| Objective:With the gradual increase of the survival rate of premature infants,the incidence of white matter injury in premature infants is also increasing year by year.Periventricular leukomalacia（PVL）is one of the main types of white matter injury in premature infants,and the location and degree of injury are directly related to the prognosis of nervous system function development in premature infants.However,there is still no effective clinical treatment for the disease.With the in-depth study of PVL by scholars at home and abroad in recent years,it has been found that astrocyte activation is a landmark feature of white matter injury in premature infants,and activated astrocytes may not be an accidental pathological reaction to the occurrence and development of PVL.It is the main participant in the process of PVL tissue damage repair.Under the condition of white matter injury,astrocytes can maintain the microenvironment around neurons and the steady state of the whole brain tissue by inhibiting toxic reaction,thus playing a key role in protecting the function of neurons.At present,studies have shown that leukemia inhibitory factor（LIF）can promote the survival of cultured astrocytes in vitro through leukemia inhibitory factor receptor（LIFR）mediated Stat3 signaling pathway.In order to protect astrocytes from reactive oxygen species damage and promote the development and maturation of oligodendrocyte precursor cells.However,the expression of LIFR and the mechanism of downstream signal response in astrocytes under different activation conditions are not clear.Therefore,we speculate that different levels of LIFR expression may play different biological effects on astrocytes through different signal transduction pathways.The purpose of this study was to find a new mechanism for regulating the function of astrocytes after PVL injury induced by hypoxia and ischemia,and to give full play to its protective effect of moderate activation,while effectively curbing the negative effects of excessive apoptosis.It provides an important research basis for the treatment of PVL.Methods:1.Animal experimental model and groups:the internationally recognized rat model of PVL brain injury in premature infants was used in vivo.SD rats on the3rd day of birth were male or female.The experimental animals were randomly divided into three groups:（1）control group（2）PVL group（3）PVL+LIF group.There were 32 rats in the control group,54 rats in the PVL group and 54 rats in the PVL+LIF group.PVL group and PVL+LIF group:the rats were ligated in the left common carotid artery and recovered for 1 hour.the rats were treated with hypoxia at37℃（O2 concentration 6%-8%）120min.From 1 day after operation,the rats in PVL+LIF group were injected intraperitoneally with LIF（50ug/kg.d,once a day,for 7days）,while the rats in control group and PVL group were injected intraperitoneally with the same dose of aseptic saline.Brain tissue was collected from rats at 3 days（HI3 days）,7 days（HI 7 days）,14 days（HI 14 days）and 28 days（HI 28 days）after HI.Sevoflurane inhalation anesthesia,local disinfection and decapitation were performed.The animal tissue specimens prepared for HE staining or immunohistochemistry were immersed in 4%paraformaldehyde and fixed in paraffin,and the animal tissue specimens prepared for Western-Blot were stored in refrigerator at-80℃.2.Cell experimental model and groups:astrocytes cultured in the same batch to the third generation were randomly divided into normal culture group（N group）and oxygen-glucose deprivation culture group（OGD group）.In addition to oxygen-glucose deprivation for 24 hours and reoxygenation for 24 hours,they were also divided into different groups according to LIF dose or SiRNA interference or not.The cells for immunohistochemistry and immunofluorescence were washed with phosphate buffer solution（PBS）three times after abandoning the culture medium,and then fixed with 4%paraformaldehyde.The cells for Real-Time PCR were lysed with Trizol total RNA extract and collected in a sterile Eppendorf tube after high pressure.The cells for Western-Blot were rinsed with PBS for 3 times,the adherent cells were digested with 0.25%trypsin and centrifuged to remove the supernatant,and collected and precipitated in aseptic Eppendorf tube,then were stored in-80℃refrigerator.3.Experimental methods and detection indexes:（1）animal experiment:the pathomorphological changes of white matter injury in neonatal rats were observed by HE staining;The expression of glial fibrillary acidic protein（GFAP）and myelin basic protein（MBP）in neonatal rat brain was detected by immunohistochemistry.Apoptosis of astrocytes in paraventricular white matter was observed by TUNEL staining;The expression of LIFR,GFAP and MBP protein in white matter of neonatal rats was detected by Western Blot method;（2）cell experiment:Primary astrocytes were identified by immunohistochemistry and immunofluorescence.The apoptosis of astrocytes in vitro was detected by TUNEL staining and Flow cytometry,and the viability of astrocytes in vitro was measured by MTT assay.The relative expression of LIF and LIFR in astrocytes cultured in vitro was detected by Real-Time PCR.The protein expressions of LIFR,Cleaved-Caspase3,Bcl2,Bax,pAKT,AKT,pStat3,Stat3,pErk1/2/2 and Erk1/2/2 in cultured astrocytes were detected by Western-Blot.GoldenTransfer TM-D transfection reagent was used to transiently transfect siRNA into astrocytes cultured in vitro to silence LIFR gene expression,and the relative expression of LIFR in successfully