The Effects Of IL-38 On LPS-induced Endothelial Injury And Its Mechanisms

SectionⅠ IntroductionSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.Sepsis is an acute and severe disease with high treatment costs and high mortality,imposing an enormous burden on the global healthcare economy.However,because the pathogenesis of sepsis affects the whole body organs and involves a wide range of mechanisms,the current research and treatments of this disease are still very limited.Endothelial cells are an important part of maintaining the structural and functional integrity of the blood vessel wall,and also the first barrier between blood and tissues.When pathogens invade the body,they can directly attack and activate endothelial cells.Therefore,endothelial cell activation plays an important role in the occurrence and development of sepsis.When endothelial cells are stimulated by pathogens,inflammation-related pathways are activated and the transcription and expression of cytokines are up-regulated,promoting the release of large amounts of pro-inflammatory cytokines to cause an inflammatory storm.These processes lead to a systemic inflammatory response and multiple organ dysfunction.Therefore,protecting endothelial cells and inhibiting endothelial inflammatory response is an important strategy for the treatment of sepsis.Interleukin-38(IL-38)was discovered back in 2001 and is classified as part of the IL-1 family.The gene IL-38 is located on chromosome 2 and encodes a protein of152 amino acids in length.IL-38 is considered to be an anti-inflammatory factor due to its amino acid sequence similarity with IL-1Ra and IL-36 Ra.Although it has been discovered more than 20 years,few studies explored IL-38.Several studies have shown that IL-38 can inhibit the expression of inflammatory factors and play an anti-inflammatory role in various inflammatory diseases,especially autoimmune diseases.In the sepsis model,IL-38 can inhibit apoptosis of macrophages and NLRP3 inflammasome,and inhibit pro-inflammatory factors of CD4+CD25+ regulatory T cells,thereby attenuating inflammation.However,whether IL-38 has anti-inflammatory effects on endothelial cells during sepsis is unclear.Therefore,in this study,an in vivo model of sepsis was constructed by intraperitoneal injection of lipopolysaccharide(LPS)into mice,and an in vitro model of inflammation was constructed by adding LPS into the medium of human umbilical vein endothelial cells(HUVECs).We used recombinant IL-38,overexpression and silence of IL-38 to explore its effects on HUVECs inflammation and its mechanisms.SectionⅡ LPS induces endothelial cell inflammation and IL-38 expression Objective:To construct sepsis models in vitro and in vivo,and to detect IL-38 expression in sepsis.Methods:1.Twelve 8-week-old C57/BL6 mice were randomly divided into control(n = 6)and septis groups(n = 6).The mice in the sepsis group were injected intraperitoneally with LPS to construct a sepsis model,and the control group was given the corresponding volume of PBS.After 24 h,the mice were executed and the serum was kept for ELISA to determine IL-1β and IL-6.A portion of the aorta was retained for HE staining to observe the pathological morphological changes and a portion for immunohist-ochemistry to evaluate the changes of intercellular adhesion molecule-1(ICAM-1)and vascular adhesion molecule-1(VCAM-1).In addition,IL-38 levels were detected by immunohistochemistry.2.HUVECs were uniformly inoculated in 6-well plates and randomly divided into control and LPS groups.When the cell density reached about 70%,the LPS group was cultured with medium containing 1ug/ml LPS,and the control group continued to be cultured with complete medium.After 24 h,cell activity was measured by cell counting kit-8(CCK-8)and cell viability changes were compared between the two groups.Changes in inflammatory factors(IL-1β and IL-6)and adhesion molecules(ICAM-1 and VCAM-1)were detected by PCR.IL-38 protein levels were detected by Western blotting.Results:1.Compared with the control group,mice in the sepsis group had reduced mobility and a significantly reduced range of motion.ELISA results revealed that serum IL-1β and IL-6 concentrations were significantly higher in the septic mice compared with the control group(both