Study On The Effect Of Recombinant Human Endostatin And Its Drug-Loaded Hydrogel On Preventing Postoperative Peritoneal Adhesion

Background: Postoperative peritoneal adhesion(PPA)is one of the most common complications after abdominal surgery,occurring in approximately 90% of abdominal surgery patients.PPA can lead to a series of troublesome complications,such as intestinal obstruction,chronic pain,and infertility.Currently,the anti-adhesion strategies mainly include physical barriers and drugs,but each has its own limitations.Therefore,finding more effective anti-adhesion drugs and developing safer and more convenient anti-adhesion strategies remains a major clinical issue.Endostatin is a small-molecule peptide with dual effects of antiangiogenesis and anti-fibrosis.Especially,recombinant human endostatin(Endostar/rhEndostatin,ES)has the same efficacy as natural endostatin with slight improvement of its structure to enhance stability,.This study evaluated the efficacy of ES in preventing PPA and explored its specific mechanism.Based on this,an ES-loaded hydrogel was prepared and its biocompatibility and anti-adhesion efficacy were evaluated.Methods:1.To evaluate the anti-adhesion efficacy of ES,we constructed rat cecum-sidewall adhesion model,set up different treatment groups(including sham surgery,control,sodium hyaluronate(HA),and different doses of an ES groups),recorded the gross adhesion scores of different groups on postoperative day(POD)7,and collected adhesion tissues to conduct hematoxylin-eosin(HE),Masson,and immunohistochemical(IHC)analysis.To investigate the molecular mechanism of ES in preventing PPA,a PPA cell model was established by inducing the HMr SV5 cell(human peritoneal mesothelial cell line)to undergo mesothelial-mesenchymal transition(MMT)with TGF-β1.2.To address the problems of short in vivo retention time and low utilization rate of ES,we used Pluronic F127 and Carboxymethyl chitosan(CMCS)as raw materials to prepare PXC(Poloxamer-CMCS)hydrogel,and further prepared PXCE(Poloxamer-CMCS-ES)drug-loaded hydrogel.Fourier transform infrared spectroscopy(FT-IR),X-ray diffraction(XRD),scanning electron microscopy(SEM),ELISA were used to study the microstructure,mechanical properties,swelling properties,self-healing properties,in vitro drug release,and other aspects of the PXCE hydrogel.3.To evaluate the anti-adhesion efficacy of PXCE hydrogel,we constructed primary and repeated-injury adhesions rat models,set up different treatment groups(including control,HA,PXC,and PXCE groups),recorded the gross adhesion scores on POD 7 and 14 for the primary adhesion model and POD 7 for the repeated-injury adhesion model,and collected adhesion tissues to conduct HE,Masson,and IHC analysis.The biocompatibility of the PXCE hydrogel was studied using CCK-8experiments,HE staining of organ sections,and hemolysis experiments.Rat serum inflammatory factors were detected using ELISA.The retention of the FITC-labeled PXCE hydrogel in the abdominal cavity was studied.The effect of the PXCE hydrogel on wound healing was studied by observing the wound healing in different groups of rats and collecting incision tissues for HE and Masson staining analysis.Results:1.ES can significantly reduce the gross adhesion scores in the rat cecum-sidewall injury model,and the high-dose ES is more effective than HA(P<0.05).ES can significantly inhibit collagen deposition,reduce adhesion band thickness,reduce the aggregation of α-SMA(+)myofibroblasts and neovascularization in adhesion tissues.By detecting the expression levels of MMT markers,it was confirmed that ES can inhibit TGF-β1-induced MMT of peritoneal mesothelial cells(PMCs);further detection of the RhoA/ROCK pathway marker p-MYPT1 and the application of the RhoA/ROCK pathway activator lysophosphatidic acid(LPA)for rescue experiments showed that ES may inhibit MMT of PMCs by inhibiting the RhoA/ROCK signaling pathway.2.Regarding material characterization,FT-IR and XRD spectra showed that ES was connected to the hydroxyl groups exposed on CMCS and F127 through hydrogen bonding.SEM showed that both PXC and PXCE hydrogels had uniform,porous,and interconnected internal structures.In addition,PXCE hydrogel exhibited excellent physical and chemical properties,including mechanical properties,swelling capacity,self-healing ability,injectability,and in vitro drug release rate.3.The PXCE hydrogel can significantly reduce the gross adhesion scores of both primary adhesion and repeated-injury adhesion model,and its efficacy is superior to that of HA(P<0.05).The PXCE hydrogel can significantly inhibit collagen deposition,reduce the thickness of the adhesion band,reduce the aggregation of α-SMA(+)myofibroblasts and neovascularization in adhesion tissues.The PXCE hydrogel has good biocompatibility,with no obvious cytotoxicity and organ toxicity,and a low hemolysis rate.The PXCE hydrogel can effectively reduce the expression levels of IL-6,TNF-α,and TGF-β1 in rat serum.The PXCE hydrogel can remain in the abdominal cavity for 7-10 days,covering the critical period of postoperative adhesion formation,and has no significant effect on rat incision healing.Conclusions:1.ES has a good therapeutic effect in preventing PPA,and its specific mechanism may be through regulating the RhoA/ROCK signaling pathway,inhibiting MMT of PMCs,and thereby suppressing the formation of PPA.2.PXCE hydrogel has a uniform porous structure and has good mechanical properties,swelling behavior,self-healing ability,injectability,and continuous release of ES.3.PXCE hydrogel has good biocompatibility and safety,and can effectively inhibit the formation of PPA induced by primary and repeated injury adhesion model.