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Objective:To study the dynamic changes of the localization and expression of nuclear transporter KPNA2 in the occurrence and development of bronchopulmonary dysplasia(BPD) in neonatal rats, and explore its role in the pathogenesis of BPD in premature infants.Methods:The BPD model of newborn SD rats was induced with 85% oxygen concentration( n=50), and the control group was inhaled with air( n=50). The lung tissue samples were collected on 1 d, 3 d, 7 d, 10 d, and 14 d, respectively, in the two groups and separated.Purification and culture of alveolar type Ⅱ epithelial cells.The distribution and expression of KPNA2 were detected by immunohistochemistry, immunofluorescence, western blot and RT-PCR. Results:Immunohistochemistry showed that KPNA2 mainly located in alveolar epithelial cells′ cytoplasm and nucleus, and BPD group was more expressive than control group.Cell immunofluorescence showed that KPNA2 in control group was mainly localized in the nucleus, and in BPD group, KPNA2 was mainly localized in the cytoplasm from 3 d to 14 d. The nuclear expression of KPNA2 was weaker than that in the control group, and the cytoplasmic expression was stronger than that in the control group.The expression trends of KPNA2 total protein, plasma protein and mRNA were basically the same.The BPD group began to increase on the 1st day ( P<0.05), and was still higher than the control group on the 14th day( P<0.05); in BPD group, KPNA2 nucleoprotein expression began to decrease on the 3rd day( P<0.01), continued to decrease to 14 days( P<0.05). Conclusion:The dysfunction of KPNA2 nuclear transport in neonatal rats exposed to hyperoxia may be an important mechanism that affects the early initiation of the DNA damage response of BPD alveolar epithelial cells.
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Objective@#To investigate the role of granulocyte-colony stimulating factor (G-CSF) on the regulation of inflammatory cytokines in neonatal hypoxic-ischemic brain damage(HIBD) rat model, and to explore the possible mechanism involved in G-CSF neuroprotective effect via the mammalian target of Rapamycin/p70 ribosomal S6 protein kinase (mTOR/p70S6K) signaling pathway.@*Methods@#A group of postnatal day 7 (P7) Sprague-Dawley rat pups (90 cases) were randomly divided into sham-operated group, hypoxia-ischemia(HI) group, G-CSF group, Rapamycin (RAP) group and control group, and the improved Rice method was used to establish a neonatal rat model of HIBD.One hour before HI induction, Rapamycin was administered intraperitoneally with a dose of 250 μg/kg, and the control group was given equal volume of ethanol injected intraperitoneally.One hour after HI, a dose of 50 μg/kg of G-CSF was injected intraperitoneally into the G-CSF group, Rapamycin group and control group.The same volume of normal saline was injected intraperitoneally into HI group and sham-operated group.Forty-eight hours after HI, Western blot was used to detect the protein levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-10, and the mTOR/p70S6K signaling pathway in brain tissue.Neuron injury of the hippocampal CA1 region and the cortex was assessed by Nissl staining, and infarct volume detected by 2, 3, 5-triphenyltetrazolium chloride staining.@*Results@#The G-CSF group and control group were associated with significantly reduced infarction volume compared to the HI group [(12.87±1.54)%, (11.90±1.31)% vs.(24.21±3.28)%], and the differences were statistically significant(P<0.05). There was an increased positive neuron cell number in the ipsilateral hemispheres of the hippocampal CA1 region in the G-CSF group and the control group [(61.00±4.90) cell/field and (61.67±6.40) cell/field] and cortex [(92.67±6.68) cell/field and (90.17±4.45) cell/field] compared with those in HI group [(42.62±4.46) cell/field and (70.83±6.97) cell/field], and the differences were all statistically significant (all P<0.05). The expression levels of TNF-α and IL-1β were significantly decreased in the G-CSF group and the control group, compared with those in HI group(TNF-α: 0.67±0.07, 0.55±0.05 vs.0.86±0.05; IL-1β: 0.65±0.06, 0.52±0.10 vs.0.86±0.06), and the differences were all statistically significant (all P<0.05). There was increased expression levels of IL-10, p-mTOR/mTOR and p-p70S6K/p70S6K in the G-CSF group and the control group, compared with those in HI group (IL-10: 0.68±0.04, 0.62±0.05 vs.0.34±0.02; p-mTOR/mTOR: 0.53±0.02, 0.51±0.01 vs.0.26±0.01; p-p70S6K/p70S6K: 0.89±0.03, 0.90±0.03 vs.0.55±0.02), and the differences were all statistically significant(all P<0.05). There was an increased infarct volume in Rapamycin group [(25.70±1.50)%], compared with the G-CSF group and the control group, and there were decreased number of positive neuron cell count in the hippocampal CA1 region [(40.67±3.50) cell/field] and cortex [(68.33±8.17) cell/field], increased expression levels of TNF-α and IL-1β (0.97±0.06 and 0.98±0.10, respectively), decreased expression levels of IL-10, p-mTOR/mTOR and p-p70S6K/p70S6K (0.21±0.02, 0.30±0.01 and 0.55±0.01, respectively) in the Rapamycin group, and the differences were all statistically significant (all P<0.05).@*Conclusions@#G-CSF may inhibit inflammatory responses after HIBD by up-regulating the mTOR/p70S6K signaling pathway in neonatal HI encephalopathy.
