| Background/ObjectiveIntraperitoneal adhesion remains one of the most challenging problems in abdominal surgery.The adhesion density,time of occurrence,and clinical manifestations are different,with no predictable pattern.In addition,adhesion diseases can become chronic medical diseases with a significant incidence but no effective treatment.Despite recent important advances in surgical techniques and anti-adhesion medications,there is no completely reliable treatment strategy.Inflammation and imbalance of fibrinolytic system are the main pathogenesis of postoperative abdominal adhesion formation.The change of macrophage function is closely related to the physiological and pathological process of abdominal adhesion and is involved in the regulation of inflammatory response and collagen deposition.JAK/STAT pathway and its upstream and downstream signals are the main way for the polarization activation of macrophages,leading to the polarization of macrophages into different phenotypes.M1 macrophage(M 1)secretes pro-inflammatory cytokines and M2 macrophage(M2)secretes anti-inflammatory cytokines.Conversion of M1/M2 can affect the severity of adhesions,so it can be regarded as an effective target for treatment.HuoXueTongFu Formula(HXTF)plays an important role in preventing and curing Intraperitoneal Adhesion.It is composed of Chinese rhubarb,Semen Persicae,Rhizoma Corydalis,Semen Raphani,Natrii Sulfas and Flos Carthami.Although it has been used clinically for more than 20 years,the molecular mechanism of its action on cells is still not fully understood.In this study,to provide scientific basis for anti-adhesive effect by HXTF,we established a RAW264.7 macrophage inflammation model and an intraperitoneal adhesion rat model to determine whether HXTF can improve adhesion by regulating the polarization state of macrophages.In addition,we analyzed the regulation of SOCS/JAK2/STAT/PPAR-? pathway by HXTF from the perspective of cell and animal,and analyzed the relationship between PPAR-? activation and the polarization state of macrophages.MethodsIn vitro? RAW264.7 macrophages were stimulated by different concentrations of HXTF-medicated serum(0,2.5%,5%,10%,20%,30%)for 24 hours,and the cell activity was measured by CCK-8;? RAW264.7 macrophages were given lipopolysaccharide(LPS)100g/mL,interferon(IFN)-y 20ng/mL,interleukin(IL)-4 20ng/mL,with or without 10%HXTF-medicated serum.After 12 hours of intervention,flowcytometry was used to detect the tendency of M1/M2 polarization,ELISA was used to detect the levels of M1/M2 related cytokines,and Westernblot was used to detect the expression of SOCS/JAK2/STAT/PPAR-y pathway related proteins expression;? RAW264.7 macrophages were given PPAR-? agonist rosiglitazone(RSG)1?mol/mL,antagonist T0070907 1?mol/mL,with or without 10%HXTF-medicated serum.After 12 hours of intervention,flowcytometry was used to detect the tendency of M1/M2 polarization,ELISA was used to detect the levels of M1/M2 related cytokines,and Westernblot was used to detect the expression of SOCS/JAK2/STAT/PPAR-y pathway related proteins expression.In vivoForty Sprague-Dawley rats were randomly divided into Sham group,IA group,HXTF group and fluvastatin(FS)group.The cecum of rats were rubbed to establish a postoperative abdominal adhesion model except for the Sham group.From the first day to the seventh day after the operation,the Sham group and the model group were administered with saline,the HXTF group was given HXTF(5.83g/kg),and the FS group was given fluvastatin(10mg/kg),samples were collected on the 8th day to observe intraperitoneal adhesions and scores.HE staining and Masson staining were used to observe local inflammation and collagen deposition in the adhesions.Westernblot and real-time PCR were used to detect SOCS/JAK2/STAT/PPAR-y pathway related factors Express the situation.ResultsIn vitro? HXTF has no effect on the cell activity of RAW264.7 macrophages.We chose 10%HXTF as the optimal experimental concentration;? The comparison results showed that the effects of HXTF and IL-4 on macrophages are similar,inducing macrophages to polarize to M2 and expressing high levels of M2-related cytokines IL-4,IL-10,IL-13 and TGF-?1(p<0.05);? HXTF reversed the M1 polarization trend induced by LPS+IFN-y,and reduced the expression of Ml-related cytokines IL-1,IL-6 and TNF-?(p<0.05);? HXTF reduced the expression of SOCS3,JAK2,STAT1,p-JAK2,and p-STAT1(p<0.05),and increased the expression of SOCS1,STAT6,PPAR-y,p-STAT6,and p-PPAR-?(p<0.05);? RSG promoted macrophage polarization to M2 and PPAR-y nuclear translocation,enhanced expression of M2-related cytokines IL-4,IL-10,IL-13 and TGF-?1(p<0.05),and activated SOCS1,STAT6,PPAR-?,p-STAT6,p-PPAR-? signals(p<0.05);? T0070907 promoted macrophage polarization to M1,inhibits PPAR-y nuclear translocation,increased expression of Ml-related cytokines IL-1,IL-6 and TNF-?(p<0.05),and activated SOCS3,JAK2,STAT1,P-JAK2,p-STAT1 signals(p<0.05);? Compared with the T0070907 group,the M1 polarization trend in the HXTF+T0070907 group was significantly reduced,the expression of M1-related cytokines decreased,the expression of M2-related cytokines increased,and PPAR-y nuclear translocation was promotedIn vivoHXTF reduced intraperitoneal adhesion,reduced the adhesion score,improved local inflammation and collagen deposition.In the adhesion tissue,it inhibited the expression of SOCS3,JAK2,STAT1,p-JAK2,p-STAT1(p<0.05),increased the expression of SOCS1,STAT6,PPAR-y,p-STAT6,p-PPAR-y(p<0.05).Conclusion? HXTF regulates the polarization of macrophages to M2;? The regulation of macrophage polarization by HXTF may activate the SOCS1/STAT6/PPAR-? pathway and inhibit the SOCS3/JAK2/STAT1 pathway;? Different activation states of PPAR-? can regulate the macrophage SOCS/JAK2/STAT/PPAR-? pathway,and affect the M1/M2 polarization of macrophage;? HXTF can promote PPAR-? nuclear translocation;?HXTF can improve the inflammatory and reduce intraperitoneal adhesions.This protective effect may be achieved by inhibiting SOCS3/JAK2/STAT1 pathway and activating SOCS1/STAT6/PPAR-? pathway. |
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