| Objective: To study the intervention effect of Terminalia chebula on H9C2 myocardial cell injury caused by aconitine,the main component of Terminalia chebula,and to clarify the toxicity of Terminalia chebula and its protective effect on the heart by two different methods.The changes in the expression of gene and protein at the cellular level revealed the mechanism by which Terminalia chebula alleviated the damage of myocardial cells caused by aconitine,the main component in Aconitum chebula,and further clarified the mechanism by which Terminalia chebula could relieve the toxicity of Aconitum chebula.Method: Experiment 1: After the H9C2 cardiomyocytes were overgrown by 80% to 90%,the culture medium was dumped and washed twice with PBS,then 0.25% trypsin of 1ml was added for digestion.The petri dishes were placed in the incubator for 2min,after which twice the amount of complete medium was added to stop digestion.The disassembled cells in the dish were blown down with a microsampler,sucked into a 15 ml centrifuge tube,and centrifuged at a speed of 1000r/min for 5min.The cell supernatant after centrifugation was poured away,and proper amount of complete medium was added for blowing.Draw 1ml of the beaten cell suspension with a microsampler and calculate with a cell counter.After adding proper amount of complete medium to dilute the cells to 1× 104 per well,H9C2 cardiomyocytes were randomly sewed into 96-well plates(100 μL/well).When the 96-well plate was filled with cells,the serum-free medium was replaced for 12 hours,and then different concentrations of aconitine were given to further screen the toxicity intensity and action time of aconitine on H9C2 cardiomyocytes,and IC50 was determined.After the concentration of aconitine injury was determined,myocardial cells were treated with aconitine injury in advance,and then treated with different concentrations of Terminalia chebula water extract and Terminalia chebula alcohol extract in 96-well plates for further culture for 6h,12 h,24h and 48 h.Then the liquid was absorbed and added into serum-free medium of 100 μL and CCK-8 of 10 μL for further culture for 2h.Two hours later,the OD value was determined by enzyme plate analyzer,and the optimal concentration and time of the two extracts were screened out.The release of LDH,CK-MB,Ca+,SOD and the content of MDA and apoptosis of cells were determined to further reveal the detoxification effect of Terminalia chekrae on aconitine in the aspects of enzyme and oxidative damage.Experiment 2: As in Experiment 1,cells were diluted to 3× 105 per well with proper amount of complete medium,and then H9C2 cardiomyocytes were randomly planted in 6-well plates(2ml/ well).When the cells were filled with 6-well plates,the serum-free medium was replaced for 12 hours,and then the aconitine was given for damage for 24 hours.After two times of washing with PBS,the water extract and alcohol extract of Terminalia chebula extracted by two different methods were used for damage protection.After administration,wash twice with precooled PBS,add 500μl RNAISO PULS to each well,and stand for 5min.The sample was absorbed by a microsampler into the EP tube and put in the tube for 5min.Then,chloroform in the volume of RNAISO Puls1/5 was a dded.After the lid was put on,it was shaken violently for 15 s.After centrifugation,carefully absorb the supernatant into the new EP tube,then add isopropanol of the same volume to be gently disturbed and placed for 10 min.After that,put it into the centrifuge and centrifuge for 10 min at a speed of 12000/min.After centrifugation,there will be precipitation at the bottom of the tube.Dispose the supernatant,add 1ml 70% ethanol,cover the lid,vibrate up and down,and then put it into the centrifuge and rotate at 12000/min for 5min.Discard the supernatant,leave the lid open for 5-10 min,volatilize the ethanol,add in 20μL RNASE-FREE WATER,and mix well.The RNA concentration of each group was measured by RNA concentration measuring instrument,and the q PCR loading volume was 3-5μL.The prepared mixture was added ℃to the eight tubes,mixed evenly with a microsampler and incubated at 42 for 2min with a thermal circulator.Add 5μl 4× ⅢHifair Supermix Plus and incubate for 5min at 25℃ ℃,15 min at 55 and 5min at 85℃.The resulting c DNA was used to prepare the mixture with a description of the reaction system.Add 2μl sample and 18μl reaction