The Study Of Hypoxia Inducible Factor-1α And Matrix Metalloproteinase-9in Severe Acute Pancreatitis Associated Lung Injury Rat Models And The Influence Of Emodin

Objectives:The purpose of our study is to investigate the expression of hypoxia inducible factor-1α andmatrix metalloproteinase-9in severe acute pancreatitis associated lung injury rat models.We alsoobserve the alternations of MMP-9, alveolar-capillary barrier function and lung edema afteradministration of the inhibitors of2-methoxyestradiol (2ME2).To explore the changes of hypoxiainducible factor-1α, matrix metalloproteinase-9, tumor necrosis factor-α and interleukin-6inLPS-stimulated alveolar epithelial cells treated with emodin.And observe the changes of all abovefacors after knockdown the gene of HIF-1α in LPS-stimulated alveolar epithelial cells.Afteradministration of emodin, which is one of major compounds of traditional medicinepieplant,observe the activity of alveolar epithelial cells and explore the therapeutic effect of Emodininterfering the injury process in vitro.Methods:Part1: Twenty male SD rats were randomly divided into2groups: sham-operated group(SO,n=10) and SAP model group (SAP,n=10).Severe acute pancreatitis was induced in10rats. Upto5%sodium taurocholate (1ml/kg) was retrogradely infused into the distal end of thebile–pancreatic duct.The10rats in the sham-operated group only underwent laparotomy.Serumamylase (AMY),PaO2and PaCO2were recorded24hours after induction of pancreatitis.TheHIF-1α and MMP-9mRNA expression level in lung tissues were detected by reverse transcriptionpolymerase chain reaction(RT-PCR).The HIF-1α and MMP-9protein level in lung tissues were detected by Western-blot method.Active HIF-1α,MMP-9and TNF-α were detected by ELISA.Thepathological scores of pancrea and lung were calculated.Part2:Forty male Sprague-Dawley rats were randomly divided into the sham-operated group(control group, n=10), in which rats were given only a sham operation, and three PALI groups (10animals in each group), in which acute pancreatitis was induced by retrograde infusion of5%sodium taurocholate (1ml/kg). The PALI groups included the following:1) untreated PALI group;2)animals treated with inhibitors of HIF-1α2-methoxyestradiol (2ME2,5mg/kg body mass)(n=10);3)animals treated with inhibitors of HIF-1α2ME2(15mg/kg body mass)(n=10). All the rats weresacrificed24h after induction. Blood was immediately taken from the abdominal aorta for bloodgas analysis.Serum amylase (AMY),PaO2,PaCO2,Evan’s blue and wet/dry ratio of lung tissues wererecorded24hours after induction of pancreatitis.The HIF-1α and MMP-9mRNA expression levelin lung tissues were detected by reverse transcription polymerase chain reaction(RT-PCR).TheHIF-1α and MMP-9protein level in lung tissues were detected by Western-blot method.The activeHIF-1α,MMP-9and TNF-α were measured by ELISA.The pathological scores of pancreas and lungwere calculated.Part3:Five dishes human alveolar epithial cells were cultured with DMEM and10%fetalbovine serum.Emodin with low dose (10μmol/L),moderate dose (20μmol/L), high dose (40μmol/L) was added to the cultured cells respectively for24hours. Lipopolysaccharide (200ng/ml)was added to LPS-stimulated group to stimulate lung injury in vitro.Activity of the cells weredetected using MTT.The HIF-1α,TNF-α and IL-6mRNA expression level were detected by reversetranscription polymerase chain reaction(RT-PCR).The HIF-1α protein level was detected byWestern-blot method. Active TNF-α and IL-6were detected with ELISA.Knockdown the gene ofHIF-1α in the epithelial cells, the protein level of HIF-1α was detected by Western-blot method,andthe protein level of TNF-α and IL-6were detected by ELISA in sh-HIF-1α and ns-sh-HIF-1αgroups.Results:PaCO2, W/D of lung tissues, Evan’s blue and serum AMY level in SAP