MiR-382-5p Targets CDK8 To Inhibit The Polarization Of M1 Macrophages In Bronchopulmonary Dysplasia And Preliminary Study On The Therapeutic Effect Of Myricetin

Bronchopulmonary dysplasia(BPD)is a chronic respiratory disease that is clinically more common in very preterm infants and very low birth weight infants.Its main feature is the growth and developmental disorder of alveolar and pulmonary blood vessels,the disease has a high mortality rate in the neonatal period,and children are prone to recurrent respiratory infections,damage to pulmonary ventilation and ventilation functions,and pulmonary hypertension,seriously affects the late survival rate and quality of life of premature infants.The pathogenesis of BPD is complex,involving multiple aspects such as heredity,preterm birth,oxygen toxicity,prenatal and postnatal infection,and mechanically ventilated lung injury,these endogenous and exogenous stimuli lead to an inflammatory cascade in the body,macrophages cells and neutrophils accumulate in the lung tissue of injured premature infants,and then damage the immature lung tissue,the abnormal repair of the lung tissue and the uncontrolled development of pulmonary blood vessels after the injury eventually lead to the occurrence of BPD.Obviously,the central link of the pathogenesis and progression of BPD is the inflammatory response caused by multiple factors,and the polarization of macrophages is involved in regulating the inflammatory response of BPD.At present,the clinical treatment of BPD is limited,and most of them stay on symptomatic treatment.With the rapid development of molecular biology,the research on BPD has gradually deepened to the level of gene transcription regulation.Micro RNAs(micro RNAs,mi RNAs)are short-sequence non-coding RNAs in the body with regulatory functions,they recognize and bind target gene m RNA through base complementary pairing,and then play the function of degrading target gene m RNA or inhibiting its translation.It can be seen that the gene encoding the protein is regulated by mi RNAs after transcription,which ultimately affects the expression of the protein.Studies in recent years have shown that mi RNAs are involved in the pathogenesis of BPD.Among them,mi R-382-5p can reduce the level of inflammatory cytokines in the body,inhibit cell apoptosis,and is abnormally expressed in the lung tissue of neonatal BPD rats.In this study,the expression of mi R-382-5p in the blood of BPD children,in the lung tissue of hyperoxia-induced BPD mice and M1-type polarized macrophages were analyzed,and its downstream targets were further searched.Cyclin dependent kinase 8(CDK8),which has been shown to activate signal transducer and activator of transcription 1(STAT1),and activation of the STAT1 pathway can promote the polarization of macrophages to the M1 type,resulting in inflammation.Myricetin belongs to flavonoids,with a wide range of biological functions,such as antibacterial,anti-inflammatory,antioxidant,anti-tumor and other effects.Studies have reported that myricetin is effective in the treatment of acute lung injury,lung cancer and other lung diseases.In this study,the pathogenesis of BPD was discussed at the molecular level,and the effect of myricetin on inflammatory cytokines in lung tissue of BPD mice was analyzed,preliminary demonstration that myricetin can reduce inflammatory response in BPD mice,provide new ideas for the prevention and treatment of BPD.Part One mi R-382-5p inhibits the polarization of M1 macrophages inbronchopulmonary dysplasia and affects the STAT1 path-wayObjective:1.To clarify that there is a phenomenon of dysregulated expression of mi R-382-5p in BPD disease.2.To demonstrate the state of macrophage polarization in BPD,and to clarify the effects of mi R-382-5p on macrophage polarization,inflammatory cytokines and STAT1 pathway in BPD from experimental animal and cellular levels.Methods:1.The expression level of mi R-382-5p in peripheral blood of premature infants with BPD44 premature infants admitted to the Department of Neonatology of the Fourth Hospital of Hebei Medical University and the First Central