| Background and ObjectivePostoperative peritoneal adhesions,which occur in the peritoneal cavity as a result of surgery or stimulation of foreign bodies,are the most frequent complication of abdominal surgery.Up to 90%of patients develop adhesions after surgery,and a considerable proportion of cases result in major short-and long-term negative consequences,including small-bowel obstruction,infertility,and chronic pelvic pain.The incidence of re-admissions directly related to adhesions varies from 5%to 20%.So far,there are two control strategies for prevention adhesion formation in clinic:one is decreasing operative injury and foreign bodies remained during the operation,another is using mechanical barrier after operation,which could prevent contact between the damaged serosal surfaces for the first few critical days.There have been a number of barriers approved by FDA for clinical use up to now,such as Seprafilm(?),Interceed(?) and ADEPT(?).While barriers have several strengths,the products still have many limitations.On the one hand,the biocompatibility and impact on wound healing are still controversial.On the other hand,it has been reported that some of them showed serious adverse reactions like peritonitis and skin fistula.In addition to meticulous surgical technique and synthetic solid barriers,several drug approaches have been reported in the prevention of adhesions including:anti-inflammatory agents,fibrinolytic agents,antibiotics.However,none of these methods has proven to be uniformly efficacious under all surgical conditions.Ultimately,there is still on drugs on the market.Thus,research and development of new drugs for prevention peritoneal adhesion is particularly important and prominent.It is widely accepted that inflammatory response,fibrinolysis system,the synthesis of fibrin and its analogs and their interactions play central roles in accelerating formation and development of postoperative peritoneal adhesions.Adhesion formation begins with trauma to the peritoneal surfaces.Immediately after surgical injury to the peritoneum there is bleeding and an increase in vascular permeability with extravasation of fibrinogen-rich fluid from the injured surfaces.Almost simultaneously,an inflammatory response occurs,with migration of inflammatory cells,release of cytokines.These inflammatory cytokines can activate the coagulation cascade that forms a complicated fibrinous reticular structure in lesion location.Moreover,they will promote the fibroblast migration and the transformation of fibroblasts into myofibroblast,which has greater capacity of synthesis and secretes collagen and ECM.Under normal conditions,these fibrinous reticular structures are resolved by fibrinolysis.However,under ischemic or inflammatory conditions,the peritoneal fibrinolytic system is suppressed and they are infiltrated with inflammatory cells and fibroblasts to organize into dense adhesions.Therefore,the aims of drug control strategy are to shift the adhesion formation to the normal healing through the prevention of one or more abnormal pathways.As a hospital preparation,Changtong oral liquid(CTOL)has been applied into clinic practice for over 10 years,which shows a good clinical efficacy on prevention and treatment of postoperative adhesion.Our previous studies indicated that CTOL presented the multi-target,multi-channel overall treatment in preventing postoperative adhesion including attenuation of inflammation,improvement of fibrinolytic activity in peritoneal cavity,inhibition of the proliferation and transformation of fibroblast and interference of peritoneum progression of fibrosis.Then different methods were used to study the extraction,characteristics,active components from the CTOL prescription,and finally 2 potential active monomers were screened which were named TANS and AESS alternatively.TANS was extracted from Salvia miltiorrhiza,and the structure of AESS was a kind of triterpenoid saponins.Based on the background above,firstly,this study is to observe the process of pathological changes by detecting some representive physiological and bio-chemical indexes at each time point,in order to demonstrate the formation process of postoperative peritoneal adhesions and determine the optimal time for drug intervention.Secondly,the pharmacodynamics and mechanisms of two potential active monomers in preventing adhesions are explored in vivo and vitro.Moreover,the optimal compatibility proportion of them is researched in vitro,and whether it will display synergistic action and show more effective in preventing adhesion formation.We expect that this research could provide new ideas for drug development and reliable experimental basis for the clinical treatment of postoperative peritoneal adhesions.Method(1)Study of the pathological changes of postoperative peritoneal adhesionsLaparotomies and creation of ischemic buttons were used to construct peritoneal adhesions rat model.Before 24 h and after 12 h,24 h,72 h,120 h,168 h,peritoneal fluid,venous blood and the ischemic tissues were collected and then detected some representive physiological and bio-chemical indexes at each time point.Paraffin slice of peritoneum tissue was prepared for Masson staining and then used for pathological detection.