group werefound significantly higher than sham-operated group (P<0.05),the level of PaO2in SAP group waslower than sham-operated group (P<0.05). The pathological scores of lung tissues and pancreatictissues were signicantly higher than the sham-operated group (P<0.05). After administration of the inhibitors of HIF-1α2ME2, the level of PaCO2, W/D of lungtissues, Evan’s blue and serum AMY in2ME2groups were found significantly lower than that inSAP group, the level of PaO2in2ME2group was higher than that in SAP group(P<0.05).Meanwhile, PaCO2, W/D of lung tissues, Evan’s blue and serum AMY level in2ME25mg/kg group were higher than that of2ME215mg/kg group, the level of PaO2in2ME25mg/kggroup was lower than that of2ME215mg/kg group (P<0.05).Active HIF-1α,MMP-9and TNF-αin2ME2groups were found significantly lower than that of SAP group using ELISA method.ActiveHIF-1α,MMP-9and TNF-α in2ME215mg/kg were lower than that of2ME25mg/kg(P<0.05).Reverse transcriptase polymerase chain reaction (RT-PCR) showed that mRNAexpressions of HIF-1α and MMP-9were upregulated significantly in the SAP group compared withthe control group (P<0.05). However, HIF-1α and MMP-9mRNA levels in lung tissues did notexhibit significant differences between the SAP group and the2ME2groups.HIF-1α was almostundetectable in the lung tissues of rats in the sham-operated group. HIF-1α and MMP-9proteinexpression showed significant increase in the SAP group compared with those in the control group(P<0.05). Compared with the SAP group, HIF-1α protein expression was significantly reduced inthe PALI+2ME2(5mg/kg) group and in the PALI+2ME2(15mg/kg) group. Moreover, MMP-9protein was weakly distinguishable in the PALI and PALI+2ME2(5mg/kg) groups(P>0.05).However, in the PALI+2ME2(15mg/kg) group, MMP-9protein expression was significantlyreduced compared with that in the PALI group.Rats in the PALI group showed significant increasein pathological scores of lung and pancreatic tissues compared with the sham-operated group(P<0.05). Compared with the PALI+2ME2(5mg/kg) group, pathological scores of lung andpancreatic tissues in the PALI+2ME2(15mg/kg) group significantly decreased (P<0.05).Activity in LPS-stimulated cells was found significantly lower than that of controlgroup.Activity,which was dependent on dose of emodin,was higher in the emodin-treated group(P<0.05). Reverse transcriptase polymerase chain reaction (RT-PCR) showed that mRNAexpressions of HIF-1α,TNF-α and IL-6were upregulated significantly in the LPS-stimulated groupcompared with the control group (P<0.05),and mRNA expression of above factors weredown-regulated in the emodin-treated group,especially in emodin40μmol/L group (P<0.05).HIF-1α protein expression showed significant increase in the LPS-stimulated group compared with that in the control group (P<0.05).The protein level of TNF-α and IL-6showed significant increasein the LPS-stimulated group compared with those in the control group (P<0.05).Protein level ofabove factors were down-regulated in emodin-treated group (P<0.05).Activity of IL-6, TNF-α inLPS-sh-HIF-1α were down-regulated compared with those in LPS-control-shRNA (P<0.05).Conclusion: HIF-1α and MMP-9play important roles in alveolar-capillary barrier disruptionand lung oedema in severe acute pancreatitis-associated lung injury rat models. HIF-1α has a majorfunction in alveolar-capillary barrier disruption and lung oedema in PALI via a molecular pathwaycascade involving MMP-9. Emodin can down-regulate the expression of HIF-1α, MMP-9andinflammatory factors in LPS-treated group. Inhibition of HIF-1α by2ME2and emodin attenuateacute lung injury. Pharmacological blockade of this pathway in patients with PALI may provide anovel therapeutic strategy.Emodin may inhibit inflammatory factors via downreguating hypoxiainducible factor-1α.