| Background:According to the epidemiology statistics, it is now clear that adhesions occur after all types of intra-abdominal procedures, regardless of surgical approach. The clinical consequences of adhesions are significant; these include bowel obstruction, infertility, pelvic pain, increased operating time due to extensive adhesiolysis, and increased morbidity. Current therapies in adhesion prevention are limited, as the pathophysiology of adhesion development has not been well understood until recently. These therapies include the tenets of good surgical technique and a variety of adhesion barriers. No one drug or anti-adhesive prevention or measures can be completely used widely in clinical application. The current occurrence of how to maximize the prevention and inhibition of peritoneal adhesion is a hotspot. Based on Our preliminary basis for many years clinical experience and research results and guided by Traditional Chinese Medicine theory. To explore and explain the mechanism of its efficacy on peritoneal adhesion, a series of experiments were designed and made on PA animal model by modern medical and biological means. Huoxue Tongfu formula can decrease FIB in plasma and celiac liquid, increase FDP density, suppress TGF-β mRNA expression in the adhesion tissue, improve exudation and fibrosis, decreased the degree of peritoneal adhesion significantly. In addition, drug serum of Huoxue Tongfu formula is able to regulate protein level of TGF-β1, FN and CTGF in order to prevent postoperative abdominal adhesion. We found Huoxue Tongfu formula in different postoperative periods had different drug effect in preliminary experiments. It is unclear that cascade process state this difference results caused by inflammatory cytokines and CD40/CD40L trigger has no contact.Objective:To establish a quantitative method for analyzing the content of Hydroxysafflor yellow A, tetrahydropalmatine, Kaempferol, Aloe Emodin, Rhein, Emodin, Chrysophanol and Physcion in Huoxue Tongfu formula by HPLC-DAD. To observe the effect of Huoxue Tongfu formula on postoperative immune activation, inflammation and fibrinolysis imbalance caused by adhesion formation via whole animal experiment combined with cell experiment starting from the regulation of peritoneal mesothelial cells of CD40/CD40L signal pathway, verify the mechanism of Huoxue Tongfu formula anti-intraabdominal adhesion is to promote the recovery of inflammatory reaction and the fibrinolytic balance through regulate CD40/CD40L system function.Methods:(1) RP-HPLC was utilized Elite Sino Chrom BP C18 (4.6 mm×250 mm,5 μm) was carried on for the qualitative identification. The mobile phase was zyrian:1% glacial acetic acid. The flow rate was 1.0 ml·min-1, the column temperature was 30℃and the detection wavelength was set at 254,280,365 and 400 nm. (2) We observed and estimated the peritoneal adhesion and general situation of rats, Elisa method was used to measure the serum level of IL-8, IL-10 and TNF-a, and reverse transcription-polymerase chain reaction for the expression of Col-I mRNA in adhesional tissue aim to observe the therapeutic effect of optimized Huoxue Tongfu formula for peritoneal adhesion and ensure the optimum dosage. (3) 150 male rats were established postoperative peritoneal adhesion model by filing operation and divided randomly into 4 groups:model group, low dose Huoxue Tongfu formula group, high dose Huoxue Tongfu formula group and SiMo decoction group, another 30 SD rats were sham operation group. Each group according to the prescribed dose by gavage for 2 weeks respectively, the animals were euthanized on days 1,3,5,7 and 14 to evaluate the adhesion severity, area of adhesion formation, gross appearance, and pathological changes. Furthermore, immunohistochemical investigations were conducted to determine the discrete influence of Huoxue Tongfu formula on peripheral blood (CD40, CD40L) and peritoneal tissue responses (CD40, CD40L, MCP-1 and MMP-9). (4) Trypsin digestion method in vitro of rat peritoneal mesothelial cells model was established, identification of groups (blank group, blank serum group, Huoxue Tongfu formula low dose group, Huoxue Tongfu formula dose groups, Huoxue Tongfu formula high dose group) using MTT for detection of serum containing of Huoxue Tongfu formula on rat peritoneal mesothelial cells. (5) Using different concentrations of lipopolysaccharide (LPS) stimulation rat peritoneal mesothelial cells at different time points, the protein and mRNA level of IL-1β,IL-6 detected respectively by ELISA and qRT-PCR. Morphological observation under scanning electron microscope in normal rat peritoneal mesothelial cells and LPS stimulated peritoneal mesothelial for the sake of seek for best PA model in vitro and optimal stimulate LPS time and dose. (6) To survey the effects of Huoxue Tongfu formula drug serum on LPS stimulated rat peritoneal mesothelial cells in CD40, CD40L, MCP-1, MMP-9 mRNA and protein expression by qRT-PCR and Western Blot.Results:(1) Methods of 8 components are in line with the requirements of the study without interference in the negative reference. (2) Compared with the model group, the medium dosage group of optimized Huoxue Tongfu formula, serum IL-8 and TNF-a levels were decreased, but the level of IL-10 were increased, while down-regulate expression of Col-1 mRNA adhesion tissues, there was significant statistical significance (P<0.05 or P<0.01). (3) Adhesion scores and pathological changes from Huoxue Tongfu formula groups were down-regulated when they were compared with that of the model groups, especially on days 3,7 after operation (P＜0.01). Compared with the model groups, the level of CD40 and CD40L peripheral blood was decreased varying degree in all of the treated groups (P＜0.05). While the level of protein expression in tissue adhesion of CD40, CD40L, MCP-1 and MMP-9 in the four treated groups is lower than in the model group (P<0.05). (4) Compared with the blank serum group, Huoxue Tongfu formula drug serum can improve RPMCs proliferation stimulating effect, and reaction time at 24 h was the most significant. (5) IL-1β and IL-6 concentration in RPMCs supernatant were measured by ELISA indicated that IL-1β and IL-6 concentration increased with the stimulation time and LPS concentration, also showed significant differences (P<0.01). The best stimulation time was 24 hours and the best stimulus concentration was 10μg/ml. Normal RPMCs manifestations under scanning electron microscopy were covered by abundant microvilli. However, LPS stimulated RPMCs appear stripping, swelling significantly, with the gap and the basement membrane. (6) LPS stimulated RPMCs on the expression of CD40, CD40L, MCP-1 and MMP-9 protein and mRNA can decreased by Huoxue Tongfu formula drug serum with a dose dependent.Conclusion:(1) The method is accpetable and accurate with good precision. The standard which established can be applied for the quality control of Huoxue Tongfu Formula. Only in this way can provide scientific basis and standard gradually on pharmacodynamics in subsequent studies. (2) The medium dosage group of optimized Huoxue Tongfu formula can suppress inflammation and relieve peritoneal adhesion by regulating the serum level of IL-8, IL-10 and TNF-α reducing the deposition of the Col-1 mRNA of extracellular matrix. (3) MCP-1, CD40, CD40L and MMP-9 may participate in the pathogenesis of postoperative peritoneal adhesion. The pharmacological mechanism of Huoxuetongfu formula improve abdominal adhesion may be effective in inhibiting CD40/CD40L activation, decreased the expression of MCP-1 and MMP-9. (4) Huoxue Tongfu formula drug serum has induced the role of Rat peritoneal mesothelial cells proliferation. (5) Rat peritoneal mesothelial cells can induce inflammation injury stimulated by lipopolysaccharide, further amplifying inflammatory response. (6) Huoxue Tongfu formula drug serum reduce the expression of CD40, CD40L and the level of MCP-1 in order to relieve inflammatory reaction, lower the expression of MMP-9 so as to reduce the collagen deposition, may as an effective measures for anti-intraabdominal adhesion. |
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