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Objective To study the expression of hypoxia-inducible factor-1a(HIF-1α) at mRNA and protein levels in the early stage of hypoxic-ischemic brain damage (HIBD) in neonatal rats and its role.Methods (1) Experiment 1:thirty-six postnatal 7-day SD rats were divided into Sham group (n =6) and model group (HIBD,n =30) according to the random table method,then the rats in the model group were divided into 5 subgroups according to the time of sacrifice after HIBD(6 h,12 h,24 h,48 h,72 h,n =6).The expression levels of HIF-1cα mRNA and protein were detected by quantitative Real-time PCR(qPCR) and Western blot,respectively.(2) Experiment 2:forty-five postnatal 7-day SD rats were randomized into 3 groups:Sham group (n =15),HIBD group (n =15) and 2-methoxyestradiol(2ME2) group(n =15).According to the experiment 1,at the time point of the highest expression levels of HIF-1 α mRNA and protein,rats were killed and the brains were collected.The location and expression of HIF-1 α protein were detected by immunofluorescence,histopathological changes of brain were observed by HE staining,brain water content was measured by dry-wet method,cell apoptosis was detected by nick end labeling(TUNEL) method.Results At the early stage of HIBD,the expression levels of HIF-1 α mRNA and protein increased at first and then decreased,and the mRNA expression level (3.38 ± 0.21) and protein expression level (2.81 ± 0.36) were the highest at 24 h after HIBD.In Sham group,HIF-1 α protein was mainly expressed in the cytoplasm,while in HIBD group it was mainly expressed in the nucleus.The number of HIF-1α staining positive cells,brain water content and apoptosis rate were significantly different among Sham group,HIBD group and 2ME2 group (all P ＜ 0.05),and which were significantly lower in 2ME2 group than those in HIBD group (all P ＜ 0.05),and the pathological changes were also less serious than those in HIBD group.Conclusions The mRNA and protein levels of HIF-1 α are the highest at 24 h after HIBD.Inhibiting the expression of HIF-1 α can ameliorate the brain damage of neonatal rats induced by hypoxia-ischemia.Therefore,it is hypothesized that HIF-1α may cause injury in the early stage of HIBD in neonatal rats.
