| Objective:(1)Clinical study: retrospective study to evaluate the clinical efficacy of the Chinese medicine of Buxuhuayu Recipe in the treatment of PBC Q i deficiency and blood stasis syndrome;(2)Experimental study: To establish a cell model for simulating apoptosis of HiBEC in patients with PBC,and to explore the mechanism by which Buxuehuayu Recipe TCM can regulate GCDC-mediated apoptosis of HiBEC based on caspase-3/9 endogenous apoptosis signaling pathway.Provide a reliable experimental basis for the clinic.Method:(1)Taking PBC qi deficiency and blood stasis syndrome from Longhua Hospital outpatients and hospitalized hospitals since 2010,the patients were divided into control group and treatment group,control group was given oral UDCA,and the treatment group was supplemented with oral UDCA and C hinese medicine once in the morning and evening,the time is 6 mo nths and 12 months.Observed and compared the improvement of UDC A response rate and TCM symptoms between the two groups.(2)In view of the good clinical efficacy of the traditional C hinese medicine in the early stage,the experiment will use GC DC as the inducer and HiBEC as the carrier to establish a cell model that simulates the apoptosis of PBC bile duct epithelial cells,with UDCA as the positive control.The concentration gradient was set up to explore the mechanism of the effect of Buxuhuayu Recipe on regulating apoptosis induced by GCDC-mediated HiBEC injury.Results:(1)According to Barcelona criteria,after 6 months of treatment,the UDCA response rate of the treatment group was 86.4% higher than that of the control group 60.0%,P=0.044,the difference was statistically significant(P<0.05);After 12 months,the response rate of two groups were compared,P=0.167,and the difference was not statistically significant(P>0.05).According to Ehime criteria,after 6 months of treatment,the UDCA response rate of the treatment group was not significantly different from that of the control group(P>0.05).And after 12 months,the treatment group response rate was 81.8% higher than the control group 52%,P=0.031,the difference was statistically significant(P < 0.05).The ALT,AST,GGT,and ALP indexes were effectively improved in the two groups after 6 months of treatment,P < 0.001,the difference was statistically significant(P < 0.05).After comparing the two groups,the improved ALP of the treatment group was found after 6 months with the control group,P=0.009,the recovery of GGT in the treatment group was better than that in the control group after 12 months,P=0.041,the difference was statistically significant(P<0.05).The Ig G of the two groups was improved compared with the previous one.The Ig G of the control group was improved for 6 months,P=0.013,and the Ig G of the treatment group was improved after 12 months,P=0.017,the difference was statistically significant(P<0.05);the two groups of Ig M was improved after 12 months of treatment.The difference between the control group P=0.018 and the treatment group P=0.032 was statistically significant(P<0.05).There was no significant difference between the two groups(P>0.05).The treatment group effect is not considered to be superior to the control group.According to the N imodiping method formula,the symptom scores of the two groups were analyzed and counted.After 6 months,the control group was effective in 8 cases,a little effective in 10 cases,ineffective in 7 cases,the effective rate s were 72%,and the treatment group was effective in 15 cases.4 cases were a little effective and 3 cases were ineffective.The effective rates were 86.4%.The difference between the two groups was P=0.023,the difference was statistically significant(P<0.05).After 12 months,the control group was effective in 11 cases and a little effective in 9 cases,5 cases were ineffective,80% were effective;18 cases were effective in the treatment group,3 cases were a little effective,1 case was ineffective,and the effective rate were 95.5%.The difference between the two groups was P=0.008,the difference was statistically significant(P<0.05);6 months later,the improvement of pruritus was effective in 12 cases in the control group,17 cases in the treatment group,P=0.038,the difference was statistically significant(P<0.05);12 months later,the improvement of the tired of fatigue,15 cases in the control group were effective,and 19 cases in the treatment group were effective,P=0.044,the difference was statistically significant(P<0.05).There was no significant difference between the two groups in the improvement of other symptoms(P>0.05).