Molecular Mechanism Study Of Changtong Oral Liquid On Intestinal Adhesions

Peritoneal adhesion is a quite common complication, especially alter generalabdominal operations. It manifests with fibrous bands joining normally separatedorgans together and/or the intestinal wall. Intra-abdominal adhesion formation andre-formation after surgeryis a significant cause of morbidity, causing such symptomsas pain, bowel obstruction, and even infertility. Although recent evidence suggeststhat a proportion of women suffering such adhesion may benefit from adhensionlysis,adhesion re-formation still remains as the greatest postoperative problem, amountingto 55ï¼…to 100ï¼…among the patients even having undergone surgical adhesionremoval according to previous studies. Up to now, a number of adjuvants have beenevaluated for their value in postoperative adhesion prevention, but currently fewherbal preparations for such purpose have been available. So it is an important trial toprevent and treat intestinal adhesion by exerting the superiority and characteristic oftraditional Chinese medicine (TCM) in treating intestinal adhesion.CTOL, prepared by Department of Pharmaceutics, Nanfang Hospital based onTCM theory, contains the ingredients of Rhubarb, Salvia miltior rhiza, Fructusaurantii immaturus and so on. As a hospital preparation, CTOL has been used to treatintestinal adhesion for many years, its curative effect clinically assured. Both clinicobservations and animal experiments indicated that CTOL could obviously preventintestinal adhesion in rat models due to its pharmacological effects of minimizingpostoperative depression of local fibrinolytic activity, reducing the severity ofpostoperative adhesions, and ultimately alleviating the pathological injuries at the siteof adhesion. On the other hand, the preparation was evidenced to ameliorate the general symptom, protect the organs and hold back the development of adhesion.However, the cytomolecular mechanism of the preparation in treating adhesionremains unclear. In this ease, we aim our research to exploration ofcytomolecular andgenie mechanism of CTOL in treating intestinal adhesion from cytomolecularpathway in an animal model, for the purpose of consolidating the laboratorialevidence on its curative effect during clinic application.Partâ… Experimental Study of CTOL on Rats' Intestinal ModelObjective: (1) To observe the effect of CTOL on postoperative adhesions byestablishing rat models of intestinal adhesions; (2) to determine and analyze thenumber of white blood cell (WBC), contents of fibrinogen (FIB), tumor necrosisfactor alpha (TNF-α), transforming growth factorβ₁(TGF-β₁), interleukin-1β,4, 6,10 (IL-1β, IL-4, IL-6, IL-10) in the blood of rats with adhesions; (3) to study theinfluence of WBC, FIB and cytokines on postoperative adhesion formation andprovide the laboratorial foundation for further studying its therapeutic mechanism.Methods: (1) Fifty-four Sprague-Dawley (SD) male rats were used in this studyand randomly divided into six equal groups (n=9): normal control; model control;Simo decoction (SMD); and three other groups at different dosages of CTOL. Exceptnormal control, all the other rats were prepared as intestinal adhesive models underthe guidance by Ellis. Normal and model control groups were orally treated throughwith distilled water; SMD was used as male control medication due to its similarefficacy in treating adhesion to assure the reliability of experimental method. CTOLwas administrated at three different dosages of 4.3g/kg, 8.6 g/kg, 17.2 g/kg, 5, 10 and20 times as much as the clinical dosages, respectively. The dose of male controlmedication was equal to the multiple of medium dose CTOL. (1) On Day 7 aftersurgery adhesion in each rat was evaluated according to the 5-grade rating scaleproposed by Nair; (2) The samples with adhesion were harvested for pathologicalobservation; (3) Additionally, blood samples from abdominal artery of all rats weresubmitted for the determination of the amount of WBC, concentrations of FIB andlevels ofTNF-α, IL-1β, TGF-β₁, IL-4, IL-6, IL-10. Results: (1) Comparison of adhesion grades between all groups: None of the ratsdied in each group within 7d after inducement of adhesions. Adhesions formed inmodel control and even grew so dense that it was difficult to remove from theinvolving organs. In contrast, animals in the four experimental groups only developeda thin, filmy and membranous