Hospital of Baoding City from September 2019 to February 2022 were selected as the research subjects.According to the 2018 NICHD diagnostic criteria for BPD,the research subjects were divided into BPD group and non-BPD group,including 21 cases in BPD group and 23 cases in non-BPD group.2-3 m L of venous blood was collected from all children one the first days after birth,and the expression of mi R-382-5p in serum was detected by RT-q PCR.2.mi R-382-5p expression and macrophage polarization state in lung tissue of BPD animal modelsFull term newborn C57BL/6mice were randomly divided into two groups:Control group and BPD group,with 18 mice in each group.By postnatal day 3(PN3),the mice in the BPD group were placed in a hyperoxia environment(85% oxygen),and the mice in the control group were placed in a normal environment(21% oxygen).Two groups of mice were sacrificed on the 5th,10 th and 15 th days of birth(PN5,PN10,PN15)and lung tissues were collected.RT-q PCR was used to detect the expression levels of mi R-382-5p in lung tissue of two groups of mice.IF double staining was used to detect the expression of the PN15 macrophage biomarker CD68 and the M1 macrophage biomarker CD86 in the lung tissues of the two groups of mice.3.Effects of overexpression of mi R-382-5p on inflammatory cytokines,macrophage polarization and STAT1RAW264.7 macrophages were routinely cultured,and infected with mi R-382-5p overexpressing lentivirus(LV-mi R-382-5p)and negative control lentivirus(LV-mi R-NC)respectively,added 20ng/m L IFN-γ and 100ng/m L LPS induces M1-type polarization of macrophages(creates inflammation).The cells were divided into four groups: Control,IFN-γ+LPS,IFN-γ+LPS+LV-mi R-NC,IFN-γ+LPS+LV-mi R-382-5p.RT-q PCR was used to detect the expression levels of mi R-382-5p in different groups to verify that overexpression of mi R-382-5p is successful.The expression levels of inflammatory cytokines TNF-α,IL-1β and IL-6 in cell supernatants were detected by ELISA.The expression of M1 macrophage biomarkers CD40 and CD86 was detected by flow cytometry.Western blot was used to detect the expression of STAT1 and p-STAT1 in cells.Results:1.The expression level of mi R-382-5p in the serum of premature infants in the BPD group(0.0080±0.0019)was significantly lower than that in the non-BPD group(0.0095±0.0022)(P=0.016).2.PN5 time point:there was no significant difference in the expression levels of mi R-382-5p in lung tissue between the Control group and the BPD group(P=0.114);PN10 time point: the expression level of mi R-382-5p in mouse lung tissue in the BPD group was lower than that in the Control group(P=0.004);PN15 time point:the expression level of mi R-382-5p in mouse lung tissue in the BPD group was lower than that in the Control group(P=0.000).Immunofluorescence double staining showed that the number of macrophages positive for biomarkers CD68 and CD86 was significantly increased in the BPD group compared with the Control group.3.The expression level of mi R-382-5p in the IFN-γ+LPS group was lower than that in the Control group(P<0.01);the expression level of mi R-382-5p in the IFN-γ+LPS+LV+mi R-382-5p group was significantly higher than that in the IFN-γ+LPS+LV-mi R-NC group(P<0.01),proved that cells overexpressing mi R-382-5p were successfully constructed.The levels of TNF-α,IL-6 and IL-1β in the IFN-γ+LPS group were higher than those in the Control group(P<0.01);The levels of TNF-α and IL-1β in the IFN-γ+LPS+LV-mi R-382-5p group were lower than those in the IFN-γ+LPS+LV-mi R-NC group(P<0.01);The levels of IL-6 in the IFN-γ+LP S+LV-mi R-382-5p group were lower than those in the IFN-γ+LPS+ LV-mi RNC group(P<0.05).Compared with the Control group,the expression levels of CD40 and CD86 in the IFN-γ+LPS group were significantly increased(P<0.01);Compared with the IFN-γ+LPS+LV-mi R-NC group,the expression levels of CD40 and CD86 in the IFN-γ+LPS+LV-mi R382-5p group were significantly decreased(P<0.01).There was no significant difference in the relative expression of STAT1 in each group(P>0.05);Compared with the Control group,the relative expression of p-STAT1 in the