| Objective: Spinal cord ischemia-reperfusion injury(SCII)is one kind of the spinal cord injury after cardiovascular surgery such as thoracoabdominal aortic aneurysm.SCII can be divided into primary injury and secondary injury according to pathology.It often leads to neurological dysfunction,even paralysis or paraplegia.The high incidence and disability rate of SCII greatly reduces the quality of life of patients,and brings huge economic losses and social burden.Although a large number of basic research and clinical intervention measures on SCII have been implemented,the ideal curative effect has not been achieved at present.Therefore,it is imperative to research the pathological process of SCII and develop new and effective treatment methods.Although the molecular mechanism of SCII is not completely clear,it is generally believed that neuroinflammatory reaction plays a key role in SCII.Traumatic spinal cord injury leads to the destruction of blood-spinal cord barrier(BSCB),axon damage,neuron demyelination,and then a series of secondary diseases,which cause serious inflammatory reaction in the damaged area.SCII causes a series of complex cascade reactions in spinal cord,including cell necrosis and programmed cell death.Programmed cell death includes apoptosis,autophagy,pyroptosis and so on.These may be important pathological mechanisms of SCII,and they are also the research hotspots in recent years.According to the previous sequencing results of our research group,we found that lnc RNA H19 and mi R-181a-5p have obvious changes after SCII in rats.Through predicting websites,we found that lnc RNA H19 and mi R-181a-5p,mi R-181a-5p and HMGB1 have binding sites.It is found that lnc RNA H19,mi R-181a-5p and HMGB1 are involved in many nervous system diseases and are related to the mechanism of pyroptosis after consulting a large number of literatures.The aim of this study was to explore the correlation between lnc RNA H19 and mi R-181a-5p in SCII,and to verify whether lnc RNA H19 and mi R-181a-5p play a role through HMGB1,and produce an effect on pyroptosis in SCII.Methods: According to the literature and previous experience of the research group,SCII model was established by clipping the aortic arch for 14 minutes.The expression of mi R-181a-5p and lnc RNA H19 in S group(sham),12 h group(SCII 12 h),24 h group(SCII 24 h),48 h group(SCII 48h)and 72 h group(SCII 72h)was detected by q RT-PCR.According to the results,we selected 48 h after SCII as the research time.Dual-luciferase reporter assay was used to verify the direct relationship between mi R-181a-5p and lnc RNA H19.Lnc RNA H19 was knocked down by intrathecal injection of si-lnc RNA H19 for three days before SCII.QRT-PCR was used to detect the effect of knockdown lnc RNA H19.Tarlov scoring system was used to verify the changes of behavior and neurological function in rats.Evans blue(EB)staining was used to detect the permeability of blood-spinal cord barrier(BSCB).TUNEL staining was used to compare the apoptosis of each group.Histopathological changes of spinal cord in rats were observed by HE staining.After intrathecal injection of mi R-181a-5p mimic for three days before SCII,the overexpressing mi R-181a-5p SCII model was established.Tarlov scoring system was used to verify the changes of behavior and neurological function in rats.The effect of mi R-181a-5p overexpression was detected by q RT-PCR.The direct relationship between mi R-181a-5p and HMGB1 was verified by double luciferase.The expression of RNA and protein of HMGB1 and the expression of TNF-α and IL-1β after mi R-181a-5p overexpression were examined by q RT-PCR and Western blotting.Immunofluorescence staining was used to detect the changes of HMGB1 expression in the anterior horn of spinal cord after mi R-181a-5p overexpression and the survival of each group of neurons.SCII models were established by intrathecal injection of si-lnc RNA H19,si-lnc RNA H19 + mi R-181a-5p inhibitor or NC si RNA for three days before SCII.The behavioral and neurological functions of sham group,SCII group,NC group,si-lnc RNA H19 group and si-lnc RNA H19 + mi R-181a-5p inhibitor group were evaluated by Tarlov scoring system.The expression changes of mi R-181a-5p and HMGB1 after si-lnc RNA H19 were verified by q RT-PCR and Western blotting.The expression of GSDMD,cleaved caspase-1,NLRP3 and IL18 in sham,SCII,NC,si-lnc RNA H19 and si-lnc RNA H19 +mi R-181a-5pin group were detected by Western blotting.Immunofluorescence