solution to the eighth tube.Real-time fluorescence quantitative PCR(RTPCR)was used to investigate the effects of Terminalia chekalia on the m RNA activity of cardiac ion channel genes SCN5A(sodium channel gene),Ry R2(calcium channel gene)and KCNJ2(potassium channel gene).Experiment 3: As in Experiment 1,cells were diluted to 3× 105 per well with proper amount of complete medium,and then H9C2 cardiomyocytes were randomly planted in 6-well plates(2ml/ well).When the cells were filled with 6-well plates,the serum-free medium was replaced for 12 hours,and then the aconitine was given for damage for 24 hours.After two times of washing with PBS,the water extract and alcohol extract of Terminalia chebula extracted by two different methods were used for damage protection.After administration,wash twice with pre-cooled PBS,add protein inhibitor cells into each well for 5min,then add RIPA lysate and scrape cells with a microsampler for half an hour.The resulting liquid is put into a 1.5ml centrifuge tube,and the whole operation is carried out on ice.After ℃that,it was placed in a precooled 4 low temperature and centrifuged at a high speed for 12000/5min.After centrifugation,the supernatant was sucked into a new EP tube and the protein was determined by BCA method.The electrophoresis solution is prepared in advance to perform electrophoresis.Remove the PAGE glue from the board and then transfer the egg whites to PVDF.It was closed at room temperature for 1h.I was diluted with the newly prepared blocking solution,and the protein antibody ratio was 1:500.I was placed at ℃4 and incubated overnight.Wash with TBST solution for three times,5min each time.The blocking solution was incubated at room Ⅱtemperature for 1h with the corresponding dilution.Wash with TBST solution for three times,5min each.Mix liquid A and B at A ratio of 1:1 and then drop into the film for 60 seconds at the same time.Then carefully absorb the chromographic solution with absorbent paper and cover it with A layer of smooth transparent paper.Put it into the cartridge and press the glue.Scan and analyze the image.Western blots were used to detect the changes of the proteins SCN5A(sodium channel protein),Ry R2(calcium channel protein)and KCNJ2(potassium channel protein)on the heart ion channel genes of Terminalia chebula to reveal the mechanism of the action of Terminalia chebula on reducing the cardiotoxicity of aconitine.Results: In Experiment 1,the damage degree of aconitine in rat H9C2 cardiomyocytes in different time periods was investigated by using enzyme plate instrument.It was found that with the increase of aconitine concentration,the morphology of cardiomyocytes was abnormal and the cell membrane was damaged.The IC50 of H9C2 cardiomyocytes induced by aconitine for 24 h and 48 h were 36.36μmol/ L and 12.15μmol/ L respectively.According to the experimental situation,the damage model of aconitine with 30μmol/ L concentration for 24 h was determined.The results of aconitine-induced H9C2 cardiomyocytes from Terminalia chebulia extracted by two methods showed that both 0.5μg/ m L of Terminalia chebulia water extract and 0.5μg/ m L of Terminalia chebulia alcohol extract had aconitine-induced toxicity at 24h(P < 0.05).The survival rates of H9C2 cardiomyocytes were 86% and 80%,respectively.0.5μg/ m L was determined as the concentration of the two extracts,and then the relevant indexes were determined.In the determination of lactate dehydrogenase(LDH),it was found that the release rate of LDH was 0.1010nmol/ m L in the control group,0.1850nmol/ m L in the aconitine group,and 0.0590nmol/ m L in the Terminalia chebulia water extract group.The LDH release rate of Terminalia chebula alcohol extract group was 0.0680nmol/ m L.Compared with the control group,the release rate of LDH in aconitine group was significantly increased(P < 0.01).Compared with the control group,the LDH release rate of Terminalia chebula extracts extracted by two methods decreased(P BBB 0 0.05).Compared with the aconitine group,there were significant differences between the two chebula extracts(P < 0.001).When detecting the creatine kinase isoenzyme(CK-MB),it was found that the release rate of CK-MB in the control group was 1.0220ng/ m L,the release rate of CK-MB in the aconitine group was 1.9465ng/ m L,and the release rate of CK-MB in the Terminalia chebula water extract group was 0.4475ng/ m L.The