Study On The Role And Mechanism Of PAPSS2 In Postoperative Abdominal Adhesion

Background:Postoperative abdominal adhesion(PAA)is one of the common complications after abdominal surgery,with an incidence of 90%-95%.The abnormal bands between these organs or between organs and abdominal wall can cause intestinal obstruction,female secondary infertility,chronic abdominal pain,etc.which increases the postoperative pain and economic burden of patients undergoing abdominal surgery.Collagen deposition and fiber formation are important molecular mechanisms of postoperative abdominal adhesion formation,however,the specific mechanism is still unclear.3′-adenosine phosphate 5′-phosphate sulfate synthase 2(PAPSS2)is one of the sulfate-modified enzymes in vivo,which is widely involved in the formation of proteoglycans,as a key extracellular matrix component.PAPSS2 has been confirmed to be widely involved in chondrogenesis,breast cancer metastasis,etc.and as a downstream molecule of Snail and TGF-β1 Signal path correlation.However,the role and mechanism of PAPSS2 in the development of abdominal adhesion have not been reported.The purpose of this study is to prove the role and mechanism of PAPSS2 in the process of abdominal adhesion through bio-information analysis and in vivo and in vitro experiments,so as to further enrich the potential mechanism of abdominal adhesion,and seek key regulatory targets for the follow-up treatment and prevention of abdominal adhesion.Aims:1.To clarify the expression of PAPSS2 in abdominal adhesions.2.To explore the role and mechanism of PAPSS2 in the formation of abdominal adhesion.Methods:1.The m RNA expression microarray data set was retrieved from Gene Expression Omnibus(GEO),and a set of altered genes and related pathways involved in the development of peritoneal adhesion were identified by bioinformatics methods.2.Fifty BALB-c mice were randomly divided into 5 groups,except for the sham operation group(Sham group only underwent abdominal surgery),the remaining forty mice received cecal scraping to establish an abdominal adhesion model.The rats were sacrificed at 1,3,5 and 7 days after operation.Another 30 mice were randomly divided into 3 groups.Each mouse was subjected to cecal scraping to establish an abdominal adhesion model,and treated with 100μL normal saline or 1×10~8p.f.u empty vector virus or PAPSS2 knockout virus,respectively.All the three groups of mice were sacrificed 7days after operation.Finally,the mouse adhesion tissue was taken out.Nair’s method was used to score by two double-blind researchers.The expression of PAPSS2 in intestinal adhesion tissues was determined by immunofluorescence(IHF),immunohistochemistry(IHC)and Western blot.HE staining,Masson staining,Sirius red picric acid staining and ELISA and other methods were used to determine the area of adhesion tissue,inflammatory infiltration and fiber increase.The expression levels of inflammation-related factors IL-6,TGF-β1 and EMT-related proteins were detected by ELISA.Finally,the data obtained from the test were statistically analyzed.3.A HMr SV5 cell line with PAPSS2 knockout was established.To investigate the biological changes of HMr SV5 cell proliferation after PAPSS2 interference when PAPSS2 was silenced by lentiviral vector.Cell scratches were used to detect cell migration and repair ability.The expression ofα-SMA,Vimentin and E-cadherin was detected by Western blotting.The integrity of mesothelial cells was detected by CK19staining.Then the data obtained from the test were statistically analyzed.Results:1.By screening the GEO database,we found that PAPSS2 was highly expressed in abdominal adhesion tissues.2.Through HE staining,Sirius red and Masson staining,the inflammation and fiber formation in abdominal adhesion tissues at different times were detected,and it was confirmed that in normal peritoneal tissues,only a small amount of inflammatory cells or fibrous tissue hyperplasia,and with the progress of time,the formation of adhesion tissue contains a large amount of inflammatory cell infiltration and fibroplasia.3.The mouse abdominal adhesion model was established,and the high expression of PAPSS2 in abdominal adhesion tissues was confirmed by IHC staining and Western blotting.Peritoneal mesothelial cells are the key cells for the formation of postoperative abdominal adhesions.The results of CK19 and PAPSS2 double immunofluorescence staining showed that PAPSS2 was highly expressed in mesothelial cells on day 3 and day7.4.The Nair’s score was lower than that of the control group after knockout of PAPSS2 in the adhesion tissue.The expression of PAPSS2 was successfully knocked out in mice.At the same time,through double immunofluorescence staining,it was found that knockout of PAPSS2 could significantly reduce postoperative intraperitoneal adhesion in mice.5.Through HE staining,it was found that the inflammation score of the PAPSS2knockout group was lower than the control group,and the levels of IL-6 and TGF-β1 in the adhesion tissue of the PAPSS2 knockout group were lower.6.Through Sirius red staining,it was found that knockout of PAPSS2 could reduce collagen deposition in adhesive tissues,and the expression ofα-SMA in adhesive tissues was detected.7.When PAPSS2 was silenced by lentiviral vector to down-regulate its expression,it was found that the peritoneal integrity of the PAPSS2 knockout group was higher than that of the control group.Wound healing assay showed that the cell migration rate of HMr SV5 cell line with low expression of PAPSS2 was decreased.At the same time,knockout of PAPSS2 attenuated TGF-β1-induced MMT in peritoneal mesothelial cells.Conclusion:This study found that PAPSS2 is highly expressed in mouse abdominal adhesion tissues,and down-regulation of PAPSS2 can reduce the formation of adhesions.The mechanism may be related to reducing inflammatory response,reducing collagen deposition,promoting peritoneal mesothelial cell repair and MMT.