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Objective To explore the change of endogenous glucocorticoids (GC) secretion after intracranial hemorrhage (ICH) of neonatal rats and the impact of Dexamethasone (DEX).Methods Ten-day-old Sprague-Dawley rat pups of both sexes were randomized into 11 groups:normal control group(CON group),sham operated group (SHAM group),ICH group(each of which was further subgrouped into 12 h group,24 h group and 72 h group according to execution time after the modeled operation),glucocorticoids receptor (GR) agonist intervene group (DEX group) and GR antagonist intervene group(RU486 group).The intracranial autologous blood injection model of ICH was employed.Neurological functional deficits was measured by neurological deficit score (NDS),the levels of cerebral homogenate GC were tested by the emission immunology method,and the pathologic change and the expression of GR in hippocampus CA1 were examined by using Nissl staining and immunofluorescence separately.Results (1) Seventy-two-hour after the modeled operation,NDS of rats in the ICH group reached (7.48 ± 2.19) scores.After intervened by DEX,NDS of rats in DEX group decreased to (3.15 ± 1.93) scores,significantly lower than in ICH group,the difference was significant (P ＜ 0.05).The necrotic neurons were found around the hematoma of rats in ICH group,while in DEX group,less necrotic neurons were found.(2)In ICH group,the GC level in cerebral homogenate climbed up to a peak of (1.359 1 ±0.308 5) μg/L at 12 h,and slowly went down.By the end of 72 h,the GC level was (0.951 0 ±0.036 1) μg/L,which was higher than those of the CON group[(0.621 3 ±0.039 3) μg/L],the difference was significant (P ＜ 0.05),while in the DEX group,the level of GC in cerebral homogenate showed no difference with statistics from CON group.(3)The mean integrated optical density (IOD) of GR in hippocampal CA1 of rats in the ICH group (1.282 4 ± 0.035 6) were much more smaller than those in the CON group (1.012 5 ± 0.027 3,P ＜ 0.05),which meant the down-regulated expression of GR.(4) No difference was found in the NDS,pathological change,GC level and GR expression between RU486 group and ICH group.DEX didn't effect the expression of GR.Conclusions ICH in neonatal rat disturbs the modulation of hypothalamus-pituitary-adrenal axis,with an increase in the GC level and less GR expression.Early application of exogenous GC helps protect the neurons.
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Objective To study the expression of hypoxia-inducible factor-1a(HIF-1α) at mRNA and protein levels in the early stage of hypoxic-ischemic brain damage (HIBD) in neonatal rats and its role.Methods (1) Experiment 1:thirty-six postnatal 7-day SD rats were divided into Sham group (n =6) and model group (HIBD,n =30) according to the random table method,then the rats in the model group were divided into 5 subgroups according to the time of sacrifice after HIBD(6 h,12 h,24 h,48 h,72 h,n =6).The expression levels of HIF-1cα mRNA and protein were detected by quantitative Real-time PCR(qPCR) and Western blot,respectively.(2) Experiment 2:forty-five postnatal 7-day SD rats were randomized into 3 groups:Sham group (n =15),HIBD group (n =15) and 2-methoxyestradiol(2ME2) group(n =15).According to the experiment 1,at the time point of the highest expression levels of HIF-1 α mRNA and protein,rats were killed and the brains were collected.The location and expression of HIF-1 α protein were detected by immunofluorescence,histopathological changes of brain were observed by HE staining,brain water content was measured by dry-wet method,cell apoptosis was detected by nick end labeling(TUNEL) method.Results At the early stage of HIBD,the expression levels of HIF-1 α mRNA and protein increased at first and then decreased,and the mRNA expression level (3.38 ± 0.21) and protein expression level (2.81 ± 0.36) were the highest at 24 h after HIBD.In Sham group,HIF-1 α protein was mainly expressed in the cytoplasm,while in HIBD group it was mainly expressed in the nucleus.The number of HIF-1α staining positive cells,brain water content and apoptosis rate were significantly different among Sham group,HIBD group and 2ME2 group (all P ＜ 0.05),and which were significantly lower in 2ME2 group than those in HIBD group (all P ＜ 0.05),and the pathological changes were also less serious than those in HIBD group.Conclusions The mRNA and protein levels of HIF-1 α are the highest at 24 h after HIBD.Inhibiting the expression of HIF-1 α can ameliorate the brain damage of neonatal rats induced by hypoxia-ischemia.Therefore,it is hypothesized that HIF-1α may cause injury in the early stage of HIBD in neonatal rats.