p < 0.05).The aortic HE staining results showed that the aortic endothelium in the control group was smooth and intact,with regular cell arrangement,uniform nuclei size,intact mid-membrane elastic membrane and elastic fibers,and neat arrangement.In contrast,the aortic endothelium of the sepsis group was rough and swollen,with irregular cell arrangement and uneven nuclei size.The elastic membrane and elastic fibers of the middle membrane were intact but disordered.Immunohistochemical results of the aorta showed that the expression levels of ICAM-1 and VCAM-1 were significantly higher in the aorta of mice with sepsis compared with the control group(both p < 0.05).In addition,immunohistochemistry suggested that IL-38 was significantly higher in the aorta of septic mice compared with the control group(p < 0.05).2.CCK-8 results showed that endothelial cell activity was significantly lower in the LPS group compared with the control group(p < 0.05).PCR results suggested that IL-1β,IL-6,ICAM-1 and VCAM-1 were significantly higher in the endothelial cells of the LPS group compared with the control group(p < 0.05).Western blotting results revealed that IL-38 was elevated in endothelial cells in the LPS group compared with the control group(p < 0.05).Conclusions:1.We successfully constructed in vivo and in vitro sepsis models,in which LPS induces endothelial inflammation and vascular injury.2.IL-38 expression was elevated in endothelial during sepsis.Section Ⅲ IL-38 inhibits LPS-induced vascular endothelial injuryobjective: To explore the effects of IL-38 on LPS-induced endothelial inflammation.Methods: 1.HUVECs in good condition were randomly divided into Control group,LPS group and IL-38 group with different concentrations(1ng/ml,10ng/ml and 20ng/ml).The IL-38 group was pretreated with the indicated concentration of IL-38 for 12 h,and then stimulated with 1ug/ml of LPS for 24 h.The optimal concentration of IL-38 was determined by CCK-8 assay,and the m RNA levels of IL-1β and IL-6 were detected by PCR.Western blotting was performed to detect protein levels of inflammation-related molecules,including IL-1β,IL-6,cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(i NOS).Dihydroethidium(DHE)staining was performed to detect changes in cellular reactive oxygen species(ROS).2.HUVECs with good condition were randomly divided into control group(CON group),empty viral vector group(VEC group)and IL-38 overexpression group(OE group).HUVECs in the OE and VEC groups were transfected with the constructed IL-38 overexpression and null-loaded adenovirus for 36 h,respectively.Transfection efficiency was detected by Western blotting.HUVECs in good condition were randomly divided into control group(CON group),sepsis group(LPS group),empty viral vector group(LPS+VEC group)and IL-38 overexpression group(LPS+OE group).IL-38 overexpression and null-loaded adenovirus were constructed and HUVECs in LPS+OE and LPS+VEC groups were transfected for 36 h,respectively.Except for the CON group,HUVECs in the other three groups were stimulated with 1ug/ml of LPS for 24 h.PCR was performed to detect m RNA levels of IL-1β and IL-6.Western blotting was performed to detect changes in protein levels of IL-1β,IL-6,COX-2 and i NOS.DHE staining was used to detect changes in cellular ROS.3.HUVECs in good condition were randomly divided into control group(CON group),negative control group(NC group)and IL-38 silencing group(Si group).HUVECs in Si and NC groups were transfected with the constructed interfering IL-38 and empty interfering fragments for 48 h,respectively.The silencing efficiency was detected by Western blotting.HUVECs in good condition were randomly divided into control group(CON group),sepsis group(LPS group),negative control group(LPS+NC group)and IL-38 silence group(LPS+Si group).IL-38 interfering fragment and negative counterpart fragment were constructed and HUVECs in LPS+Si group and LPS+NC group were transfected for 48 h,respectively.Except for the CON group,HUVECs in the other three groups were stimulated with 1ug/ml of LPS for 24 h.Western blotting was performed to detect protein levels of IL-1β,IL-6,COX-2 and i NOS.4.Eighteen 8-week-old C57/BL6 mice were randomly divided into control group(CON group,n = 6),virus-vec group(VEC group,n = 6)and overexpression IL-38 group(OE group,n = 6).Mice in OE group and VEC group were injected with overexpressing and null viruses,respectively.After 28 days,the mice were executed and the aorta was retained for IL-38 transfection efficiency detection by Western blotting.Forty 8-week-old C57/BL6 mice were randomly divided into control group(n=10),sepsis group(n=10),empty viral vector group(n=10)and overexpressed IL-38 group(n=10).Mice in the IL-38 overexpression group and the empty viral vector group were injected with IL-38-overexpression virus and empty virus in the tail vein,respectively.After 28 days,the control group was injected intraperitoneally with saline and the rest three groups were injected intraperitoneally with LPS to simulate sepsis.Mice were executed 24 h after LPS injection,and serum was collected to detect serum IL-1β and IL-6 levels by ELISA.The aorta was retained.A portion of the remaining aorta was stained with HE to observe the morphological changes of aortic pathology,and a portion stained with immunohistochemistry to detect the changes of adhesion molecules(ICAM-1 and VCAM-1).In addition,protein levels of IL-1β,IL-6,COX-2 and i NOS were detected by Western blotting.Results: 1.CCK-8 results showed that the viability of endothelial cells was significantly decreased after LPS stimulation,while pretreatment with different concentrations of recombinant IL-38 significantly increased cell viability(p < 0.05).But no significant difference between different concentration groups was observed,that is there was no concentration gradient effect.PCR results revealed that pretreatment with different concentrations of recombinant IL-38 significantly decreased IL-1βand IL-6 levels(p < 0.05),and the effect of 10 ng/ml IL-38 had the most pronounced effect.Therefore,we used this concentration of IL-38 to treat the cells in all subsequent experiments.Western blotting results showed that the LPS group significantly promoted IL-1β,IL-6,COX-2 and i NOS protein levels compared to the control group.Recombinant IL-38 significantly inhibited IL-1β,IL-6,COX-2 and i NOS protein levels compared to the LPS group(p < 0.05).DHE staining yielded a similar trend.Compared to the control group,the LPS group produced more ROS,while recombinant IL-38 inhibited the production of ROS after pretreatment(p < 0.05).2.Compared with the CON and VEC groups,IL-38 was significantly higher in the OE group(p < 0.05),suggesting successful viral transfection of IL-38.PCR results suggested that IL-1β and IL-6 were significantly higher in both the LPS and LPS+VEC groups compared with the CON group,while both were significantly lower in the LPS+OE group(p < 0.05).Western blotting yielded similar results,with IL-1β,IL-6,COX-2 and i NOS proteins significantly elevated in the LPS and LPS+VEC groups,compared to the CON group.These four proteins were significantly lower in the LPS+OE group compared to the LPS and LPS+VEC groups(p < 0.05).DHE staining showed that,compared to the CON group,the LPS and LPS+ VEC group had elevated ROS,which was significantly lower in the LPS+OE group(p < 0.05).3.Compared with the CON and NC groups,IL-38 was significantly lower in the Si group(p < 0.05),suggesting successful silencing of IL-38.Western blotting results suggested that IL-1β,IL-6,COX-2 and i NOS proteins were significantly elevated in the LPS and LPS+NC groups compared with the CON group,and these four proteins were further elevated in the LPS+Si group(p < 0.05).4.Western blotting showed that IL-38 was significantly higher in the OE group compared with the CON and VEC groups(p < 0.05),suggesting successful viral transfection of IL-38.ELISA results suggested that IL-1 β and IL-6 were significantly higher in the LPS and LPS+VEC groups compared with the CON group.While IL-1β and IL-6 in the LPS+OE group was significantly lower than that in the LPS group and LPS+ VEC group(p < 0.05).HE staining revealed that the aortic endothelium in the LPS and LPS+VEC groups was not smooth,with irregular cell arrangement,disorganized mesenteric elastic membrane and elastic fibers,and inhomogeneous nucleus compared with the CON group.In contrast,the aortic injury in the LPS+OE group was alleviated.Immunohistochemical staining showed that ICAM-1 and VCAM-1 were