(2)Model establishment: CCK-8 and FCM results: HiBEC apoptosis was mainly induced by 1.0 m M/L GCDC,and the apoptosis rate increased with the increase of GC DC concentration.The apoptotic rate was 8.41±1.04% in the 0.5 m M/L GCDC group,16.2±1.26% in the 1.0 m M/L GCDC group,and 33.86±0.65(%)in the 1.2 m M/L GCDC group.The differences between the blank groups were statistically significant(P<0.05).WB results: cleaved-caspase 3 was up-regulated in the 1.0 m M/L and 1.2 m M/L GCDC group,and the difference was statistically significant(P<0.05).Among them,1.2 m M/L GCDC group Caspase-9 up-regulated compared with blank,the difference between the two groups was statistically significant(P<0.05);the up-regulation of cleaved-caspase 9 in the 1.0 m M/L GCDC group was statistically significant(P<0.05);the anti-apoptotic protein Bcl-2,there were different degrees of expression reduction in each group,and the difference was statistically significant(P<0.05).In addition,1.0 m M/L and 1.2 m M/L GCDC could up-regulate the expression of PDC-E2,and the difference was statistically significant(P <0.05).Mechanism of TCM: CCK-8 and FCM results: compared with the model group apoptotic rate of 16.21±1.26(%): high concentration of 0.2 mm/L UDCA group apoptotic rate increased by 28.82 ± 0.85(%),the difference was statistically significant(P <0.05);the middle and low concentrations of UDCA group and TCM high,medium and low concentration groups and combination group can reduce the apoptosis rate of HiBEC in different degrees,the difference was statistically significant(P <0.05);Compared with the low concentration UDCA group,the UDC A group with moderate concentration of 0.02 mm/L had better inhibition of HiBEC apoptosis,and the difference was statistically significant(P <0.05).The high,medium and low concentrations of TCM were among the three groups.For comparison,the effect ordering: medium concentration TCM group> high concentration TCM group=low concentration TCM group,the difference was statistically significant(P <0.05);medium concentration 0.02 mm/L UDCA group and medium concentration 1 mg/ml TCM group,there was no significant difference between the two groups(P>0.05).The difference between the combination group and the 0.02mm/L UDC A group was stat istically significant(P<0.05).WB results: compared with the model group: the expression of total protein of Caspase-3: the difference of medium concentration of 1 mg/ml TCM group was statistically significant(P<0.05);activated cleaved-caspase 3: high concentration of 0.2 mm/L UDCA group> The combination group = medium concentration 0.02 mm/L UDCA group,the difference was statistically significant(P <0.05);Caspase-9 total protein expression: combination group > medium concentration 1mg / ml TCM group = medium concentration 0.02 mm/L UDCA The difference was statistically significant(P<0.05);activated cleaved-caspase 9: combination group > medium concentration 1 mg/ml TCM group = medium concentration 0.02 mm/L UDCA group > high concentration 0.2 mm/L UDCA group = low concentration In the 0.002 mm/L UDCA group,the difference was statistically significant(P<0.05);Bcl-2: high,medium and low concentrations in the TCM group and the treatment group were all adjusted to different degrees of Bcl-2,the difference was statistically significant.(P<0.05),medium concentration 1 mg/ml TCM group= treatment group>high concentration 5 mg/ml TCM group.Conclusion:(1)The Chinese medicine of Buxuhuayu Recipe can accelerate the UDCA response process of qi deficiency and blood stasis type PBC to a certain extent,and promote the recovery of liver function ALP,GGT,etc.,regulate immunity and improve the clinical symptoms of patients.(2)A cell model for mimicking apoptosis of biliary epithelial cells in PBC patients can be established when the concentration of GCDC is 1.0 m M/L.It was confirmed by experiments that TCM has a certain regulatory effect on the apoptosis model of GCDC-mediated HiBEC injury,which is expressed by down-regulating the expression of caspase-3/9 and its activated cleaved form,and the mitochondria-associated anti-apoptotic protein Bcl-2.The expression has a certain up-regulation effect,and the effect of the combination with UDCA is more prominent.Therefore,the mechanism of action of TCM to regulate HiBEC apoptosis may be closely related to the endogenous pathway of caspase-3/9. |
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