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Objective To explore the change of endogenous glucocorticoids (GC) secretion after intracranial hemorrhage (ICH) of neonatal rats and the impact of Dexamethasone (DEX).Methods Ten-day-old Sprague-Dawley rat pups of both sexes were randomized into 11 groups:normal control group(CON group),sham operated group (SHAM group),ICH group(each of which was further subgrouped into 12 h group,24 h group and 72 h group according to execution time after the modeled operation),glucocorticoids receptor (GR) agonist intervene group (DEX group) and GR antagonist intervene group(RU486 group).The intracranial autologous blood injection model of ICH was employed.Neurological functional deficits was measured by neurological deficit score (NDS),the levels of cerebral homogenate GC were tested by the emission immunology method,and the pathologic change and the expression of GR in hippocampus CA1 were examined by using Nissl staining and immunofluorescence separately.Results (1) Seventy-two-hour after the modeled operation,NDS of rats in the ICH group reached (7.48 ± 2.19) scores.After intervened by DEX,NDS of rats in DEX group decreased to (3.15 ± 1.93) scores,significantly lower than in ICH group,the difference was significant (P ＜ 0.05).The necrotic neurons were found around the hematoma of rats in ICH group,while in DEX group,less necrotic neurons were found.(2)In ICH group,the GC level in cerebral homogenate climbed up to a peak of (1.359 1 ±0.308 5) μg/L at 12 h,and slowly went down.By the end of 72 h,the GC level was (0.951 0 ±0.036 1) μg/L,which was higher than those of the CON group[(0.621 3 ±0.039 3) μg/L],the difference was significant (P ＜ 0.05),while in the DEX group,the level of GC in cerebral homogenate showed no difference with statistics from CON group.(3)The mean integrated optical density (IOD) of GR in hippocampal CA1 of rats in the ICH group (1.282 4 ± 0.035 6) were much more smaller than those in the CON group (1.012 5 ± 0.027 3,P ＜ 0.05),which meant the down-regulated expression of GR.(4) No difference was found in the NDS,pathological change,GC level and GR expression between RU486 group and ICH group.DEX didn't effect the expression of GR.Conclusions ICH in neonatal rat disturbs the modulation of hypothalamus-pituitary-adrenal axis,with an increase in the GC level and less GR expression.Early application of exogenous GC helps protect the neurons.
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Objective To investigate the effect of docosahexaenoic acid (DHA) on the body weight growth and lipid metabolism of neonatal rats during lactation.Methods The specific pathogen free Sprague-Dawley neonatal rats were randomly assigned into 4 groups (high-dose group,medium-dose group,low-dose group and control group) by random number table method.The rats in 3 experiment groups received intragastric administration with DHA 600 mg/kg,300 mg/kg and 100 mg/kg,respectively,while the control group were given 9 g/L saline,totally for 21 days.Body weight was monitored and compared among groups on postnatal day 1,7,14 and 21.And body weight growth rates at each time point were calculated.The serum concentrations of high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol(LDL-C),triglyceride (TG) and total cholesterol were measured and compared at 6-week and 8-week ages.The pathological and histological changes in the heart,the large vessel and the liver were observed at same time.Results The mean body weight of the neonatal rats were significantly different among 4 groups on postnatal day 7,14 and 21 (F =17.334,4.159,6.485,all P ＜ 0.01).Comparisons were made between every 2 groups,the low-dose group was higher than the control group on postnatal day 7 [(21.60 ±0.89) g vs.(18.57 ± 0.76) g] and day 21 [(58.52 ±6.62) g vs.(53.01 ± 11.75) g];the medium-dose group was lower than the control group on postnatal day 7 [(14.23 ±0.49) g vs.(18.57 ±0.76) g] and lower than the low-dose group on postnatal day 21 [(52.47 ±8.18) g vs.(58.52 ±6.62) g];the high-dose group was lower than the low-dose group on postnatal day 7[(16.13 ± 1.02) g vs.(21.6 ±0.89) g],and it was lower than the control group and the low-dose group on postnatal day 14[(31.69 ± 1.77) g vs.(37.60 ± 1.32) g and (36.24 ±0.84) g],and lower than all the other 3 groups on postnatal day 21 [(45.9 ± 13.17) g vs.(53.01 ± 11.75) g,(58.52 ±6.62) g and (52.47 ±8.18) g];all the differences above were statistically significant (all P ＜ 0.05).During the first and the second week after birth,there were significant differences in the mean body weight growth rate among 4 groups (F =8.369,8.331,all P ＜ 0.01),but there was no significant difference during the third week (F =0.603,P ＞ 0.05).Compared with 2 groups,the mean body weight growth rate of the low-dose group was higher than that of the control group in the first week [(184.96 ± 63.16) ％ vs.(141.02 ± 72.07) ％],but which was lower than that of the control group in the second week [(72.60 ± 35.37) ％ vs.