adhesion or even no adhesions at all. The severity ofadhesion in treatment groups was significantly less than that of model control. (2)Comparison of plasma FIB level and WBC count: Compared to that of normal group,FIB level and WBC count of model group were significantly increased (P＜0.01). FIBand WBC in all experimental groups were evidently decreased (P＜0.05), compared tomodel group. (3)Comparison of serum cytokine levels: The proinflammatory cytokinelevels of TNF-α, TGF-β₁, IL-1βand IL-6 in model control group were significantlydifferent from those of normal control group (P＜0.01), although theanti-inflammatory cytokines IL-4 and IL-10 were not (P＞0.05). However, thecytokine levels showed no marked differences between normal control group andSMD group as well as CTOL groups. The serum concentrations of TNF-αand IL-6 inrats of CTOL groups were significantly decreased (P＜0.05 or P＜0.01), compared tocontrol groups. The serum TGF-β₁ and IL-1βlevels in two CTOL groups of largerdosages were considerably lower than those of model control group (P＜0.05 orP＜0.01), but those of 4.3g/kg CTOL group showed no evident reduction. In addition,serum IL-4 and IL-10 levels in all CTOL groups tended to elevate, but the incrementwas not statistically significant (P＞0.05). No statistical difference was noted incytokine levels among the CTOL groups (P＞0.05). The results demonstrated theefficacy of SMD was similar to that of CTOL.Conclusions: CTOL can evidently prevent rat intestinal adhesion in rat models,reduce the severity of postoperative adhesions, and improve the pathological injuriesof adhesive spots. On the other hand, CTOL is found to decrease plasma WBC andFIB concentration and serum levels of proinflammatory cytokines such as TNF-α,IL-1β, TGF-β₁ and IL-6. However, it has no significant impact on serum levels ofanti-inflammatory cytokines IL-4 and IL-10 in rats with postoperative adhesions. This experiment showed that CTOL could treat and protect intestinal adhesions again.Partâ…¡Effect of CTOL on Proliferation and Cell Cycle of NF and AFObjective: To explore the effect of CTOL on proliferation and cell cycle ofcultured fibroblasts from normal peritoneum and adhesion, and prepare thelaboratorial foundation for further study on its molecular therapeutic mechanism.Methods: (1) Preparation of medication serum: Twenty SD male rats wererandomly divided into 4 groups: normal serum group, 3 CTOL groups (low, mediumand high dose). After administration of medication at the different dosages for 7 days,serums were obtained from the abdominal arteries of all rats. (2) In vitro culture offibroblast: The adhesion tissues were harvested from intestinal adhesion ratsundergoing laparotomy, meanwhile the normal parietal peritoneal tissues were fromtheir anterior abdominal walls at least 2 cm away from the midline incision. Theprepared tissues were cultured for two weeks until outgrowth of fibroblasts. Whenconfluence was reached, the fibroblasts subcultured with trypsinization at 1:3 splitratio and then transferred to 75cm² culture flasks for further culturing in standardmedia with 10 percent FBS. In our study, the passage 3～8 cells were used in order tomaintain comparability. (3) Purity and identification of fibroblasts: Because of theirsensitivity to trypsinization, pure fibroblasts were gained by repeated trypsinizationand passages. Mouse anti-Vimentin were used as first antibody of immunochemicalidentities of fibroblasts and Phosphate buffered saline (PBS) replacing Vimentin inthe blank controls.(4) Proliferation assay of fibroblasts:â‘ MTT method: The cellswere seeded on 96-well plates at a density of approximately 5×10³ cells/well in 10percent FBS. To induce differentiation, the basic culture medium was replaced withDMEM with 1ï¼…FBS, penicillin (100 U/ml), and streptomycin (100μg/ml), in whichthe growth factors were withdrawn. Then the fibroblasts cultured in DMEM wasdivided into four groups: serum normal control group, CTOL groups (high dose, medium dose and low dose). Cultured for 24, 48, 72 and 96 hours respectively, theproliferation of fibroblasts was observed with MTT assay.