| Purposes:This study through the network pharmacology method to predict the effect of berberine prevention and treatment of atrial fibrillation targets,biological process and signaling pathways;a single-center,prospective clinical study of berberine in the prevention of early onset atrial fibrillation after off-pump coronary artery bypass grafting was conducted;and through animal experiments,tissue samples transcriptome sequencing and analysis,to observe the cardioprotective effect of berberine on inhibiting fibrosis,alleviating atrial remodeling and preventing atrial fibrillation in rabbit model of myocardial fibrosis induced by rapid atrial pacing through TLR4/ NF-?B,PPAR,AMPK signaling pathway,to further clarify the mechanism of inflammation induced atrial fibrosis in the occurrence and maintenance of atrial fibrillation,and to provide new insights and theoretical support for the exploration of the prevention and treatment effect and mechanism of berberine on atrial fibrillation,and for the clinical application of preventing early onset of atrial fibrillation after cardiac surgery.Materials and methods:1.Retrieve Berberine action targets through Gene Cards?Swiss Target Prediction and TCMSP databases.Screen atrial fibrillation-related disease targets by Dis Ge NET,OMIMD and TTD databases.The co-acting targets were input into the String platform to construct a protein-protein(PPI)interaction network model.Then,GO analysis and KEGG signal pathway enrichment analysis were performed on the corresponding targets using David platform and Cytoscape software to analyze the target and pathway of berberine in the treatment of atrial fibrillation.2.We randomly assigned 200 patients with sinus rhythm to undergo elective simple off-pump coronary artery bypass grafting and to receive perioperative berberine(1.2 g/day)or placebo,followed by dynamic electrocardiogram monitoring for 7 days postoperatively.The primary outcome was the incidence of atrial fibrillation lasting longer than 30 seconds during the 7 days after surgery.Secondary outcomes included onset time of atrial fibrillation lasting more than 30 seconds within 7 days after surgery,maximum ventricular rate during atrial fibrillation,mean ventricular rate during atrial fibrillation,atrial fibrillation load,blood biomarkers,and heart rate variability(HRV)related measures.SPSS 23.0 statistical software was used for analysis.The enumeration data were statistically described by frequency and percentage.If the measurement data conform to the normal distribution,the mean ± standard deviation will be expressed,while the non-normal distribution will be statistically described by the median(P25,P75)or the maximum and minimum value.For the statistical analysis of major outcome indicators,Kaplan-Meier curve was used to describe the incidence of dependent outcome events when they were recorded,Log-rank test was used to compare the differences between the two groups,and ?~2 test was used to compare the incidence between the two groups.Equilibrium analysis of baseline data,intergroup comparison of secondary outcome indicators and safety indicators between the two groups were performed using nonparametric Wilcoxon rank sum test.For classified data,the chi-square test is used.Statistical tests were double-tailed,and the test level ?=0.05,that is,when P<0.05,the difference was statistically significant.3.30 ordinary male New Zealand white rabbits with the body weight of 2.8-3.2kg.They were randomly divided into 5 groups:(1)left atrial rapid pacing group(RAP group,n=6);(2)Left atrial rapid pacing + berberine group(RAP+BBR,n=6);(3)Sham operation group(SHAM,n=6);(4)Sham operation + berberine group(SHAM+BBR,n=6);(5)Blank Control group(Control,n=6).Provide humane care according to the 3R principles for animal use.Adaptable feeding under normal conditions for 1 week,cage feeding,free diet,drinking water.Rabbits in RAP+BBR group and SHAM+BBR group were given100mg/(kg·d)berberine by gavage before surgery,once a day,for 1 week.The other groups were given normal saline gavage,once a day,for 1 week.Then the model was made,the left chest was opened,the skin pouch was made and the implant was placed.Four electrode wires were extended through the subcutaneous tunnel.Two electrocardiogram acquisition electrodes were sutured and placed subcutaneously under the left upper limb and the right upper arm for observation of ECG.Two stimulation electrodes were sutured to the left atrium and left atrium respectively.Postoperatively,penicillin sodium was given 400,000 units per day for 3 consecutive days,intramuscular injection to prevent infection.3 days later,the animal telemetry stimulation system was turned on to collect and stimulate signals through wireless transceiver;Power Lab physiological log was used to continuously monitor body surface I lead ECG signals,and special computer program stimulation software