release rate of CK-MB in Terminalia chebula alcohol extract group was 0.4365ng/ m L.Compared with the control group,the release rate of CK-MB in aconitine group was significantly increased(P < 0.01).Compared with the control group,the release rate of CK-MB in Terminalia chebula water extract group was significantly decreased(P < 0.01).Compared with the control group,the release rate of CK-MB in Terminalia chebula alcohol extract group was decreased(P < 0.05).Compared with the aconitine group,the release rate of CK-MB in both extracts was significantly decreased(P < 0.001).In the detection of calcium ions,it was found that the calcium ions in the control group were 1.0455mmol/ L,the calcium ions in the aconitine group were 2.2650mmol/ L,the calcium ions in the water extract of Terminalia chebulia were 1.1585mmol/ L,and the calcium ions in the alcohol extract of Terminalia chebulia were 1.2315mmol/ L.Compared with the control group,calcium ions were significantly increased in the aconitine group(P < 0.001).Compared with the control group,the release rate of calcium ions in the water extract of Terminalia chebula was significantly increased(P < 0.01),and the release rate of calcium ions in the alcohol extract of Terminalia chebula was significantly increased(P < 0.001).Compared with aconitine,the Ca release rate of the two Terminalia chebula extracts was significantly decreased(P < 0.0001).In the detection of malondialdehyde(MDA),the MDA of the control group was 0.5491nmol/ m L,the MDA of the aconitine group was 1.2920nmol/ m L,the MDA of the water extract group was 0.6783nmol/ m L,and the MDA of the alcohol extract group was 0.7267nmol/ m L.Compared with the control group,MDA release rate in aconitine group increased and had no difference(P> 0.05).Compared with the control group,the MDA release rate of the two Terminalia chebula extracts increased to different degrees and there was no difference(P > 0.05).Compared with aconitine,the MDA release rate of the two Terminalia chebula extracts was significantly decreased and had no difference(P > 0.05).In the detection of superoxide dismutase(SOD),the SOD release rate in the control group was 0.4180U/ m L,the SOD release rate in the aconitine group was 0.0205U/ m L,the SOD release rate in the water extract group was 1.0490U/ m L,and the SOD release rate in the alcohol extract group was 1.0925U/ m L.Compared with the control group,the SOD release rate of aconitine group was significantly decreased(P < 0.05).Compared with the control group,the SOD release rate of both Terminalia chebula extracts was significantly increased(P < 0.0001).Compared with aconitine group,the SOD release rate of two Terminalia chebula extracts was significantly increased(P < 0.0001).In the detection of mitochondrial membrane potential and cell apoptosis,it was found that the apoptosis rate was 0% in the control group,4.7% in the aconitine group,2.9% in the water extract group and 1.4% in the alcohol extract group of Terminalia chebula.Compared with the control group,the apoptosis rate of aconitine group was increased and there was no difference(P BBB 0 0.05).Compared with the control group,the apoptosis rate of the two extracts increased and there was no difference(P > 0.05).Compared with aconitine group,the apoptosis rate of the two Terminalia Terminalia Extracts groups had different degree of apoptosis,and there was no statistical significance(P BBB 0 0.05).Compared with aconitine group,the apoptosis rate of the two Terminalia chebulia extract groups decreased to different degrees and had no difference(P BBB 0 0.05).Experiment 2: The expression of SCN5 a gene in the aconitine group was significantly increased compared with the control group(P < 0.01)when q PCR was used for real-time fluorescence quantitative detection of heart ion channel related genes.Compared with the control group,SCN5 a gene in the two extract groups was down-regulated to varying degrees,and there was no difference(P > 0.05).Compared with aconitine group,SCN5 A gene of both Terminalia chebula extracts was significantly down-regulated(P < 0.01).KCNJ2(potassium ion channel gene)gene was detected in the aconitine group compared with the control group,and the expression of KCNJ2 gene was decreased with no difference(P > 0.05).Compared with the control group,the expression of KCNJ2 gene in the two Terminalia chebula extract groups was increased