hrs, cardiac cells were tained positive toα-Sarcomeric actin and negative for Fibronectin. The purity of myocardial cells was close to 95%.2.2 Effect of HA on the beating rate of cardiac cellsThe beating rate changes of cardiac cells after co-incubation with different concentration of HA were observed under microscope in 5, 15, 30 and 60min.after culturing 4 days, the beating rate of normal cardiac cells was near 50~70 time/min. The effect of HA on the beating rate of cells was time and dose dependent. 6~60 M/L of HA increased the beating rate of cells during 5~30min. The stopped beating of cells occurred when the dose of HA was over 60 M/L . 2.3 Effect of HA on the survival rate of cardiac cellsThe survival rate of cardiac cells after co-incubation with different concentration of HA was observed with MTT. Compared with control, survival rate of cells was decreased 15% after co-incubation of 120 M/L of HA in 60min.2.4 Effect of HA on the release rate of LDH of cardiac cellsThe increasing of release rate of LDH can be used to reflect the changes of cell permeability and cell injury. During 15~60min after medication, the release rate of LDH increased with the dose increasing of HA, but no time- dependence.2.5 Effect of HA on the morphology of cardiac cellsWith the dose and time increasing, it can be found that under microscope, cardiac cells appeared to be larger with vacuolar changes in cytoplasm like"cauliflower",pseudopodia reducing and cell shrinkage after co-incubation with HA. Some cardiac cells died of losing wall and floated in the culture medium. Summary: The cell injury induced by HA is related with the dose of HA and the time of co-incubation. 6~60 M/L of HA increased the beating rate of cells during 5~30min. Over 30min, the beating rate of cells decreased, and the release rate of LDH, the survival rate of cardiac cells increased after co-incubation with HA. The stopped beating of cells occurred when the dose of HA was over 60 M/L, and the release rate of LDH and the survival rate of cardiac cells increased continuously and badly.3 Effect of HA on expression of mRNA of calcium regulated proteins of cardiac cellsCalcium overload and calcium regulating disorder in cardiac cells are the main mechanism of arrhythmia. The abnormal expression and dysfunction of L-type calcium channel, sodium channel, CaM, RyR2 and NCX will damage the calcium steady in cardiac cells, which will lead to arrhythmia.3.1 Effect of HA on the fluorescent intensity of the Ca2+ in cardiac cellsCombined with Fluo-3-AM, the concentration of free Ca2+influenced by HA in cardiac cells can be detected by flow cytometry. The results show that 30~120Μ/L of HA increased the concentration of free Ca2+ , and the calcium overload in cardiac cells happened in 15min after co-incubation with HA(P<0.01). 3.2 Effect of HA on expression of L-type calcium channelα1C,SCN5A & NCX mRNA in cardiac cellsReal-time PCR is used to detect the expression of L-type calcium channelα1C, SCN5A and NCX mRNA in cardiac cells after co-incubation with HA, and then the changes of these above mRNA during different time(5min,15min,60min) were compared. The expression of L-type calcium channelα1C, SCN5A and NCX mRNA of cardiac cells increased after co-incubation with 60Μ/L and 120Μ/L of HA in 15min(P<0.05),compared with control. The mRNA expression of L-type calcium channelα1C increased after co-incubation with 60Μ/L of HA in 5min(P<0.05) and the mRNA expression of SCN5A increased after 30min(P<0.05).3.3 Effect of HA on expression of CaM mRNA of cardiac cellsReal-time PCR is used to detect the expression of CaM mRNA in cardiac cells after co-incubation with HA, and then the changes of CaM mRNA during different time(5min,15min,60min) were compared. The expression of CaM mRNA of cardiac cells increased after co-incubation with 30~120Μ/L of HA in 15min(P<0.01),compared with control, no dose and time-dependence. The mRNA expression of CaM increased after co-incubation with 30Μ/L of HA in 15min(P<0.01)and the mRNA expression of CaM increased after 15~60min(P<0.01).3.4 Effect of HA on expression of RyR2 mRNA of cardiac sarcoplasmic reticulumReal-time PCR is used to detect the expression of RyR2 mRNA in cardiac sarcoplasmic reticulum after co-incubation with HA, and then the changes of RyR2 mRNA during different time(5min,15min,60min) were compared. The expression of RyR2 mRNA of sarcoplasmic reticulum increased after co-incubation with 60Μ/L and 120Μ/L of HA in 15min(P<0.05),compared with control, no time-dependence.Summary: The expression of L-type calcium channelα1C,SCN5A, NCX, CaM and RyR2 mRNA in cardiac cells increased after co-incubation with 60Μ/L and 120Μ/L of HA(P<0.01 or P<0.05).The increasing expression of mRNA of these calcium regulated proteins will enhance activities of these calcium regulated proteins and increase the concentration of free Ca2+in cardiac cells through CICR. The calcium overload in cardiac cells happens, then. In addition, the threshold concentration of HA regulating these calcium regulated proteins is near 60Μ/L. The expression of mRNA of these calcium regulated proteins will increase when the dose of HA is over this threshold concentration (P<0.01 or P<0.05).The result shows that HA may induce arrhythmia through Ca2+/CaM pathway in cardiac cells.4 Effect of HA on expression of Connexin43 in cardiac cellsThe expression of Cx43 in cardiac cellls was observed after co-incubation with HA through Western-Blot. The results show that the expression of Cx43 in cardiac cells decreased after co-incubation with HA in 15min.This effect seems to be dose-dependent.Summary:HA can decrease the expression of Cx43 in cardiac cells, and the effect is dose-dependent. The decreasing expression of Cx43 will increase the susceptibility to arrhythmia. But it also may be a self-protective reaction of cardiac cells.Conclusion:1.The safest processed Fuzi has been selected through the safety evaluation, which has the special effect of RYFR.2.HA can be used as the new standard to evaluate the quality of the processed Fuzi3.HA can influence the beating rate, the release rate of LDH and the survival rate of cardiac cells.4.HA can lead to calcium overload in the cardiac cells, which will result in arrhythmia5.HA can lead to the increasing expression of mRNA of L-type calcium channel, SCN5A, NCX, CaM and RyR2.6.HA can lead to the decreasing expression of Cx43, which will increase the susceptibility of arrhythmia.and is a self-protective reaction of cardiac cells7.AC, MA and HA can lead to arrhythmia, and influence the beating rate, the release rate of LDH and the survival rate of cardiac cells.8.The mechanism of arrhythmia induced by AC is that AC has the activating effect on the sodium channels of cardiac cells and AC can increase expression of mRNA of L-type calcium channel, SCN5A, NCX, CaM and RyR2. The mechanism of arrhythmia induced by HA is similar to AC. And it is observed that HA can activate L-type calcium channel of cardiac cells, and decrease the expression of Cx43.Innovation:1.It is first time to find the safest processed Fuzi which has the special effect of RYFR through safety evaluation. HA can be used as the new standard to evaluate the quality of the processed Fuzi.2.It is first time to observe the time-toxicity and dose-toxicity relationship of HA inducing cardiotoxicity.3.It is first time to research the effect of CJIC in arrhythmia induced by HA.
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