was used to deliver intermittent(stimulation for 2 s,pause for 2 s)high frequency(frequency 20 Hz)supra threshold(intensity 2 m A,pulse width 1 ms)stimulation for 2 weeks.RAP+BBR group and SHAM+BBR group were given berberine intragastric gavage(the same dosage and method as before)for 2 weeks.The other groups were given normal saline gavage(same as before operation).During the whole experiment,all rabbits were awake and free to move,and their eating and sleeping habits were not changed.Rabbits in each group were sacrificed 2 weeks after surgery,and the whole left atrium tissue was extracted,washed with ice normal saline,and redundant tissues such as thrombus and cellulose were removed.Partial atrial tissue were immobilized by 4% paraformaldehyde immersion,and wax blocks were embedded by dehydration in the later stage.Some atrial tissues were frozen by liquid nitrogen and stored in a refrigerator at-80? for later use.Beijing Capitalbio technology co.,LTD.was commissioned to conduct transcriptome sequencing of m RNA from tissue samples of rabbit myocardial fibrosis model induced by rapid atrial pacing under different conditions by using high-throughput sequencing technology(total transcriptome sequencing)as the main means.Differential gene expression,gene function and signal pathway enrichment analysis were performed on the sequencing results to provide experimental basis for the possible mechanism of berberine in the prevention and treatment of atrial fibrillation.Atrial paraffin sections were detected by immunohistochemistry,Masson staining and H-E staining.Western blot analysis was performed on atrial tissues,including the protein expressions of p-p65,p65,IKB?,TLR4,PPAR-?,p-AMPK,AMPK and ?-SMA.SPSS 23.0 statistical software was used.Data in each group were represented as(?)±s.One-way analysis of variance was used for comparison between groups,with P < 0.05 considered statistically significant.Results:1.Network pharmacology to predict the target and pathway of berberine in preventing atrial fibrillationBased on network pharmacological method,we screened a total of 50 targets for the interaction between berberine and atrial fibrillation,and the core targets such as IL-6,TNF,VEGFA,NOS3,MMP9 and STAT3 were more important.A total of 1644 GO items(P<0.05)were obtained by GO function enrichment analysis on 50 selected berberine-atrial fibrillation co-action targets in David database,including 1544 biological process(BP)items,31 cell composition(CC)items and 69 molecular function(MF)items.KEGG pathway enrichment analysis was performed,and 128 signaling pathways were screened(P<0.05),of which 30 were directly related to atrial fibrillation.It can be seen that several biological functions are closely related to the occurrence and development of atrial fibrillation,mainly involving the regulation of berberine through inflammatory response,oxidative stress,atrial fibrosis,angiogenesis and other biological processes,and play a role in the treatment of atrial fibrillation by regulating TNF,PI3K-Akt,MAPK,IL-17,HIF-1,VEGF,TLR,TGF-? and other signaling pathways.2.A single-center,prospective clinical study of berberine in preventing early onset of atrial fibrillation after off-pump coronary artery bypass grafting2.1 Baseline characteristics Of the 200 patients included in the final analysis of this study,all completed the trial as required.Demographic data and preoperative variables of the two groups were summarized as shown in Table 1,and the differences were not statistically significant.Two groups of patients with preoperative heart rate variability index difference has no statistical significance.2.2 Main Results The incidence of atrial fibrillation after OPCAB was 20%(20 cases)in the berberine group,35%(35 cases)in the placebo group and control group(P=0.018).After OPCAB,the risk of POAF within 7 days in the berberine group was 0.5 times that in the placebo group;95% confidence interval: 0.29 to 0.87;P = 0.0143.Compared with placebo group,the incidence of atrial fibrillation after cardiac surgery in berberine group was significantly reduced,reducing the risk of POAF by at least 13%.2.3 Secondary results and POAF related measurements The longest postoperative duration of atrial fibrillation,the maximum ventricular rate during atrial fibrillation and the mean ventricular rate during atrial fibrillation in the two groups were with statistically significant(P<0.05),and the berberine group was better than the placebo group.The amount of amiodarone in atrial fibrillation patients was different between the two groups with statistically significant(P =0.013),and the amount of berberine group was less than that of placebo group.All patients treated with amiodarone regained sinus rhythm.Blood biomarker measurement.Two time points were selected at about 48 hours and on the seventh day after surgery.Preoperative hs CRP,IL-6,NLR,BNP,hs-TNT levels