â‘¡BrdUimmunofluorescent staining: Fibroblasts in desired condition were mechanicallydissociated after digestion by 0.25ï¼…trypsin for 2min. Then, the dissociatedfibroblasts were seeded on 24-well plates in 5×10⁵ cells/well. According to theexperimental design, the media were treated by adding different treatments, such asCTOL medium (0.5μl/ml), normal rat serum (0.5μl/ml), PAI-1(1ng/ml), tPA (1ng/ml),respectively. Untreated cells were used as control. Twenty four hours after thetreatment with medication, 10μM bromodeoxyuridine (BrdU) was applied to theculture media for 3 hours to label the nuclei of cells that had differentiated, and thenimmunocytochemistry was performed to detect BrdU positive cells after fixation with4ï¼…PFA. (5) Specimen preparation for transmission electron microscopy (TEM): Thecells in desired condition were plated in 75cm2 flasks at a density of approximately2.5×10⁶ cells/well in 10 percent FBS. Then the fibroblasts cultured in DMEM weredivided into three groups: 10ï¼…FBS control group, rat serum normal group, CTOLgroup (medium dose). Cells were cultured respectively for 24, 48hour. Aftertrypsinization and washing with PBS, the collected cells were fixated with 2.5ï¼…glutaraldehyde and then send for observation of their ultrastructure by TEM. (6) Cellcycle: The cells in desired condition were plated in 75cm² flasks at a density ofapproximately 2.5×10⁶ cells/well in 10 percent FBS. Then the fibroblasts cultured inDMEM were divided into three groups: rat serum normal group, CTOL groups(medium dose and high dose). Cells were cultured respectively for 12, 24, 48 hours.After trypsinization, centrifugation and washing with PBS, the collected cells werefixed in 70ï¼…alcohol and placed overnight at 4℃. After staining for 30 min with PIdyeing liquor, their cell cycles were analyzed by flow cytometer.Results: (1) Growth velocity: Fibroblasts from normal peritoneum andadhesions grew easily in vitro, but those fibroblasts from adhesions grew faster thanthose from normal peritoneum. (2)Identification of fibroblasts: Vimentin expressionspreviously used a marker for fibroblasts were visible in all fibroblasts whose cytoplasm was stained brown. (3) Proliferation of fibroblasts:â‘ MTT method: Thecorrelation between medication dosages and their OD value negative, but it wasreverse for the correlation between medication dosages and their inhibitory rates.Compared with rats serum normal group of NF, CTOL different dosage groups coulddecrease their OD values at hours 24 and 48 during the experiment, but only thedifference was statistically significant at hours 72 and 96 (P＜0.01). Compared withrats serum normal group of AF, CTOL different dosage groups all could significantlydecrease their OD values at hours 24, 48, 72 and 96 (P＜0.05). Additionally, inhibitoryrate of low dose CTOL group to AF was higher than to normal ones at the same timepoint (P＜0.01). However, inhibitory rate of CTOL at a high dose to AF was higherthan to normal one only at hour 72 (P＜0.05), and so it was with medium and highdose CTOL groups at 96 hour (P＜0.05).â‘¡BrdU immunofluorescent staining: Innormal cultures without treatment, some 21.02±1.87ï¼…of AF incorporated BrdU inhour 4 indicated by immunocytochemistry. When CTOL medium, PAI-1 and tPAwere added to the cultures, the fraction of BrdU positive cells was significantlychanged (p＜0.001) (CTOL medium, 12.76±1.72ï¼…; PAI-1, 24.44±1.44ï¼…; tPA,9.54±5.64ï¼…). However, positive cells of normal rats' serum had no significant change.(4) Ultrastructure of fibroblasts: The ultrastructures of fibroblasts both from normalperitoneum and adhesions were basically uniform with TEM. The cell nucli with oneor two obvious nucleoli were round or oval in shape. It was indicated that CTOLmedium might deform AF as dilated rough endoplasmic reticulum, distendedmitochondria and increased lysosome were observed, but no obvious damage ofmembrane structure was found. However, CTOL medium and normal rats' serumexerted no influence on the normal formations of NF. (5)Cell cycle: Compared withrats' serum normal group of NF, medium dosage CTOL could increase the proportionof NF in the G₁ stage at hours 12, 24, 48, but only high dosage CTOL didsignificantly in statistics at different time points (P＜0.001). Medium and high doseCTOL could not markedly reduce the proportions of cell at S and G₂ stages.Compared with rats' serum normal group of AF, medium and high dosage CTOLcould both increase the proportion of AF at the G1 stage at hours12, 24, 48 significantly (P＜0.01). But CTOL could not markedly reduce the proportions of cellsat S and G₂ stages.Conclusions: CTOL at medium and high doses as well as tPA could evidentlyrestrain the proliferation of fibroblasts. However, PAI-1 accelerated the