(2)Pharmacodynamic and mechanism evaluation of TANSPeritoneal adhesions rat models were randomly divided into 6 groups(n=10):dexamethasone(Dex)group(5 mg/kg/d),TANS group(8.0 mg/kg/d,4.0 mg/kg/d,2.0 mg/kg/d),vehicle group(normal saline)and blank control group via tail vein injection after 12h for once a day.At day 7,the animals were killed and the degree of adhesion was quantified.Collected the peritoneal fluid and determined the levels of tPA,PAI-1 and tPA activity.Collected the ischemic tissues in each experimental group,and placed one half in 4%paraformaldehyde fixing solutions after longitudinally section of ischemic tissues used for pathological and immunohistochemical detection.The other half of the tissues was divided into three portions.One part was prepared for tissue homogenate and was used to measure Hyp level.The other two parts was frozen at-80℃ for RT-PCR,western blot and other experiments.Adhesive fibroblast(AFB)induced by TGF-β1 was established:AFB cells were induced by different concentrations of TGF-β1 for 24 h,and then detected the optic density and expression of PAI-1 and α-SMA.TANS with different concentrations was added to the AFB cells for 24 h to detect the optic density for cellular proliferation inhibition rate,in order to gain the optimal concentration gradient.Then high,medium and low doses of TANS were added into AFB cells induced by TGF-β1 and cultured for 24 h.Western blot were performed to detect the expression levels of p-Smad2/3,p-Smad4,PAI-1,Collagen-I and α-SMA.The secretion of PAI-1 and α-SMA in the supernatant were detected by ELISA.Kartogenin,the specific activitor of Smad was used to block the effect of TANS and then detected the changes of proteins level.(3)Pharmacodynamic and mechanism evaluation of AESSPeritoneal adhesions rat models were randomly divided into 6 groups(n=10):Dex group(5 mg/kg/d),AESS group(2.0 mg/kg/d,1.0 mg/kg/d,0.5 mg/kg/d),vehicle group(normal saline)and blank control group via tail vein injection after 12 h for once a day.At day 7,the animals were killed and the degree of adhesion was quantified.Collected the peritoneal fluid and determined the levels of tPA,PAI-1 and tPA activity.The plasma was collected through the abdominal aorta under anesthesia,and determined the levels of FIB,The ischemic tissues were frozen at-80℃ for RT-PCR,western blot and other experiments.The normal human peritoneal mesothelial cells(HMrsV5)were induced by different concentrations of IL-1β for 12 h,24 h,48 h and 72 h,and then detected the and level of tPA and its activity.AESS with different concentrations was added to the HMrsV5 cells for 24 h to detect the optic density,in order to gain the safe concentrations.Then the different doses of AESS were added into HMrsV5 cells induced by IL-1β and cultured for 24 h.Western blot were performed to detect the expression levels of p-MYPT1 and tPA.The secretion of tPA and its activity in the supernatant were detected.The specific agonists of Rho kinase LPA was used to block the AESS effect and then detected the changes of proteins level.In order to clarify whether AESS showed a specific inhibition effect on Rho kinase,firstly we compared the efficacy between AESS and Hydroxyfasudil,C3 exoenzyme by culturing with HMrsV5 cells induced by IL-1β.Moreover,we investigated whether AESS inhibited the activation of Rho kinase through mevalonate(MVA)biosynthesis pathway,which is one of the most important way to activate the Rho kinase.MVA,Geranylgeranyl pyrophosphate(GGPP)and Farnesyl pyrophosphate(FPP)were used to interfere the AESS effect and then detected the changes of protein levels.