staining was used to detect the co-localization of HMGB1 and three kinds of cells in spinal cord,and the co-localization and quantification of HMGB1 and NLRP3.After knockdown HMGB1,q RT-PCR,Western blot,and immunofluorescence were used to detect the protein expression levels of NLRP3 and Cleaved caspase-1,as well as the co-location and quantification of HMGB1 and NLRP3.Results: Compared with sham group,the RNA expression level of lnc RNA H19 was significantly increased at 48 hours after SCII.The RNA expression level of mi R-181a-5p decreased significantly at 48 hours after SCII.The results of Dual-luciferase reporter assay confirmed that there was a direct binding between lnc RNA H19 and mi R-181a-5p,and spearman showed a negative correlation between them.In Tarlov scoring system,the score of SCII group was significantly lower than that of sham group,and the score of si-lnc RNA H19 group was significantly higher than that of SCII group.The results of EB experiment showed that the EB permeation in SCII group was significantly higher than that in Sham group,while the EB permeation in si-lnc RNA H19 group was significantly lower than that in SCII group.The results of TUNEL showed that the number of TUNEL positive cells in SCII group was significantly higher than that in sham group,while that in si-lnc RNA H19 group was significantly higher than that in sham group.After knockdown lnc RNA H19,the number of TUNEL positive cells decreased significantly compared with SCII group.Compared with sham group,the RNA expression level of mi R-181a-5p decreased significantly at 48 hours after SCII.Intrathecal injection of mi R-181a-5p mimic can effectively over-express mi R-181a-5p.The results of double luciferase confirmed the direct binding relationship between mi R-181a-5p and HMGB1.The results of q RT-PCR showed that the expression of HMGB1 RNA decreased significantly after mi R-181a-5p overexpression.Western bloting showed that the expression of HMGB1,TNF-α and IL-1β protein decreased significantly after mi R-181a-5p overexpression.The expression of HMGB1 in the anterior horn of spinal cord after the overexpression of mi R-181a-5p by immunofluorescence staining was consistent with the result by Western bloting.After SCII,neurons in the anterior horn of spinal cord were damaged and their number was reduced.The survival rate of neurons was significantly increased after overexpression of mi R-181a-5p.After knockdown lnc RNA H19,the expression of mi R-181a-5p increased significantly,while the expression of HMGB1 decreased significantly.Immunofluorescence assay confirmed that HMGB1 was co-localized with neurons and microglia,but not astrocyte.After knockdown lnc RNA H19 and inhibiting mi R-181a-5p,the expression levels of HMGB1,GSDMD,cleaved caspase-1,IL18,NLRP3 were higher than those in si-lnc RNA H19 group.The results of immunofluorescence staining showed that HMGB1 was co-localized with NLRP3,and the expression levels of HMGB1 and NLRP3 were consistent in each group.Conclusions: 1.The expression of lnc RNA H19 increased within 72 hours after SCII,and reached its peak at 48 hours after SCII.The expression level of mi R-181a-5p decreased within 72 hours after SCII,and reached the lowest level at 48 hours after SCII.There is a direct regulation relationship between them,and it is negatively correlated.After SCII,severe motor dysfunction of hind limbs appeared in rats,apoptosis of motor neurons in anterior horn of spinal cord increased,and BSCB was destroyed.Knockdown lnc RNA H19 alleviated SCII-induced nerve injury.2.The expression of HMGB1 increased within 72 hours after SCII,and reached its peak at 48 hours after SCII.mi R-181a-5p is directly bound to HMGB1.Overexpression of mi R-181a-5p can inhibit the expression of HMGB1.HMGB1 is the direct target of mi R-181a-5p.Overexpression of mi R-181a-5p can inhibit the expression of inflammatory factors IL-1β and TNF-α by regulating HMGB1,which alleviates nerve injury caused by SCII.3.Pyroptosis occurd in microglia after after SCII.Knockdown lnc RNA H19 inhibited the expression of pyroptosis-related proteins,while mi R-181a-5p inhibitor reduced the effect of si-lnc RNA H19 on pyroptosis after SCII.Knockdown HMGB1 inhibited NLRP3 and Cleaved caspase-1 protein expression.In conclusion,lnc RNA H19 can regulate microglia pyroptosis in SCII through mi R-181a-5p/HMGB1.This result may be a new and effective therapeutic target for SCII. |
PDF Full Text Request