(103.20 ± 40.11) ％];the medium-dose group was lower than the low-dose group at the first week [(116.78 ± 51.59) ％ vs.(184.96 ± 63.16)％],but higher than the low-dose group and lower than the control group at the second week[(139.93 ± 67.4) ％ vs.(72.60 ± 35.37) ％ and (103.20 ± 40.11) ％];the high-dose group was lower than the low-dose group in the first week [(137.33 ± 34.42) ％ vs.(184.96 ± 63.16) ％] and lower than that of the medium-dose group in the second week [(98.22 ± 65.86) ％ vs.(139.93 ± 67.4) ％];all these differences were statistically significant (all P ＜ 0.05).At 6 weeks of age,the mean serum concentrations of total cholesterol,TG and LDL-C were not significandy different (F =1.899,1.450,2.581,all P ＞ 0.05) among 4 groups,but the mean concentration of HDL-C was statistically different (F =7.801,P ＜ 0.01).In detail,the mean concentration of HDL-C in medium-dose group was higher than that of the control group,the low-dose group and the high dose group [(1.66 ± 0.08) mmol/L vs.(0.97 ± 0.16) mmol/L,(1.20 ± 0.09) mmol/L and (0.82 ± 0.09) mmol/L,all P ＜ 0.05],and which in the high-dose group was lower than that in the low-dose group (P ＜ 0.05).At 8-week age,the mean serum concentrations of HDL-C,LDL-C and total cholesterol were not significantly different among 4 groups (F =0.935,0.300,1.299,all P ＞ 0.05),but the mean concentration of TG was significantly different (F =2.875,P ＜ 0.05).The mean concentration of TG in the medium-dose group was lower than that in the control group [(0.98 ± 0.11) mmol/L vs.(1.36 ± 0.09) mmol/L,P ＜ 0.05].There were 5 (15.62％) neonatal rats in the high-dose group which were found to have adipose tissue accumulation around the large vessel walls and the heart and were confirmed by histological examination.The liver cells in these rats were found to have mild fatty changes.No similar changes were found in the other groups.Conclusions Neonatal rats supplemented with DHA during lactation can affect their body weight growth and lipid metabolism.Supplemented with high dose may bring risks,while moderate dose may bring benefits.
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Objective To explore the effect of exogenous recombinant human erythropoietin (rhEPO) on neuronal apoptosis in neonatal rats after hyperoxia brain injury.Methods Thirty neonatal Wistar rats were randomly divided into 3 groups by random number table method:rhEPO treatment + 800 mL/L hyperoxia group (group A),9 g/L saline +800 mL/L hyperoxia group (group B),9 g/L saline + air group (group C).Group A was given subcutaneous injection of rhEPO 1 000 IU/kg for 5 days.Group B and group C received the same dose of 9 g/L saline.Group A and group B were continuously exposed to atmospheric pressure hyperoxia model cabin to maintain the oxygen concentration in the container (800 ± 30) mL/L for 5 days.During the course of the experiment,the general situation and weight changes in rats were observed.After 5 d,all rats were sacrificed and brain tissues were taken.Neuronal apoptosis in hippocampal structural region of the newborn rats was observed by terminal deoxynucleotidyl transferase dUTP nick and labeling(TUNEL) staining.Immunohistochemical method was used to detect the expression of 5-lipoxygenase in hippocampal structural region of newborn rats.Results The weight gain and brain weight of group B were lower than those of group C,the weight gain and brain weight of group A were higher than those of group B,and the differences were statistically significant(F =11.179,8.140,all P ＜ 0.05).In group A and group B were found that the neuronal nucleus of the hippocampal neurons was partially contracted,deeply dyed,and the neuronal arrangement was loose,even with local neuron deletions and focal necrosis,but in group A neuron density was higher with less necrosis than that in group B.The neuronal cells in hippocampal structural region were neat and intact in group C.The number of TUNEL positive cells in hippocampal structural region of group B[(6.20 ± 1.93) number/high power field] was significantly higher than that in group C [(1.80 ± 0.79) number/high power field],the number of TUNEL positive cells in hippocampal structural region of group A [(4.20 ± 1.32) number/high power field] was significantly lower than that in group B,and the difference was statistically significant (F =23.912,P ＜ 0.05).The number of 5-lipoxygenase positive cells in group B [(6.90 ± 1.29) number/high power field] was significantly higher than that in group C [(1.00 ± 0.67) number/high power field],the number of 5-lipoxygenase positive cells in group A [(5.60 ± 0.97)number/high power field] was significantly lower than that in group B,and the difference was statistically significant (F =95.044,P ＜ 0.05).Conclusion rhEPO has a protective effect on neonatal rats with hyperoxia brain injury,and alleviates brain cell apoptosis caused by hyperoxia brain injury,which may interfere with the 5-lipoxygenase pathway.