transfected astrocytes was detected by Real-Time PCR.The protein expression of LIFR,Bcl2,Bax,pAKT,AKT,pStat3,Stat3,pErk1/2and Erk1/2/2 in transfected astrocytes was detected by Western-Blot.Results:In vivo experiments:1.HE staining showed that the peri-ventricular white matter of HI 3d rats in PVL group showed disordered arrangement of nerve cells and widened space,loose white matter structure and sieve necrosis.2.On the HI 7d,cell edema was obvious and irregular,nuclear pyknosis,periventricular white matter edema,tissue looseness,punctate,stripe and cystic necrosis.On the HI 14d,empty cysts were found in the white matter of the brain,the structure of the white matter around the ligated lateral ventricle was blurred and sparse,the nerve fibers were disordered,the arrangement of cells was disordered,and glial cell proliferation could be seen.The pathological changes of HI 28d were similar to those of HI 14d.The performance of PVL+LIF group was similar to that of PVL group at each time point,but the degree of brain injury was relatively mild.2.GFAP immunohistochemistry and Western-Blot showed that the number of GFAP positive cells in periventricular white matter of PVL group and PVL+LIF group was higher than that of control group,but the increase of GFAP positive cells in PVL+LIF group was more obvious than that in control group（P<0.05）.On the HI 14d,the number of GFAP positive cells in periventricular white matter of PVL group and PVL+LIF group were also higher than control group,but the number of GFAP positive cells in PVL+LIF group was lower than that in PVL group（P<0.05）.3.MBP immunohistochemistry and western-blot results showed that:positive MBP immunohistochemical staining was observed in corpus callosum,striatum,internal capsule and external capsule of each group of rats at HI 7d,HI 14d and HI 28d,and the positive staining was gradually enhanced over time.However,MBP positive staining was weaker in PVL group than in control group at each time point（P<0.05）.Although the MBP positive staining of PVL+LIF group was weaker than that of the control group,its expression was enhanced compared with that of PVL group at the corresponding time point（P<0.05）.4.the Western-Blot results of Bax/Bcl2 showed that apoptosis occurred in PVL group and PVL+LIF group at HI 3d,HI 7d,HI 14d and HI 28d.However,the level of apoptosis in PVL+LIF group was lower than that in PVL group at the same time（P<0.05）.5.the Western-Blot results of LIFR showed that the expression of LIFR in periventricular white matter of PVL group and PVL+LIF group were higher than that of control group on the HI 3d,but PVL+LIF group was more significant than that in control group（P<0.01）.6.The level of astrocyte apoptosis was observed by GFAP and TUNEL immunofluorescence co-staining.The results showed that the number of astrocyte apoptosis in periventricular white matter of PVL group was gradually higher than that of control group from HI 3d（P<0.05）.The number of astrocyte apoptosis in PVL+LIF group was also higher than that in control group,but compared with PVL group,the number of astrocyte apoptosis in PVL+LIF group decreased,especially on HI 14d and HI 28d（P<0.05）.In vitro experiments:1.Oxygen-glucose deprivation and reoxygenation successfully induced apoptosis of astrocytes,and the expression of LIFR was increased.2.The expression of Bcl2 decreased,the expression of Cleaved-Caspase3 increased,the expression of p-Akt/Akt and p-Stat3/Stat3 decreased,and the expression of p-Erk1/2/Erk1/2/2 increased.2.Under the condition of OGD-R,the apoptosis of astrocytes was improved after LIF intervention,especially at the dose of LIF 30ng/ml,the effect of inhibiting apoptosis was the most obvious.3.The expression of LIFR in astrocytes increased gradually after LIF treatment under OGD-R condition,and the expression level of LIFR reached the peak at LIF 30ng/ml.4.Under the condition of OGD-R,the expression of Cleaved-Caspase3 decreased,the expression of Bcl2 increased,the ratio of p-Akt/Akt and p-Stat3/Stat3 increased,and the ratio of p-Erk1/2/Erk1/2/2 decreased in astrocytes treated with LIF.5.SiRNA interference successfully inhibited the expression of LIFR.6.After siRNA silenced LIFR expression,the activity of astrocytes decreased significantly under OGD-R condition,and the apoptosis of N group and OGD group was significantly higher than that of undisturbed group.7.the expression level of Bcl2 in SiRNA interfered OGD group was further lower than that in OGD group,and the expression level of Bax in OGD+SiRNA group was further higher than that in OGD group.8.The ratio of P-Akt/Akt and P-Stat3/Stat3 in OGD+SiRNA group was further lower than that in OGD group.On the contrary,the ratio of P-Erk1/2/Erk1/2/2 in OGD+SiRNA group was higher than that in OGD group.Conclusion:1.LIF can significantly improve the pathological changes of PVL rats,and has the effect of protecting the brain of PVL model rats from hypoxic-ischemic brain injury and protecting myelination.LIF plays a different role in the activation of astrocytes in different periods after HI,and can protect and repair the nervous system by inhibiting astrocyte apoptosis.2.LIF could significantly increase the activity of astrocytes and inhibit the apoptosis of astrocytes under the condition of oxygen-glucose deprivation.LIFR is the key to inhibit astrocyte apoptosis.3.LIFR can inhibit astrocyte apoptosis by regulating Stat3,Akt,Erk1/2 and other signal transduction pathways to increase the expression of Bcl2. |
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