growth of AF.AF were more sensitive to medication than normal ones, coming out to bedose-dependent.It was demonstrated in the present study that CTOL regulates theproliferation and differentiation of AF, which may have implications for clinicalutilization of CTOL to prevent peritoneal adhesions.Partâ…¢mRNA Expression of Cytokines by Real-Time Quantitative PCRObjective: To determine the response of AF and NF to CTOL rats' serum byreal-time quantitative PCR.Methods: (1) Collection of fibroblasts: The fibroblasts in right condition fromnormal peritoneum and adhesion tissues were plated in 75cm2 flasks at a density ofapproximately 2.5×106 cells/well in 10 percent FBS. The fibroblasts cultured inDMEM were divided into three groups: normal rats' serum, CTOL medium dose andCTOL low dose. After the cells in the three groups were cultured respectively for 48hour, they were washed with D-Hanks, prepared for extraction of total RNA. (2)TotalRNA extraction: Total RNA was isolated from all the cells after treatment for 48hours using TIANGEN RNAprep reagent kits according to manufacturer'sdescription. (3)TaqMan probes and primers: PCR primers and TaqMan probes forIL-1β, IL-6, and IL-10, TGF-β1, TGF-β2, TIMP-1, MMP-1, TPA, PAI-1, INF-γ, andβ-actin were designed using Primer Express 2.0 Software program. (4) Reversetranscription: RT reactions were carried out on each RNA sample in 0.2ml PCRreaction tubes using TIANGEN reverse transcription reagents. Each reaction tubecontained 2μg total RNA in a volume of 15.5μl containing 10μM Oligo dT (18),10mM dNTTs, RNA-free H20. The reactions were kept warm at 65℃for 5 min, andthen placed in ice water for 5 min, followed by adding 40μ/μl of RNase inhibitor, 10×AMV Reaction Buffer, DTT (IM), and Reverse Transcriptase (AMV) in PCRreaction tubes. In this way, RT reactions sustained at 37^C for 1 hour and then at 70℃for 15 min. Finally, the RT reaction mixture was placed at 4℃for immediatePCR amplification, or just stored at -20℃for future use. (5)PCR reactions: Aider 31rounds of the procedures including denaturalization for 30 seconds at 93℃, annealingfor 30 seconds at 56℃, extending for 30s at 72℃, the products by reversetranscription were stored for another 5 min at 72℃and finally prepared at 4℃readyfor PCR reactions, which for the following performed in 0.2ml PCR reaction tubescontaining a volume of 25μl containing 10×Buffer, 2.5 mM dNTPs, 25μM Primer 1,25μM Prime, cDNA (RT reaction) template and 5U/μl Taq pfu polymerase. (6)Construction of standard curve: To determine the absolute copy numbers of the targettranscript, PCR products for IL-1β, IL-6, IL-10, TGF-β1, TGF-β2, TIMP-1, MMP-1,tPA, PAI-1, INF-γ, andβ-actin were used to generate a standard curve. PCR productswere purified using TIANquick Midi kit according to the manufacture's instructions.PCR amplification sizes were respectivelyβ-actin 106bp; IL-1β141 bp IL-6 94bp;IL-10 155bp; INF-γ141bp; PAl 195bp; TGF-β1 130bp; TGF-132 183bp; TIMP1129bp; MMP-1 81bp. The copy numbers of DNA template were calculated accordingto the molecular weight of the target gene and then converted into the copy numberbased upon the Avogadro's number (1 mol=6.022×1023 molecules). Every PCRproduct was serially tenfold diluted at a range of 10⁵～10¹⁰, respectively. Each samplewas treated in triplicates, and the fluorescence intensity ratio of the amount ofreporter dye emission to the quenching dye emission and threshold cycle (CT) valueswere averaged from each reaction. (7) TaqMan real-time quantitative PCR: Real-timePCR was performed in a 96-Well Reaction Plate, each well containing 1μl of each RTproduct or 1μl of each DNA template dilution, 5×PCR Buffer, 25 mM dNTPs, 25μMprimers forward and reverse, 10pmol/ul TaqMan Probe, 5U/ul Hotstart Taq DNApolymerase, and sterile distilled water in a total volume of 25ul, under theamplification conditions of 40 rounds of the procedures including denaturalization 3min at 93℃, annealing 45 seconds at 93℃and extending 60 seconds at 55℃. All reactions were performed in MJ Research(R) DNA Engine OPTICON 2 SequenceDetection System for the test samples, standards, and no template controls were runin triplicates using the equence Detector V 1.6 program. The fluorescence intensityDR and CT were averaged from the values obtained in each reaction. A "standardcurve" was constructed by plotting the CT vs. the known copy numbers of thetemplate in the standard. According to the standard curve, the