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Objective To discuss whether Omega-3 fish oil fat emulsion has the potential protective mechanism for 7-day-old rats with hypoxic-ischemic brain damage (HIBD).Methods One hundred and sixty-eight 7-day-old SD rats were randomly divided into 4 groups:group A (sham group),group B (Omega-3 fish oil fat emulsion group),group C (normal fat emulsion group),group D (model group),and there were 42 cases in each group.Neonatal HIBD was induced by the method of Rice.Rats were sacrificed at 1 d,3 d and 7 d after the surgery.Hippocampus was removed for Real-time PCR and Western blot test to detect Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) mRNA and protein expression.TUNEL staining comparison was done among different groups to observe the number of cellular apoptosis.Results HE staining of hippocampus CA1 area in 3 d showed that brain tissues in group A maintained normal structures;those in group D had much more brain cells with severe edema than other groups;TLR4 and NF-κB mRNA and protein expression levels in group D were higher than those in group A in 1 d (all P ＜0.05);TLR4 and NF-κB expression levels of mRNA and protein in group B (4.89 ± 0.51,9.30 ± 1.53;1.15 ±0.10,1.44 ± 0.14) were lower than those in group C (17.58 ± 2.50,20.13 ± 1.00;2.56 ± 0.10,2.82 ± 0.09) and group D (15.94-± 2.52,26.21 ± 3.00;2.34 ± 0.11,4.51 ± 0.36) in 3 d (all P ＜ 0.05),and compared with group A (6.30 ± 1.52,5.32 ± 1.06;1.32 ± 0.10,2.42 ± 0.14),there was significant difference (all P ＞ 0.05);TLR4 and NF-κB mRNA and protein expression levels in group B were lower than those in group C and group D in 7 d(all P ＜0.05),and compared with group A there was no significant difference (all P ＞ 0.05).The apoptotic cell number of brain tissues in 3 d:group B (13.67 ±2.52) were lower than those in group C (27.67 ±2.52) and group D (41.00 ±3.61) (all P ＜0.05),and compared with the group A (6.00 ±2.00),the difference was not statistically significant (P ＞ 0.05).Conclusions Omega-3 fish oil fat emulsion plays an important role in protecting neonatal rats with HIBD.The mechanisms were likely to reduce TLR4,NF-κB and cell apoptosis levels.