were not significantly different between berberine group and placebo group(P>0.05).The levels of Hs CRP and IL-6 in the berberine group 48 hours and 7 days after surgery,and the levels of TNF-?,NLR and BNP in the berberine group 48 hours after surgery were significantly lower than those in the placebo group,with statistical significance(P<0.05).The SOD level of berberine group was significantly higher than that of placebo group 48 hours after operation,and the difference was statistically significant(P<0.05).The Hs-Tn T of the berberine group at 48 hours after surgery and the NLR of the berberine group at 7 days after surgery were different from those of the placebo group with no statistically significant(P>0.05).Postoperative general conditions.The two groups were similar in ventilator assist time,ICU stay time,blood transfusion volume and postoperative LVEF value,without significant difference(P>0.05).Heart rate variability(HRV).Comparison of postoperative HRV indexes between the two groups showed that the mean levels of SDNN,SDANN,RMSSD,TP,LF,HF and VLF in the berberine group were significantly higher than those in the placebo group(P<0.05).The difference in LF/HF between the two groups was not statistically significant(P>0.05),but the difference in PNN50 between the two groups might be statistically significant(P=0.067).There were significant differences in ?SDNN,?SDANN,?RMSSD and ?HF between the two groups(P<0.05),that is,the values of HRV indexes after surgery in the two groups were lower than those before surgery(with statistically significant).The reduction was more significant in the placebo group than in the berberine group.HRV indexes were calculated by repeated measurement.SDNN and SDANN were significantly different between the two groups 24-48 hours after surgery(P<0.05),and the berberine group was higher than the control group.VLF,LF and HF were significantly different between the two groups on the7 th day after operation(P<0.05),and the berberine group was higher than the control group.RMSSD and PNN50 levels were similar between the two groups without significant difference(P>0.05).2.4 Adverse reactions and complications In terms of safety,6 patients in the berberine group had slight adverse reactions(gastrointestinal reactions,constipation),which did not affect their life and treatment.There were no abnormal changes in blood routine,urine routine and liver function after taking the medicine.There were no significant differences in IABP utilization rate,secondary thoracotomy,perioperative myocardial infarction and cerebrovascular accident rates between the two groups(P>0.05).There was difference in the incidence of arrhythmias other than atrial fibrillation between the two groups with statistically significant(P=0.047),and the berberine group was significantly lower than the placebo group.3.Study on the effect and mechanism of berberine on atrial remodeling in rabbit model of myocardial fibrosis induced by rapid atrial pacing3.1 Effect of berberine on atrial structural remodeling in rabbit model of myocardial fibrosis induced by rapid atrial pacingImmunohistochemistry and Western Blot were used to detect the expression of ?-SMA protein in left atrial tissues of rabbits in each group.Masson staining and H-E staining were used to compare the degree of atrial fibrosis in each group.Immunohistochemistry shows varying degrees of brown or yellow spots in atrial myocytes,indicating immunohistochemical positivity for ?-SMA.The positive expression of?-SMA was the most obvious in the RAP group,the number of cells expressing ?-SMA was significantly increased,and the cytoplasm was brown-yellow.Compared with the RAP group,the positive staining of ?-SMA was significantly reduced in the RAP+BBR group.The effects of SHAM and SHAM+BBR were similar and lower than those of RAP+BBR group.There was only a small amount of positive staining in the control group.Western Blot analysis showed that the expression of ?-SMA protein in RAP+BBR group was significantly lower than that in RAP group(P<0.05).Masson staining of atrial tissues in each group showed blue collagen fibers and red myocardial cells.The area of myocardial fibrosis increased in RAP group.The myocardial interstitial fibrosis in RAP+BBR group was significantly less than that in RAP group.The fibrosis degree of SHAM group was similar to that of SHAM+BBR group.In the Control group,the myocardial fibers were arranged neatly and the nucleus size was normal.The quantitative analysis of collagen volume fraction(CVF)showed that the myocardial interstitial fibrosis in each group was significantly increased compared with the Control group(P<0.001),and the number of collagen fibers in ARP+BBR group was significantly decreased compared with RAP group(P<0.001).Both groups were higher than that in SHAM group and SHAM+BBR group(P<0.001),and the number of collagen fibers in SHAM and