copy numbers for allunknown samples were obtained automatically. (8)Normalization of target genemRNA levels to that ofβ-actin: The absolute copy number of target gene andβ-actinmRNA in each sample was calculated based on its CT value with its positive templateDNA standard curve. The absolute copy number of target gene e.g. IL-1βmRNA wasthen normalized toβ-actin to minimize variability in the results due to differences inthe RT efficiency and RNA integrity among test samples.Results: (1) CTOL inhibited IL-1βand IL-6 expression in NF and AF, but to agreater extent inAF. AF showed significantly higher levels of IL-1β(274ï¼…; P＜0.01)and IL-6 mRNA (91ï¼…; P＜0.001) than those of NF. There was no evident change inthe mRNA levels of IL-1βand IL-6 in NF in response to low-dosage CTOL, but therewas a marked decrease in AF, respectively 19ï¼…(P＜0.05), 32ï¼…(P＜0.01). Mediumdosage CTOL could reduce the mRNA levels of IL-1β(68ï¼…; P＜0.05), (20ï¼…; P＜0.05)and IL-6(68ï¼…;P＜0.001), (47ï¼…; P＜0.01) in NF and in AF. Medium dose CTOLmarkedly decreased IL-1βand IL-6 mRNA levesl in NF, with an even more potentimpact on AF. On the other hand, CTOL was dose dependent. (2) CTOL inhibitedTGF-β1 and TGF-β2 expression in NF and AF, but to a greater extent in AF. Thebasal mRNA levels of TGF-β1 (52ï¼…; P＜0.001) and TGF-β2(30ï¼…; P＜0.001) werehigher in AF, as compared to NF. There was no evident change in the mRNA levels ofTGF-β1 in NFI and AF in response to low dose CTOL. However, There was adecrease in the mRNA levels for TGF-β2 (P＜0.01 or P＜0.05) in NF and AF inresponse to low-dosage CTOL. Medium dosage CTOL could inhibit the mRNA levelsof TGF-β1 (44ï¼…, P＜0.001; 26ï¼…, P＜0.05) and TGF-β2 (50ï¼…, P＜0.001; 31ï¼…, P＜0.001)in NF and AF. CTOL markedly decreased TGF-β1 and TGF-β2 mRNA in NF, with an even further increase in the ration in AF. (3) CTOL increased the ration ofMMP-1/TIMP-1 expression in cultured AF but not influenced NF. The basal mRNAlevels of MMP-1 (78ï¼…; P＜0.001) were higher in adhesion, as compared to peritonealfibroblasts. There was a decrease in the mRNA levels for MMP-1(17ï¼…, P＜0.05; 13ï¼…,P＜0.01) in NF and AF in response to CTOL low. However, medium dose CTOL coulddecrease the mRNA levels for MMP-1(27ï¼…; P＜0.01)in NF, and increase the mRNAlevels for MMP-1 (17ï¼…; P＜0.001) in AF.The basal mRNA levels of TIMP-1 (18ï¼…;P＜0.05)were higher in AF, as compared to NF. There was no evident change in themRNA levels of TIMP-1 in NF and AF in response to low dosage CTOL. However,medium dosage CTOL could decrease the mRNA levels of TIMP-1 in NF and in AF,respectively 17ï¼…(P＜0.05), 41ï¼…(P＜0.001).There was an increase in the basalration of MMP-1/TIMP-lmRNA levels (53ï¼…) in AF, as compared to peritonealfibroblasts. However, medium dosage significantly increased this ration in adhesions,even if not affecting the ration in peritoneal fibroblasts. (4) CTOL increased the rationof tPA/PAI-1 expression in cultured AF and NF: AF showed significantly lower levelsof tPA mRNA (52ï¼…; P＜0.001) than those of NF. There was no evident change in themRNA levels of tPA in NF and AF in response to low dose CTOL. However, CTOLmedium treatment could increase the mRNA levels of tPA in NF and inAF,respectively, (26ï¼…, P＜0.001; 95ï¼…, P＜0.001). In contrast, AF displayed significantlyhigher levels of PAI-1 mRNA (131ï¼…; P＜0.001) than those from NF. There was noevident change in the mRNA levels for PAI-1 in NF in response to low dose CTOL.However, there was an evident change in AF (15ï¼…, P＜0.01). Medium dose CTOLcould decrease the mRNA levels of PAI-1 in NF and in AF, respectively (28ï¼…,(P＜0.01; 60ï¼…, P＜0.001).As a result, AF were at a significantly lower level (79ï¼…;P＜0.001) of tPA/PAI-1 mRNA ratio than those from NF. Medium dose CTOLmarkedly enhanced (43ï¼…) the tPA/PAI-1 mRNA ratio in NF, with an even furtherincrease in the ration (79ï¼…) in AF. (5) IL-10, INF-γmRNA levels: There was noevident difference in the mRNA levels of IL-10 in NF and AF. Low dose CTOL hadno marked effect on the mRNA levels of IL-10 in NF and AF. Medium dose CTOLcould increase the mRNA levels of IL-10 in AF (43ï¼…, P＜0.001), but no effecton those in NF. The mRNA levels of INF-γwere not detected inNF and AF.Conclusion: In this study it was found that CTOL induced molecular changes inboth NF and AF and the effect of CTOL was more profound on AF, which indicatesthe mechanism with which CTOL prevents intestinal adhesion in molecular levels.