Studies Of Pharmacodynamic Action Evaluation And Mechanism On Weitong Injection For Postsurgical Abdominal Adhesions

BackgroundPost-surgical adhesion is a quite common complication,especially after general laparotomy.It manifests with fibrous bands joining normally separated organs together with the intestinal/abdominal wall.Intra-abdominal adhesion formation and re-formation after surgery is a significant cause of morbidity,such as pain,bowel obstruction,even infertility.According to previous studies,although recent cases suggests that a proportion of people suffering such adhesion may benefit from adhesiolysis,adhesion re-formation still remains as the most serious post-surgical problem,the probability ranging from 55%to 95%among the patients even those who have had undergone surgical adhesion removal.Up to now,a number of TCM (Traditional Chinese Medicine) treatment and herbal preparation have been evaluated for their value in post-surgical adhesion prevention.So it is necessary to exert the superiority and characteristic of TCM in treating post-surgical adhesion.WTI(Weitong Injection),developed by Department of pharmaceutics,Nanfang Hospital,is a pure herbal injectable powder for anti-adhesion.It bases on the study of Changtong Oral Liqud and contains components from herb,which can improve microcirculation of blood,decrease inflammatory reaction and exudation.It is another important new drug for post-surgical adhesion.ObjectiveTo evaluate and explore the pharmacodynamic action and mechanism of WTI with the following experiments:To observe the effect of WTI on post-surgical adhesions by establishing rat experimental models with intestinal adhesions.To detect the main components of WTI by HPLC.To explore the effect of WTI and its components on proliferation and cell cycle of cultured fibroblasts from normal and adhesive peritoneal tissues.To detect synthesis product and mRNA expression of cytokines from NFB and AFB.Methods1.Pharmacodynamic action of WTIRats were received laparotomy and prepared as intestinal adhesion model.WTI was intravenously injected for 7 days.Dexamethasone were used as positive control. Blood routine examination was conducted.Intraperitoneal adhesions were graded by adhesion score on day 7.Tissues from normal and adhesive tissues were collected to prepare pathological sections.2.Detection of components in WTIComponents of WTI were determined by HPLC.The mobile phase of D was phosphate buffer solution:methanol(95:5).The mobile phase of Q was 1% phosphoric acid:acetonitrile(60:40).Flow rate was 1mL·min^-1.The UV detective wavelength was 220nm.Chromatographic column was Luna^? 5u C18(2) 100A. Column temperature was room temperature. 3.Effect of WTI on proliferation and cell cycle of NFB and AFBThe normal and adhesive peritoneal tissues were harvested from adhesion model of rots.The prepared tissues were cultured for 1～2 weeks until outgrowth of NFB and AFB.The cells of 3～5 passage were seeded on 96-well plates at a density of approximately 5×10³ cells per well in 10%FBS.Then each kind of FB was divided into 5 groups:WTI,D standard,Q standard,D+Q standard and dexamethasone group. Each group had 6 different drug concentration ranging from 0～40μg·mL^-1.The dosage of D+Q standard group was based on the ratio of D and Q in WTI(D:Q=1:10). Cultured for 72hr,the proliferation of NFB and AFB were detected by MTT assay.The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10⁵ cells per dish in 10%FBS.Then each kind of FB was divided into 6 groups:blank,WTI,D standard,Q standard,D+Q standard and dexamethasone group.The dosage of D+Q standard group was based on the ratio of D and Q in 10μg·mL^-1WTI(D:Q=1:10).The drug concentration of other drug groups was 10μg·mL^-1.Cultured for 72hr,the cell cycle of NFB and AFB were analyzed by FCM.The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10⁵ cells per dish in 10%FBS.Then each kind of FB was divided into 2 groups:blank and WTI group.The drug concentration of WTI group is 10μg·mL^-1.Cultured for 72hr,then cells were collected to observe the ultramicrostructure by transmission electron microscope.4.Effect of WTI on cytokines mRNA expression and collagen synthesis of NFB and AFBThe cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10⁵ cells per dish in 10%FBS.Then each kind of FB was divided into 5 groups:blank,WTI,D standard,Q standard,D+Q standard and dexamethasone group.The dosage of D+Q standard group was based on the ratio of D and Q in 10μg·mL^-1WTI(D:Q=1:10).The drug concentration of other drug groups was 10μg·mL^-1.Cultured for 72hr,cells were collected to extract total RNA.Then the RT reaction was carried out on each RNA sample to produce cDNA.Real-time PCR was performed in a reaction plate containing RT products(cDNA),TaqMan fluorescent probe,relative primer and other PCR reagents.C_T of each sample was recorded. According to the standard curve ofβ-actin and each target gene,the copy number of original templates(β-actin,TGF-β₁,TGF-β₂,MMP-1,TIMP-1)were obtained.Finally, the absolute copy number of target gene was normalized to that ofβ-actin.On the other hand,collected the supernatant of cells and determine HyP by base hydrolysis-chromatometry.Results1.WTI could significantly reduce the level of adhesion,lower WBC and NEU counting,recover PLT counting to the preliminary level.WTI could also lower HyP concentration in traumatized tissues.Pathological sections showed the inflammation level of WTI groups were much lower than model group.Peritoneal and cecal wall of WTI groups were also fixed.Such effect of WTI were better than dexamethasone.2.D and Q were the main components of WTI.The methods built up for HPLC were fit for the assaying of these components.The content of D was 4.99%.The content of Q-A and Q-B were 14.98%and 11.42%respectively.3.IC₅₀ of all AFB groups were lower than NFB groups.Dexamethosone groups had the highest IC₅₀ among all AFB groups.Compared with blank groups,all drug groups had higher G₀/G₁ proportion and lower S,G₂/M proportion.Compared with all drug groups of NFB,all drug groups of AFB had higher G₀/G₁ proportion and lower S, G₂/M proportion.Transmission electron microscope picture showed WTI could change the ultramicrostructure of AFB.4.Compared with blank group,the mRNA level of TGF-β₁,TGF-β₂,MMP-1, TIMP-1 of all drug groups were lower.Among the AFB groups,MMP-1/TIMP-1 mRNA ratio of drug groups were higher than blank group,and there was no significant difference between NFB drug groups and NFB blank group.Compared with blank groups of AFB,drug groups of AFB had lower HyP in the supernatant,and there was no significant difference between NFB drug groups and NFB blank group.Conclusions1.WTI could reduce excess synthesis of collagen in traumatized peritoneum and decrease fibrosis level.WTI could also reduce inflammatory reaction and improve the high blood viscosity caused by trauma.2.D and Q were the main components of WTI.3.Both WTI and its main components could regulate the cell cycle of FB and restrain the proliferation of FB.And AFB was more sensitive to medication than NFB.4.Both WTI and its main components could lower mRNA expression level of TGF-β₁ and TGF-β₂ in AFB to restrain the proliferation of FB.They could also regulate MMP-1/TIMP-1 mRNA ratio in AFB to decrease synthesis of collagen,which lead to the inhibition of excess fibrosis.