(4)Study of optimal compatibility proportion and synergistic action for WIEThe screened concentration range of TANS and AESS were mixed all and constituted 30 intersecting groups.AFB and HMrSV5 cells were incubated with all of these intersecting groups for 24 h to detect the optic density respectively,in order to find a group that showed a better synergistic interaction on proliferation inhibition rate for AFB cells,and had on obvious cytotoxicity for HMrSV5 cells.Peritoneal adhesions rat models were randomly divided into 9 groups(n=12):Dex group(5 mg/kg/d),AESS group(2.0 mg/kg/d),TANS group(8.0 mg/kg/d),WIE groups(3.5 mg/kg/d,1.75 mg/kg/d,0.9 mg/kg/d),vehicle group(normal saline)and blank control group via tail vein injection after 12h for once a day.At day 7,the animals were killed and the degree of adhesion was quantified.Collected the peritoneal fluid and determined the levels of tPA,PAI-1 and tPA activity.The plasma was collected through the abdominal aorta under anesthesia,and determined the levels of FIB.Collected the ischemic tissues in each experimental group,and placed one half in 4%paraformaldehyde fixing solutions after longitudinally section of ischemic tissues used for pathological and immunohistochemical detection.The other half of the tissues was divided into three portions.One part was prepared for tissue homogenate and was used to measure Hyp level.The other two parts was frozen at-80℃ for RT-PCR,western blot and other experiments.Result1.The pathological changes of postoperative peritoneal adhesions(1)Statistical analysis was performed by repeated measures analysis of variance,Manuchly ’s sphericity test meet the requirements.Inflammatory factor levels in the peritoneal fluid:The expression levels of IL-1β at each time point after operation were significant higher than preoperative level(P<0.001);The expression of TGF-β1 at 12h after operation had no significant differences compared with baseline level(P=0.144),the other time point TGF-β1 levels were significant higher than the preoperative(P<0.001).(2)Peritoneal fibrinolytic system:Compared with baseline level(preoperative level),tPA and its activity were significantly increased(P<0.01),but the tPA and its activity at other time point were significantly decreased except the 24h time point(P<0.01).However,the PAI-1 secretion levels were significant lower than the base line(P<0.01).(3)The contents of FIB and D-dimer in plasma:FIB and D-dimer showed a similar trend,the levels of FIB and D-dimer in plasma at each time point after operation were significantly increased compared with the preoperative level.(4)The collagen formation in peritoneum tissue:Compared with preoperative level,the content of Hyp in peritoneal ischemia was significantly increased(P<0.05).Masson staining in peritoneal ischemia showed that IOD level at 72 and 168h after operation were significant higher than the base line and 24h time point after operation(P<0.05).2.Pharmacodynamic and mechanism evaluation of TANS(1)The Kruskal-Wallis test was used to identify the differences in the adhesion grades between the groups.Compared with the vehicle group,all drug administrated groups witnessed a significant decrease in adhesion level(P<0.05).Compared with DEX group,H-TANS group significantly decreased the scores(P=0.024),but M-TANS and L-TANS groups had no significant differences.The experimental data presented normal distribution in each group,so used one-way ANOVA for multiple comparisons between groups.H-,M-and L-TANS groups significantly decreased PAI-1 level,and significantly increased the tPA activity compared with the vehicle group(P<0.05),but showed on effect on tPA expression and secretion(P>0.05).Each TANS groups could significantly decreased the contents of Hyp in peritoneal ischemia compared with the vehicle group(P<0.01).Western blot and RT-PCR results showed that each TANS groups decreased PAI-1,Collagen-I and α-SMA protein and mRNA levels compared with the vehicle group(P<0.05),and the p-Smad2/3 protein level was also significantly decreased(P<0.05).Immunohistochemistry results showed that the expressions of Collagen-I and α-SMA were very low and p-Smad2/3 expression was poor in blank control,but all of them were obviously increased in vehicle group.Each TANS groups could decrease the expressions of them compared with the vehicle group.(2)TANS concentrations in the 3.8～13 μmol/L showed a good proliferation inhibition rate for AFB cells,and we chose 3.8 μmol/L,5.0μmol/L and 6.3 μmol/L as the low,medium and high dose group.The inducing conditions of AFB cells indicated that AFB cells were induced by 10ng/mL of TGF-β1 for 24h showed a good effect on increasing PAI-1 and α-SMA secretions and promoting the proliferation,and could increase the p-Smad2/3,p-Smad4,PAI-1,α-SMA and Collagen-I expressions in AFB cells compared the blank group(P<0.05).H-,Mand L-TANS groups could significantly decreased these protein levels in AFB cells induced by TGF-β1(P<0.05).But this regulatory effect could be blocked by the specific activitor of Smad,the secretion levels of PAI-1 and α-SMA was significantly increased compared with H-TANS group(P<0.01).3.Pharmacodynamic and mechanism evaluation of AESS(1)Compared with the vehicle group,all drug administrated groups witnessed a significant decrease in adhesion level(P<0.05).Compared with DEX group,H-AESS group significantly decreased the scores(P<0.05),but M-AESS and L-AESS groups had no significant differences.H-,M-and L-AESS groups significantly increased tPA level,and significantly increased the tPA activity compared with the vehicle group(P<0.05),but showed on effect on PAI-1 expression and secretion(P>0.05).FIB contents in plasma of AESS treated groups were significantly decreased compared with the vehicle group.Western blot and RT-PCR results showed that p-MYPT1 and D-dimer levels in each AESS groups was significantly decreased(P<0.05)and the protein and mRNA levels were significantly increased compared with the vehicle group,but showed on significant difference in PAI-1 and its mRNA expressions(P>0.05).