| Objective:The Hirudo extract was prepared by PBS water extraction,then separated and purified.The stability of each part of the Hirudo extract was preliminary analyzed by HPLC(high performance liquid chromatography,HPLC),and the effects of the separated and purified Hirudo extract on the retinal pigmented epithelial,RPE were observed for screening the active parts of Hirudo extract that promote RPE apoptosis.By detecting the activity of p38 mitogen-activated protein kinases(MAPK)in RPE cells after the intervention of the effective parts of Hiruto extract,it is further explored that the mechanism of promoting the apoptosis of RPE cells in the p38 MAKP signal transduction pathway.UPLC-MS(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS)was used to detect the chemical composition of the the Hirudo extract and determine the effective parts of it for promoting the apoptosis of RPE cells.Abnormal proliferation and migration of RPE cells is the main cause of the development of proliferative vitreoretinopathy(PVR).Optical coherence tomography(OCT)and other observation were used to observe the clinical effects of Hirudo compound preparation “Haikun Huayu Tablets” on PVR through collecting the clinical cases.It is further suggested that Hirudo may achieve anti-proliferation effects by promoting RPE apoptosis.Methods:The first part:1.Preparation,separation and purification of Hirudo extract: 3 batches of Hirudo extract were prepared by PBS water extraction,separated and purified by D-101 macroporous adsorption resin according to the polarity size,with 6 parts of Hirudo extract in each batch.2.Preliminary analysis of Hirudo extract by HPLC: HPLC analyzes the stability of each part of Hirudo extract after separation and purification.3.Preliminary screening of the effective parts of Hirudo extract: Six parts of Hirudo extract after separation and purification were taken,and 4 concentrations were set for each part,2mg/m L,0.5mg/m L,0.125mg/m L and 0.03125mg/m L.Each part of Hirudo extract of different concentrations was co-cultured with RPE cells for 48 h,and the effect of drugs on RPE cells apoptosis was detected by flow cytometry,RT-PCR and western blot.The effective parts and optimal concentration of Hirudo extract were screened initially.All cells in the control group were cultured only in the medium with PBS.4.Study on the effect of effective parts of Hirudo extract on apoptosis of RPE cells and related molecular mechanism: The effective parts of the screened Hirudo extract were co-cultured with RPE cells for 48 h.The apoptosis rate of RPE cells was detected by flow cytometry,and the expression of apoptotic protein Caspase3 and p38 MAPK signal transduction pathway protein markers in RPE cells were detected by RT-PCR and western blot.All cells in the control group were cultured only in the medium with PBS.5.The composition detection of the effective parts of Hirudo extract: The main chemical composition of the effective parts of Hirudo extract tested by UPLC-MS.The second part:Retrospective clinical case-control study.Collected the completely medical information of patients who went to the Department of Ophthalmology,Hospital of Chengdu University of Traditional Chinese Medicine from January 2019 to January2021 and diagnosed with rhegmatogenous retinal detachment,underwent retinal detachment reduction(simple scleral external pressure + condensation),and whose TCM syndrome type was consistent with the syndrome of dampness-heat stasis.The treatment group was given Haikun Huayu Tablets(with Hirudo as the main drug)combined with glucocorticoid for 30 days after operation,and the controlled group was only given glucocorticoid intervention for 30 days after operation.The general data of the patients were analyzed,including age,gender,preoperative blood pressure,and intraocular pressure(IOP).OCT examination was used to observe whether the anterior macular membrane appeared after 30 days of intervention,the changes of best corrected visual acuity(BCVA),and whether the retinal was detached again,and compared the scores including TCM syndrome scale and Life Quality for Diseases with Visual Impair(SQ-VI)scale.Results:The first part1.Preparation,separation and purification of Hirudo extract: The mother liquor of Hirudo extract was separated and purified into 6 parts by D-101 macroporous adsorption resin,which were named in descending order of polarity as Hirudo extract A(unabsorbed part),Hirudo extract B(extracted part with ultrapure water),Hirudo extract C(20% ethanol elution part),Hirudo extract D(50% ethanol elution part),Hirudo extract E(70% ethanol elution part),Hirudo extract F(90% ethanol elution part).2.Preliminary analysis of Hirudo extract by HPLC: HPLC analyzed 3 batches of Hirudo extracts mother liquor,then separated and purified each part.The result showed that the peak extraction time and peak area of the mother liquid and each part of the chromatographic were about the same,among which the Hirudo extract F(90%ethanol elution part)had the best repeatability.3.Preliminary screening of effective parts of Hirudo extract: Through flow cytometry apoptosis detection technology,combined with RT-PCR and western blot preliminary screening.Compared with the control group,0.125mg/m L of Hirudo extract F(90% ethanol elution part)had the most obvious apoptotic effect on RPE cells with the highest Caspase3 m RNA and protein content expression.4.Study on the effect of effective parts of Hirudo extract on the apoptosis of RPE cells and its related molecular mechanism: 0.125mg/m L Hirudo extract F(90% ethanol