and there was no difference(P > 0.05).Compared with aconitine group,the expression of KCNJ2 gene in Terminalia chebulia water extract group was increased(P < 0.05).Compared with aconitine group,the expression of KCNJ2 gene in Terminalia chebulia alcohol extract group was increased and there was no difference(P > 0.05).In the detection of Ry R2(calcium ion channel gene)gene,it was found that the expression of Ry R2 gene in the aconitine group was decreased compared with the control group and no difference was found(P > 0.05).Compared with the control group,the expression of Ry R2 gene in Terminalia chebulia water extract group was increased and there was no difference(P > 0.05).Compared with the control group,the expression of Ry R2 gene in Terminalia chebulia alcohol extract group was increased(P < 0.05).Compared with aconitine group,the expression of Ry R2 gene in Terminalia chebulia water extract group was increased(P < 0.05).Compared with aconitine group,the expression of Ry R2 gene in Terminalia chebulia water extract group was increased and there was no difference(P BBB 0 0.05).The expression of Ry R2 gene in Terminalia chebula alcohol extract group was significantly increased(P < 0.01).Experiment 3: Western blotting was used to determine the ion channel proteins associated with arrhythmia.Compared with the control group,the expression of SCN5 a protein was up-regulated in the aconitine group(P < 0.05).Compared with the control group,the expression of SCN5 a protein in the two Terminalia chebula extract groups was up-regulated to different degrees without any difference(P > 0.05).Compared with aconitine group,the expression of SCN5 a protein in both Terminalia chebulia extract groups was decreased to different degrees(P Bb0 0.05).In the detection of KCNJ2(potassium ion channel gene)protein,it was found that the expression of KCNJ2 protein was down-regulated in the aconitine group compared with the control group(P < 0.05).Compared with the control group,the expression of KCNJ2 protein in the two Terminalia chebula extract groups was decreased to different degrees and there was no difference(P > 0.05).Compared with the aconite group,the expression of KCNJ2 protein in the two Terminalia chebulia extract groups was up-regulated to different degrees and there was no difference(P > 0.05).In the detection of Ry R2(calcium ion channel gene)protein,it was found that compared with the control group,the aconitine group could down-regulate the expression of Ry R2 protein with no difference(P > 0.05).Compared with the control group,the expression of Ry R2 protein in the two Terminalia chebulia extract groups was up-regulated to different degrees and there was no difference(P > 0.05).Compared with aconitine group,the expression of Ry R2 protein in Terminalia Terminalia water extract group was up-regulated,and there was no difference(P > 0.05).Compared with aconitine group,Terminalia chebulia alcohol extract group could up-regulate the expression of Ry R2 protein(P < 0.05).Conclusion: Terminalia chebula extract has protective effect on myocardial cells induced by aconitine.The mechanism of reducing cardiotoxicity may be to decrease the release rate of LDH,CKMB and increase the release rate of SOD to protect the heart.In terms of oxidative damage,Terminalia chebula can reduce the release rate of MDA to protect the heart.In the aspect of cell apoptosis,Terminalia chebula can reduce the rate of apoptosis to protect the heart.In terms of genes,aconitine can up-regulate the expression of SCN5 a gene and down-regulate the expression of KCNJ2 gene and Ry R2 gene to damage cardiomyocytes.Terminalia chebula extract can down-regulate the expression of SCN5 A gene and up-regulate the expression of KCNJ2 gene and Ry R2 gene to protect cardiomyocytes.In terms of protein,aconitine can up-regulate the expression of SCN5 a protein and down-regulate the expression of KCNJ2 protein and Ry R2 protein to damage cardiomyocytes.Terminalia chebula extract can down-regulate the expression of SCN5 A protein and up-regulate the expression of KCNJ2 protein and Ry R2 protein to protect cardiomyocytes.It is further concluded that the protective mechanism of Terminalia chebula on cardiomyocytes is related to the reduction of oxidative damage of cardiomyocytes,the regulation of ion channels and apoptosis. |
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