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Objective To explore the effects of bilirubin on myeloid differentiation factor 88 (MyD88)and interleukin-1 receptor associated kinase-4(IRAK-4).Methods Seven-day-old Sprague Daw-ley rats (clean grade),male or female,weighing 12.0 to 15.0 g,were randomly assigned to 6 groups.There were normal saline group(Ⅰ),lipopolysaccharide(LPS)control group (LPS,Ⅱ),15 mg /kg bilirubin con-trol (free-LPS)group (Ⅲ),15 mg /kg group (Ⅳa),30 mg /kg group (Ⅳb)and 50 mg /kg group (Ⅳc), and then subsequently divided into 2 h,5 h and 24 h subgroups in each groups.Some of the 200 newborn rats died amid the experiment.Finally a total of 144 were involved in the analysis of results,and 8 rats in each subgroups.Newborn Sprague Dawley rats were administered at various doses of bilirubin (15 mg /kg, 30 mg /kg and 50 mg /kg respectively)intravenously;1 h after injection,the rats were administered LPS intrap-eritoneally at a dose of 1 mg /kg;MyD88 and IRAK-4 were detected by immunohistochemistry at 2 h,5 h and 24 h after the injection of bilirubin.Results (1 )LPS could stimulate the expression of MyD88 and IRAK-4 in spleen cells (qMyD88 2 h =49.89,qMyD88 5 h =139.54,qIRAK-4 2 h =7.93,qIRAK-4 5 h =24.30,qIRAK-4 24 h =6.97 ,P 0.05).Effects of medium and high concentration of bilirubin on LPS stim-ulation MyD88 were inhibitory(qⅣb 2 h =42.87,qⅣc 2 h =51.38,qⅣb 5 h =103.61 ,qⅣc 5 h =1 15.44,qⅣb 24 h =1.18,qⅣc 24 h =1 1.66,P <0.01 ).(4)Effects of low,medium and high concentration of bilirubin on LPS stimulation IRAK-4 were inhibitory(qⅣa 2 h =9.52,qⅣb 2 h =14.39,qⅣc 2 h =25.55,qⅣa 5 h =38.83,qⅣb 5 h =54.62,qⅣc 5 h =60.51 ,qⅣa 24 h =2.41 ,qⅣb 24 h =1.47,qⅣc 24 h =7.61 ,P <0.01 ).(5)The inhibition of biliru-bin to MyD88 and IRAK-4 was observed at 2 h,strengthened at 5 h,disappeared at 24 h in low-mid concen-trations of bilirubin(P <0.01 )while still visible at 24 h in high concentration of bilirubin.(6)There was neg-atively correlation between the expression level of MyD88,IRAK-4 and bilirubin concentration(rsMyD88 2 h =-0.86, rsMyD88 5 h =-0.92,rsMyD88 24 h =-0.53,rsIRAK-4 2 h =-0.82,rsIRAK-4 5 h =-0.86,rsIRAK-4 24 h =-0.57,P <0.01).(7) Under the effect of bilirubin and LPS,there were positively correlation between the expression levels of MyD88 and IRAK-4 of spleen cells(r2 h =0.77,r5 h =0.9,r24 h =0.67,P <0.01).Conclusion Bilirubin could inhibit the expression of MyD88 and IRAK-4.As the concentration of bilirubin increasing,its inhibition is more obvious and prolonged.The mechanism that bilirubin affects immune function of newborn rat may be related to regulation of expression of MyD88 and IRAK-4 at toll-like receptor 4 signal pathway.
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Objective To investigate the role of receptor for advanced glycation end - products nuclear factor - κB(RAGE - NF - κB)signaling pathway in the lipopolysaccharide - induced acute lung injury(ALI)in neo-natal rats. Methods Thirty - two SD rats were divided into 4 groups by complete randomization method(8 cases in each group).(1)Lipopolysaccharide(LPS)group was given intraperitoneal injection of 9 g/ L saline and 3 mg/ kg LPS 1 h later.(2)Bortezomib group was given intraperitoneal injection of Bortezomib(0. 2 mg/ kg)and 3 mg/ kg LPS 1 h later.(3)Anti - RAGE mAb group was given intraperitoneal injection of anti - RAGE mAb(15 mg/ kg)and 3 mg/ kg LPS 1 h later.(4)Control group was given 9 g/ L saline was given at each time point. All the rats were sacrificed and observed 24 h later. Levels of tumor necrosis factor(TNF) - α in the plasma and bronchoalveolar lavage fluid(BALF) were detected by enzyme linked immunosorbent assay. RAGE and NF - κB levels in tissue homogenates were detected by Western blot and mRNA levels were detected by reverse transcription - polymerase chain reaction. The pathological assessment of the lung tissues was performed by HE staining. Results (1)Among 4 groups,there were significantly differences in TNF - α in serum and BALF(F = 150. 