| BackgroundPostsurgical adhesions are fibrous scar tissue formations,or fibrin matrices,that from between tissues or organs following injury associated with surgical procedures.Postoperative adhesion formation has been a problem since the advent of intra-abdominal manipulation.Peritoneal adhesions are a near inevitable occurrence after laparotomy and a major cause of both patient and physician misery.Intra-abdominal adhesion formation after surgery is a significant cause of morbidity,such as bowel obstruction, infertility and pain.Unfortunately,clinical strategies and therapies aimed at alleviating or controlling adhesion formation have been largely inadequate in their address of both ongoing human suffering and economic cost.Recently,a number of TCM(Traditional Chinese Medicine) treatment and herbal preparation have been evaluated for their value in postoperative adhesion prevention.So it is necessary to exert the superiority and characteristic of TCM in treating postoperative adhesion.WTI(Weitong Injection),developed by Department of pharmaceutics,Nanfang Hospital,is a herbal injectable powder for anti-adhesion.It bases on the study of Changtong Oral Liqud and is composed by D and Q,which can improve microcirculation of blood,decrease inflammatory reaction and exudation.It is a important new drug for postoperative adhesion.ObjectiveWe plan to observe the effects of D and Qon NFB and AFB in vitro,analyse synergistic and antagonistic effect of combined D and Q in vitro.To found out the optimal dose of D combined with Q and investigate the influence of optimal combination on FB.Method1 Culture and Identification of Fibroblasts from rats normal peritoneum and adhesionThe prepared tissues were cultured until outgrowth of fibroblasts.When confluence was reached,the fibroblasts were subcultured with trypsinization at 1:3 split ratio.The passage 3-8 cells were used in order to maintain comparability.Cellular morphologies were observed by inverted phase contrast microscopy,and identified by viment in immunostaining.2 Effect of Principal Constituents of WTI on proliferation and cell cycle of NFB and AFBNFB and AFB were seeded on 96-well plates at a density of approximately 4×103 cells per well in 10% FBS.Then each kind of FB was divided into 3 group:D,Q and D+Q group.Each group had 6 different drug concentration ranging from 0～128μg/ml. The dosage of D+Q group was based on the ratio of D and Q in WTI(D:Q=1:1).Cultured for 72hr,the proliferation of NFB and AFB were detected by MTT assay.The median-effect principle and MTT method were used in analysis the effect of combined drugs. The cells of 3～8 passage were seeded on 24-well plates at a density of approximately 3×104 cells per well in 10% FBS.Then each kind of FB was divided into 2 group:D and Q.Cultured for 72hr,we collected the supernatant of cells and determine Hyp by base hydrolysis-chromatometry.The cells of 3～8 passage were seeded on 24-well plates at a density of approximately 3×104 cells per well in 10% FBS. Each kind of FB was divided into 2 group:D and Q. D and Q was separately added into plates after 72hr. Cultured for 48hr,detected the effects of drugs on the lactate dehydrogenase(LDH) activity.NFB and AFB were seeded on 6-well plates at a density of approximately 1×105 cells per well in 10% FBS. Each kind of FB was divided into 2 group:D and Q. Cultured for 72hr,the in vitro cell scratch wound model was set up to observe the effect of D and Q(48ug/ml) on the migration of FB after 24hr of culture.3 Study on optimal component ratio of WTIMTT assay was used to detect NFB and AFB cell viability, the proliferation inhibition rates in FB was applied to measure the effect of drugs. Six groups of different ratios and doses were analyzed by the weighted modification method.Based on the analysis,five ratioes and doses were selected to analyzed the interaction-of D and Q.4 Effect of Optimal combination on proliferation and cell cycle of NFB and AFBNFB and AFB were seeded on 96-well plates for 24hr in condition of 37℃, 95% humidity,and 5% CO2,then culture medium in every well was removed,culture medium containing optimal combination was added into wells,and 96-well was cultured again for 72hr.Absorbance value of each well was determined by MTT colorimetric method.The cells of 3～8 passage were seeded on 24-well plates, culture medium containing optimal combination was added into wells after 72hr.Cultured for 48hr,detected the effects of drugs on the lactate dehydrogenase(LDH) activity.The cells of 3～8 passage were seeded on 6-well plates. Cultured for 72hr,the in vitro cell scratch wound model was set up to observe the effect of optimal combination (48μg/ml) on the migration of FB after 24hr of culture.The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×106 cells per dish in 10% FBS.Then each kind of FB was divided into 2 groups:blank,optimal combination group.Cultured for 72hr,the cell cycle of NFB and AFB were analyzed by FCM.The cells of 3～8 passage were seeded on 24-well plates, Then each kind of FB was divided into 2 groups:blank,optimal combination group.Cultured for 72hr,The quantitative enzyme-inked test kit was used to assay the secretion of TNF-α,IFN-γ,IL-2.Result1 Fibroblasts from normal peritoneum and adhesions grew easily in vitro,but fibroblasts from adhesions grew faster than those from normal peritoneum. Immunostaining of viment in was positive in cultured fibroblasts.2 Effect of Principal Constituents of WTI on proliferation and cell cycle of NFB and AFBD and Q alone or in combination inhibited the proliferation of FB significantly in dose-dependent manner.The combination of D with Q had synergistic effect on the growth of FB when the inhibiton ratio of NFB was more than 0.6 or inhibition ratio of AFB was more than 0.65.Compared with drug groups of NFB,drug groups of AFB had higher HyP in the supernatant,and there was no significant difference between two drug groups and blank group.With increasing of Q concentration,LDH leakage rate of NFB and AFB gradually increased,the difference of 48 and 96μg/ml concentration Q group and blank group of AFB was significant.The rates of NFB's migration at bland group,D group and Q group were 8.7%±2.2%,2.2%±1.4% and 2.7%±0.3%, The rates of AFB's migration at bland group,D group and Q group were 19.7%±1.6%,7.6%±1.6% and 6.0%±1.4%,respectively,indicating that D and Q could significantly reduce the migration of NFB and AFB.3 Study on optimal component ratio of WTID and Q owned a strong additive effect.At the ratio of 9:2,D and Q significantly decreased the proliferatin of NFB and AFB.4 Effect of Optimal combination on proliferation and cell cycle of NFB and AFBOptimal combination significantly inhibited the cell viability of NFB and AFB in a concentration-dependent. When concentration of optimal combination was 24～96μg/ml, there was no statistical differences about LDH leakage rate between-combination group and blank group.The rates of NFB's migration at bland group and optimal combination group were 7.7%±1.5% and 2.6%±1.7%, The rates of AFB's migration at bland group and optimal combination group were 30.0%±7.1% and 2.4%±1.1%, respectively, indicating that combination group could significantly reduce the migration of NFB and AFB.Compared with blank groups,drug groups had higher G0/G1 proportion and lower S proportion,but G2/M proportion was no statistical differences.The secretory amount of TNF-αand IFN-γinduced by different concentration of FBincreased gradually.But the secretory amount of IL-1 showed no significant difference between blank and drug group.Conclusion1 Acquired fibroblasts can be cultured in stable condition in vitro,and sufficient ad reliable target cells can be obtained for the study of the mechanisms of intestinal adhesions at molecular level.2 D and Q could inhibit the proliferation and migration of fibroblasts,depressed cell activity,but D and Q does not influence collagen synthesis of FB. Combination therapy of D with Q may take synergistic inhibitory effect on the proliferation of FB.3 D and Q at a ratio of 9:2 results in a optimal combination.4 Optimal combination could regulate the cell cycle of FB and restrain the proliferation and migration of FB.It could also reduce the secretory amount of TNF-a and IFN-y. |
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