(2)The maximum safe concentration to HMrSV5 cells for 24h treatment of AESS was 26μmol/L.After different concentrations of IL-1β induced HMrSV5 cells for 12h,24h and 48h,the levels of tPA and its activity results showed that the best inducing condition was 10ng/mL IL-1β induced for 24h.Compared with the blank control group,IL-1β could significantly increase the p-MYPT1 expression(P<0.01)and significantly decrease tPA expression and secretion(P<0.01).When treated with each doses of AESS,p-MYPT1 expression was significantly decreased and tPA expression was significantly increased(P<0.01).However,after added into LPA,the effect of AESS was reversed,tPA secretion and tPA activity in the supernatant were significantly decreased(P<0.05).Compared with model group,H-AESS,Hydroxyfasudil and C3 exoenzyme could decrease the p-MYPT1 and tPA expression,and significantly increase tPA secretion and tPA activity in the supernatant(P<0.01).When MVA,GGPP and FPP were added into AESS treated group respectively,we found that MVA and GGPP could reverse the AESS effect,and the tPA secretion and tPA activity in the supernatant were significantly decreased(P<0.05),but FPP showed on efficacy.4.Optimal compatibility proportion of WIE and synergistic action for WIE(1)Optimal compatibility proportion results showed that:when 5.0μmol/L TANS and 26μmol/L AESS were matched,this intersecting group exhibited a better synergistic interaction on proliferation inhibition rate for AFB cells,and had on obvious cytotoxicity for HMrSV5 cells.(2)Compared with the vehicle group,all drug administrated groups witnessed a significant decrease in adhesion level(P<0.05).Compared with DEX group,TANS-group and AESS group,H-WIE and M-WIE groups significantly decreased the scores(P<0.05),but L-WIE groups had no significant differences.H-,M-and L-WIE groups significantly increased tPA level and tPA activity compared with the vehicle group(P<0.05),and significantly decreased PAI-1 expression and secretion(P<0.05).tPA activity level in H-and M-WIE groups were significant higher than other groups.FIB contents in plasma and Hyp contents in peritoneal ischemia of WIE treated groups were significant lower than the other groups.Masson staining in peritoneal ischemia showed that H-and M-WIE groups showed a better effect on improving the degree of fibrosis than other groups,and L-WIE group was similar to H-TANS and H-AESS groups.Western blot and RT-PCR results showed that each WIE groups could both decrease p-Smad3 and p-MYPT1 significantly(P<0.05),significantly decrease the protein levels of PAI-1,Collagen-I,α-SMA,D-dimer and their mRNA expression(P<0.01),and significantly increase tPA protein and mRNA expression compared with the vehicle group,Immunohistochemistry results showed that the expression of Collagen-I and D-dimer were very low,p-Smad3 and p-MYPT1 expressions were poor in blank group,but all of them were obviously increased in vehicle group.Each WIE groups could decrease the expressions of them compared with the vehicle group.Conclusion(1)The study of pathological changes for postoperative peritoneal adhesion indicates that we could drug treatment could be initiated at 12 h time point after operation.(2)TANS and AESS show different pharmacodynamic and mechanism for prevention of adhesions,but shows a synergistic effect on peritoneal fibrinolytic system.TANS could delay the fibrosis of peritoneal ischemia and decrease PAI-1 expression by inhibition of TGF-β/Smad signaling pathway.AESS shows a activation of fibrinolytic system directly by increasing the expression and secretion of tPA,and this effect may be mediated by Rho/Rho-kinase signal pathway.(3)Optimal compatibility proportion of WIE is that the concentration ratio of TANS and AESS is 5:26.(4)WIE could inhibit TGF-β/Smad and Rho/Rho-kinase signal pathway simultaneously,which shows a synergistic effect on peritoneal fibrinolytic system.And it exhibits a better effect on preventing postoperative peritoneal adhesion compared with two monomers. |
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