IFN-γ+LPS group increased(P<0.01);Compared with the IFN-γ+LPS+LV-mi R-NC group,the relative expression of p-STAT1 in the IFN-γ+LPS+LV-mi R-382-5p group decreased(P<0.01).Summary: The expression levels of mi R-382-5p in the peripheral blood of BPD premature infants and in the lung tissue of BPD neonatal mice were decreased;The numbers of macrophages and their M1-type polarization in lung tissue of BPD mice increased;The expression of mi R-382-5p decreased after inducing M1-type polarization of macrophages.Overexpression of mi R-382-5p could inhibit the polarization of M1-type macrophages and inhibit the release of inflammatory cytokines;Overexpression of mi R-382-5p can down-regulate the expression level of p-STAT1.Part Two mi R-382-5p targets CDK8 to regulate STAT1-mediatedpolarization of M1 macrophages in bronchopulmonarydys-plasiaObjective: The target regulation relationship between mi R-382-5p and CDK8 was explored,and the effect of mi R-382-5p on macrophage M1 polarization was clarified by targeting CDK8 and regulating STAT1.Methods:1.Validation of the targeting relationship between mi R-382-5p and CDK8The targeting relationship between mi R-382-5p and CDK8 was clarified by dual-luciferase reporter gene assay.2.The effect of overexpression of mi R-382-5p on CDK8mi R-382-5p overexpressing lentivirus(LV-mi R-382-5p)and negative control lentivirus(LV-mi R-NC)infected RAW264.7 macrophages respectively,adding20ng/m L IFN-γ and 100ng/m L LPS induces M1-type polarization of macrophages.The cells were divided into four groups: Control,IFN-γ+LPS,IFN-γ+LPS+LV-mi R-NC,IFN-γ+LPS+LV-mi R-382-5p.The levels of CDK8 in cells were detected by RT-q PCR and Western blot.3.Expression of CDK8 in lung tissue of BPD animal modelFull term newborn C57BL/6 mice were randomly divided into two groups: Control group and BPD group,with 18 mice in each group.By postnatal day 3(PN3),the mice in the BPD group were placed in a hyperoxia environment(85% oxygen),and the mice in the control group were placed in a normal environment(21% oxygen).Two groups of mice were sacrificed on PN5,PN10,and PN15 days,and 6 C57BL/6 mice were randomly selected from each group,and the lung tissues of the mice were collected.IF double staining was used to detect the expression and co-localization of CDK8 and macrophage biomarker CD68 at PN15 time point in the lung tissues of the two groups of mice.4.The rescue experiment demonstrates the effect of CDK8 on mi R-382-5p-mediated RAW264.7 M1-type polarizationRAW264.7 macrophages were cultured and infected with CDK8 overexpressing lentivirus(LV-CDK8)and its negative control lentivirus(LV-NC).The cells were divided into three groups: Control,LV-NC,and LV-CDK8.The expression of CDK8 was detected by RT-q PCR to verify the effect of overexpressing CDK8 lentivirus in infecting cells.RAW264.7 macrophages were cultured and infected with mi R-382-5p overexpressing lentivirus(LV-mi R-382-5p)and CDK8 overexpressing lentivirus(LV-CDK8)/mi R-382-5p overexpressing lentivirus(LV-mi R-382-5p)and CDK8 overexpressing negative control lentivirus(LV-NC),adding20ng/m L IFN-γ and 100ng/m L LPS induced M1-type polarization of macrophages.The cells were divided into four groups:IFN-γ+LPS,IFN-γ+LPS+LV-mi R-382-5p,IFN-γ+LPS+LV-mi R-382-5p+LV-NC,IFN-γ+LPS+LV-mi R-382-5p+LV-CDK8.RT-q PCR detection of IL-6 expression in cells.Flow cytometry detection of CD40 expression in cells.Results:1.The dual luciferase reporter gene experiment showed that the luciferase activity in the mi R-382-5p mimics+CDK8 3’UTR WT group was significantly lower than that in the NC mimics+CDK8 3’UTR WT group(P<0.01).The luciferase activity of mi R-382-5p mimics+CDK8 3’UTR Mut group and NC mimics+CDK8 3’UTR Mut group did not change significantly.2.The expression of CDK8 in the IFN-γ+LPS group was up-regulated at the m RNA and protein levels compared with the Control group(P<0.01);The expression of CDK8 in the IFN-γ+LPS+LV-mi R-382-5p group was down-regulated at the m RNA and protein levels compared with the IFN-γ+LPS+LV-mi R-NC group(P<0.01).3.Immunofluorescence double staining showed that on the 15 th day after