significantly higher in the LPS group and LPS+VEC group than in the CON group,while ICAM-1 and VCAM-1 levels decreased in the LPS+OE group but were still higher than in the CON group(p < 0.05).Western blotting results revealed that IL-1β,IL-6,COX-2 and i NOS was shown similar trends in expression changes in all groups(p < 0.05).Conclusions:(1)Recombinant IL-38 inhibits LPS-induced endothelial inflammatory response and injury(2)Overexpression of IL-38 inhibits LPS-induced endothelial inflammatory response and injury(3)Silence of IL-38 enhances LPS-induced endothelial inflammatory response and injury(4)In vivo experiments demonstrated that overexpression of IL-38 alleviated the endothelial inflammatory response to sepsis and reduced vascular injury.Section Ⅳ IL-38 inhibits LPS-induced endothelial injury dependent on MAPK/NFκB pathwaysObjective: To investigate the mechanism of IL-38 inhibition of LPS-induced endothelial injury.Methods: 1.HUVECs in good condition were randomly divided into control group(CON group),sepsis group(LPS group),empty viral vector group(LPS+VEC group)and IL-38 overexpression group(LPS+OE group).IL-38 overexpression and null-loaded adenovirus were constructed and HUVECs in LPS+OE and LPS+VEC groups were transfected for 36 h,respectively.Except for the CON group,HUVECs in the other three groups were stimulated with 1ug/ml of LPS for 24 h.Protein levels of p-p38/p38 and p-p65/p65 were detected by Western blotting.2.HUVECs in good condition were randomly divided into control group(CON group),sepsis group(LPS group),negative control group(LPS+NC group)and IL-38 silence group(LPS+Si group).IL-38 interfering fragment and negative counterpart fragment were constructed and HUVECs in LPS+Si group and LPS+NC group were transfected for 48 h,respectively.Except for the CON group,HUVECs in the other three groups were stimulated with 1ug/ml of LPS for 24 h.Protein levels of p-p38/p38 and p-p65/p65 were detected by Western blotting.3.HUVECs were randomly divided into four groups: sepsis group(LPS group),IL-38 silencing group(LPS+Si-IL38 group),p38 inhibitor group(LPS+SB203580 group)and IL-38 silencing + p38 inhibitor group(Si-IL38+ SB203580 group).when HUVECs in LPS+Si-IL38 group and Si-IL38+ SB203580 group reached 30%-50% density,Si-IL38 interfering fragment was added to HUVECs for transfection.After 48 h,10u M of SB203580 was added to the HUVECs in LPS+SB203580 group and Si-IL38+ SB203580 group.after treatment for 2h,the original medium was aspirated and discarded.All four groups added medium containing 1ug/ml LPS and continued to incubate for 24 h.Western blotting was performed to detect changes of protein levels in IL-1β,IL-6,COX-2,i NOS,p-p38/p38 and p-p65/p65.Results: 1.Compared with the CON group,p-p38/p38 and p-p65/p65 were significantly higher in the LPS group and LPS+VEC group.And p-p38/p38 and p-p65/p65 were lower in the LPS+OE group than in the LPS group and LPS+VEC group(p < 0.05).2.Compared with the CON group,p-p38/p38 and p-p65/p65 were significantly higher in the LPS and LPS+NC groups.After interfering with IL-38,p-p38/p38 and p-p65/p65 were further elevated in the LPS+Si group than in the LPS group and LPS+NC group(p < 0.05).3.Protein levels of IL-1β,IL-6,COX-2,i NOS,p-p38/p38 and p-p65/p65 were significantly elevated in the LPS+Si-IL38 group compared to the LPS group.These proteins were decreased in the LPS+ SB203580 group compared to the LPS+ Si-IL38 group.Furthermore,these proteins were all elevated in the Si-IL38+ SB203580 group compared to the LPS+ SB203580 group(p < 0.05).Conclusions: IL-38 inhibition of LPS-induced endothelial injury is dependent on MAPK and NFκB pathways.Section Ⅴ SummaryIn this study,we constructed a sepsis model in vivo and in vitro to verify the anti-inflammatory effect of IL-38 on endothelium using three strategies: recombinant IL-38,overexpression and silence of IL-38.Using rescue experiments,we further explored the mechanism by which IL-38 inhibits LPS-induced inflammation in endothelial cells and found that IL-38 exerts anti-inflammatory effects in sepsis dependent on MAPK and NFκB pathways.In conclusion,this study explored the role of IL-38 on endothelial injury in sepsis and its mechanisms,providing a new idea and strategy for the treatment of sepsis.