elution part)was used to intervene RPE cells for 48 h.Compared with the control group,the OD value of RPE cells by flow apoptosis was 72.5967±9.8656,P=0.028 < 0.05,showing a statistically significant difference.Compared with the control group,the expression of Caspase3 and p38 MAPK protein m RNA in RPE cells was significantly increased by RT-PCR,Caspase3 was 3.1200±0.9923,P=0.026 < 0.05,and p38 MAPK was 1.5333±0.2103,P=0.038 < 0.05,which showed statistically significant difference.Compared with the control group,the expressions of Caspase3 and p38 MAPK protein in RPE cells was significantly increased by western blot,Caspase3 was 4.2170±0.7067,P=0.003 < 0.01,which was a significantly statistical difference.p38 MAPK was1.6322±0.0924,P=0.011 < 0.05,which also showed an obviously statistical difference.5.Composition detection of the effective parts of Hirudo extract: UPLC-MS detected 11 kinds of small molecule compounds in the Hirudo extract F.It was preliminarily speculated that compound 1 might be spermidine based on the literature study and the binding relative molecular weight of secondary ion fragments.The second part1.General data analysis: The age,gender,left or right eyes,blood pressure at baseline,and IOP of the eyes at baseline of the patients were analyzed,and there was no significant statistical difference among the groups(P > 0.05).2.OCT examination of the incidence of anterior macular membrane: The percentage of anterior macular membrane in the treatment group(6.7%)was less than that in the control group(30%)at 30 days after operation,P=0.042 < 0.05,there was a significant statistical difference.3.BCVA: Baseline time and 30 days after operation BCVA(Log MAR)of the treatment group were 0.9311±0.7201,0.5607±0.4769;the control group were1.0125±0.5604 and 1.0298±0.6707.Comparing the BCVA(Log MAR)between the treatment group and the control group at baseline,P=0.627 > 0.05,no statistically significant difference.30 days after operation,the BCVA(Log MAR)of the treatment group was smaller than that of the control group(P=0.003 < 0.01),showing a statistically significant difference.The BCVA(Log MAR)of the treatment group was less than the baseline time at 30 days after operation,P=0.00 < 0.01,which had a statistically significant difference.The BCVA(Log MAR)was less than the baseline time in the control group 30 days after operation(P= 0.871> 0.05),no statistically significant difference.4.Retinal detachment: 30 days after operation,1 case(3.3%)of retinal detachment in the treatment group and 6 cases(20%)in the control group,P= 0.044<0.05,there was a statistically significant difference.5.TCM syndrome scale score: The scores of TCM syndrome scale in the treatment group at baseline time and 30 days after operation were 6.93±2.48,4.87±2.24,while the control group were 6.77±1.83,6.23±2.01.Comparing the scores of TCM syndrome scale between the treatment group and the control group at baseline time,P=0.768>0.05,showing no significant statistical difference.The scores of TCM syndrome scale in the treatment group were less than those in the control group 30 days after operation,P=0.016 < 0.05,with statistically significant differences.The scores of TCM syndrome scale in the treatment group 30 days after surgery were less than the baseline time,P=0.00 < 0.01,showing a very significant statistical difference.The scores of TCM syndrome scale in the control group 30 days after surgery were less than the baseline time,P=0.009 < 0.01,with statistically significant difference.6.SQL-VI scores: The SQL-VI scores of the treatment group at baseline time and30 days after operation were 164.93±44.99 and 200.03±50.87,while the control group were 155.00±41.92 and 164.87±47.87.The comparison of SQL-VI scores between the treatment group and the control group at baseline showed P=0.380 > 0.05,showing no statistically significant difference.The SQL-VI scores of the treatment group were greater than that of the control group 30 days after operation,P=0.008 < 0.01,with statistically significant difference.The SQL-VI scores of the treatment group 30 days after operation was greater than the baseline time,P=0.00 < 0.01,showing a very significant statistical difference.In the control group,the SQL-VI scores 30 days after operation was greater than the baseline time,P=0.011 < 0.05,showing a statistically significant difference.Conclusion1.Its stable to extract the Hirudo through PBS water,separate and purify the Hirudo extract through D-101 macroporous adsorption resin.2.After separation and purification,the Hirudo extract F(90% ethanol elution part)showed the best repeatability and stability by HPLC analysis,and the chemical composition was single.3.0.125mg/m L Hirudo extract F(90% ethanol elution part)could promote the apoptosis of RPE cells and was an effective part of Hirudo extract.4.0.125mg/m L Hirudo extract F(90% ethanol elution part)promoted RPE cell apoptosis by up-regulating p38 MAPK signal pathway of transducing.5.UPLC-MS preliminarily suggested that the compound promoting the apoptosis of RPE cells in the effective part of Hirudo extract might be Spermidine,and further research is needed to identify the structural formula of the compound.6.The intervention of Hirudo compound preparation “Haikun Huayu Tablets”combined with glucocorticoid can affect the occurrence and development of PVR after retinal detachment to a certain extent,and its anti-proliferation mechanism may be that Hirudo promotes RPE cell apoptosis,which needs further verification. |
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