birth,compared with the Control group,the CD68-positive macrophages in the lung tissue of the mice in the BPD group increased significantly,and CDK8 was localized in the macrophages and the expression level increased.4.In rescue experiment:There was no significant difference in the expression level of CDK8 m RNA between the LV-NC group and the Control group(P>0.05).The expression level of CDK8 m RNA in the LV-CDK8 group was higher than that in the LV-NC group(P<0.01).Indicates that the overexpressed CDK8 lentivirus successfully infects cells.The expression level of IL-6 m RNA in the IFN-γ+LPS+LV-mi R-382-5p group was lower than that in the IFN-γ+LPS group(P<0.01);The expression level of IL-6 m RNA in the IFN-γ+LPS+LV-mi R-382-5p+LV-CDK8 group was higher than that in the IFN-γ+LPS+LV-mi R-382-5p+LV-NC group.Compared with the IFN-γ+LPS group,the expression level of CD40 in the IFN-γ+LPS+LV-mi R-382-5p group decreased(P<0.01);Compared with the IFN-γ+LPS+LV-mi R-382-5p+LV-NC group,the expression level of CD40 in the IFN-γ+LPS+LV-mi R-382-5p+LV-CDK8 group increased(P<0.01).Summary: mi R-382-5p has a targeting relationship with CDK8;The expression of CDK8 increased after the induction of M1 polarization in macrophages,and overexpression of mi R-382-5p could inhibit the increased expression of CDK8;The number of macrophages in the lung tissue of BPD mice increased,CDK8 was localized in macrophages,and the expression level was increased;mi R-382-5p inhibits M1-type polarization of macrophages by directly targeting CDK8.Part Three Preliminary study on the therapeutic effect of myricetin onbronchopulmonary dysplasiaObjective: By observing the inflammatory indicators of lung tissue in the Control group,BPD group and myricetin group at different times,it was preliminarily confirmed that myricetin can reduce the inflammatory response of BPD,providing a new direction for the treatment of BPD.Methods: The full-term newborn C57BL/6 mice were randomly divided into three groups: Control group,BPD group,and myricetin treatment group,with 18 mice in each group.By postnatal day 3(PN3),the mice in the BPD group and myricetin group were placed in a hyperoxia environment(85%oxygen),and the mice in the control group were placed in a normal environment(21%oxygen).Mice in the myricetin group were given myricetin100 mg/kg gavage treatment on the day before PN5,PN10,and PN15 were sacrificed.The three groups of mice were sacrificed at time points PN5,PN10,and PN15 respectively.6 C57BL/6 mice were randomly selected from each group,and the levels of TNF-α,IL-6 and IL-1β in lung tissue were detected by ELISA.Results: At PN5 time point,there was no significant difference in the levels of TNF-α,IL-6 and IL-1β among the three groups(P>0.05);At PN10 time point,the levels of TNF-α,IL-6 and IL-1β in the myricetin group were higher than those in the control group,and lower than those in the BPD group(P<0.01);At PN15 time point,the levels of TNF-α,IL-6 and IL-1β in the myricetin group were higher than those in the control group,and lower than those in the BPD group(P<0.01).Summary: Myricetin can reduce the inflammatory response in the lung tissue of BPD mice by reducing the levels of TNF-α,IL-6 and IL-1β.Conclusions:1.The expression of mi R-382-5p is down-regulated in the peripheral blood of BPD premature infants.2.Animal experiments showed that the expression of mi R-382-5p was down-regulated in the lung tissue of BPD neonatal mice;The numbers of macrophages and their M1-type polarization in lung tissue of BPD mice increased,and the expression level of CDK8 in macrophages increased.3.Cell experiments confirmed that overexpression of mi R-382-5p can reduce the inflammatory response of M1 macrophages,inhibit the polarization of M1 macrophages and the activation of STAT1 pathway.mi R-382-5p has a targeting relationship with CDK8.mi R-382-5p downregulates CDK8 expression by targeting CDK8,thereby inhibiting STAT1 pathway activation,inhibiting STAT1-mediated M1 macrophage polarization,and alleviating BPD inflammatory injury.4.Myricetin can reduce the inflammatory response in the lung tissue of BPD mice.