SHAM+BBR groups was similar(P>0.05).Effect of berberine on left atrial mass/body weight ratio(LAM/BM)of rabbits in each group.The LAM/BM of RAP group was significantly higher than that of other groups(P<0.001),and the LAM/BM of RAP+BBR group was significantly lower than that of RAP group(P<0.001).There was no significant difference between SHAM and SHAM+BBR group(P>0.05),but there was significant difference between SHAM and Control group(P<0.001).HE staining showed that the myocardial fibers in the Control group were neatly and densely arranged,with clear nuclei and reduced ECM,while the myocardial fibers in the RAP group were atrophied,disorderly arranged and infiltrated with a large number of inflammatory cells.RAP+BBR group significantly reduced the pathologic changes compared with RAP group,and the degree of pathological changes in SHAM group was similar to that in SHAM+BBR group,which was between RAP+BBR group and Control group.3.2 Transcriptome sequencing and analysis of tissue samples from rabbit model of myocardial fibrosis induced by rapid atrial pacingThrough pair comparison of gene function enrichment analysis between RAP group and RAP+BBR group,it was found that berberine can have an intervention effect by causing changes in functions and pathways such as inflammation,metabolism,cell growth and development.In the signaling pathway analysis,176 different signaling pathways were found,and the related signaling pathways with the smallest Corrected p-value were selected,including PPAR-? signaling pathway,AMPK signaling pathway,cancer signaling pathway,PI3K-Akt signaling pathway,fatty acid metabolism,NF-?B signaling pathway,m TOR signaling pathway,TNF signaling pathway,and JAK-STAT signaling pathway,etc.It is particularly active in the regulation of PPAR-? signaling pathway and AMPK signaling pathway.It not only provides a new research point to clarify the mechanism of action of berberine,but also provides a reference for looking for a new therapeutic target of atrial fibrillation.3.3 Effects of berberine on inflammation signaling pathway TLR4/NF-?B,PPAR-?,AMPK in rabbit model of myocardial fibrosis induced by rapid atrial pacingImmunohistochemistry shows varying degrees of brownish-yellow or black spots in atrial myocytes,indicating positive immunohistochemistry of p-65,p-AMPK.In RAP group,the positive expression of P-65 was obvious,and the number of expression cells was significantly increased,and the nuclear black cytoplasm was brownish yellow.The positive expression of p-AMPK was obvious,and the number of cells was significantly increased,and the nuclear blue cytoplasm was brownish yellow.Compared with RAP group,RAP+BBR group were all positive,and the positive staining of nuclear and cytoplasm was significantly reduced.The effects of SHAM and SHAM+BBR were similar and lower than those of RAP+BBR group.In the control group,only a small amount of positive staining was observed.Western Blot analysis showed that the protein expression of p-p65 and TLR4 in RAP+BBR group was significantly lower than that in RAP group(P<0.05),and the protein expression of IKB? was significantly higher than that in RAP group(P<0.05).The protein expressions of PPAR-? and p-AMPK in RAP+BBR group were significantly higher than those in RAP group(P<0.05).Conclusion:1.Based on the network pharmacology method,the functional material basis of berberine and the molecular mechanisms of action on atrial fibrillation were predicted.Many potential therapeutic targets of berberine in the treatment of atrial fibrillation have been discovered.It shows that berberine plays a role in the treatment of atrial fibrillation from multi-target and multi-pathway,the pharmacological mechanism of berberine for prevention and treatment of atrial fibrillation was systematically revealed.which provides new ideas and scientific basis for its basic research and clinical application.It also provides a scientific basis for the modernization research of precise therapeutic targets of traditional Chinese medicine.2.Berberine can significantly reduce the incidence of newly developed atrial fibrillation after simple off-pump coronary artery bypass transplantation,improve the onset time of atrial fibrillation,maximum ventricular rate during atrial fibrillation,average ventricular rate during atrial fibrillation,atrial fibrillation load,inhibit inflammatory cytokines,and regulate the indexes of heart rate variability of patients.3.Berberine can effectively inhibit the production and release of inflammatory transmitters through TLR4/NF-?B,PPAR-?,AMPK signaling pathways,alleviate inflammatory response,reduce myocardial fibrosis and hypertrophy of cardiomyocytes,improve atrial structural remodeling in rabbit model of myocardial fibrosis induced by rapid atrial pacing,and thus have the effect of preventing atrial fibrillation. |
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