70,P ﹤ 0. 001;F = 165. 83,P ﹤ 0. 001). Levels of TNF - α in LPS group were significantly higher than those of two pretreatment groups(all P ﹤ 0. 05).(2)Western blot figures il-lustrated that the concentrations of RAGE mRNA and NF - κB in anti - RAGE mAb group and bortezomib group were lower than those of the LPS group.(3)Reverse transcription - polymerase chain reaction analysis showed that there were significant differences in the expression of RAGE mRNA and NF - κB mRNA among 4 groups(F = 175. 14,P ﹤0. 05;F = 188. 65,P ﹤ 0. 05). Levels of RAGE mRNA and NF - κB mRNA in the LPS group were significantly higher than those of two pretreatment groups(all P ﹤ 0. 05).(4)Lung injury score differences among 4 groups were statistical-ly significant(F = 106. 01,P ﹤ 0. 001). Pathological changes in two pretreatment groups reduced compared to those of the LPS group(all P ﹤ 0. 05). Conclusions RAGE - NF - κB signaling pathway regulates the LPS - induced ALI in neonatal rats. Anti - RAGE mAb and Bortezomib both have a protective effect on LPS - induced ALI.
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Objective To investigate anti-inflammatory effect of recombinant human erythropoietin(rhEPO) on bronchopulmonary dysplasia in newborn rats exposed to hyperoxia.Methods Ninety-six Wistar newborn rats were randomly divided into 4 groups after birth:room air-exposed control group,room air-exposed rhEPO treated group,hyperoxia-exposed group,and the hyperoxia-exposed rhEPO treated group.The last two groups were exposed to oxygen,FiO2 =850 mL/L,room air-exposed rhEPO treated and hyperoxia-exposed rhEPO treated group received rhEPO 2 400 IU/kg subcutaneously at birth,30 minutes' before oxygen exposure and 2 d after birth.The isodose of 9 g/L saline was given in the same way in room air-exposed controls and hyperoxia-exposed pups.Rats from each group were sacrificed on day 3,7 and 10.Lung histology was observed under microscope,and mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil hemoattractant-1 (CINC-1) were determined with reverse transcriotion-polymerase chain reaction(RT-PCR).Results Under microscope,in the hyperoxia-exposed group,inflammatory cell influx was detected in the lungs on the 3rd day and there was marked neutrophlic infiltrate on the 7th day.Alveolar enlargement and fibrosis were evident on the 10th day.At the same time,the histopathological changes were improved greatly in the lungs of hyperoxia-exposed rhEPO treated pups compared with the hyperoxia-exposed pups.MCP-1 and CINC-1 mRNA expression increased in hyperoxia-exposed pups,compared with room air-exposed controls especially on the 7th day [(0.94 ± 0.45) vs (0.21 ± 0.03),P ＜ 0.001 ; (1.26 ± 0.29) vs (0.26 ± 0.06),P ＜ 0.001].MCP-1 and CINC-1 mRNA expression were greatly depressed in the hyperoxia-exposed rhEPO treated pups compared with the hyperoxia-exposed pups especially on the 7th day.[(0.65 ± 0.07) vs (0.94 ± 0.45),P＜0.05;(0.83±0.07) vs (1.26±0.29),P＜0.05].Conclusions The therapy of rhEPO (2 400 IU/kg) therapy can reduce lung inflammatory cell infiltration and alveolar fibrin deposition in newborn rats with hyperoxic lung injury,and it can restrain MCP-1 and CINC-1 mRNA expression.The anti-inflammatory mechanism of rhEPO is related to inhibition of MCP-l and CINC-1 mRNA expression.
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Objective To investigate the dynamic expression of mRNA and protein of surfactant protein C (SPC),E-cadherin (E-cad),N-cadherin (N-cad) and α-smooth muscle actin (α-SMA) and elucidate the significance of epithelial-mesenchymal transition (EMT) in a newborn rat model of Bronchopulmonary dysplasia (BPD).Methods A newborn rat model of BPD in hyperoxia was established,and a control group exposed to air was established.Lung tissue was collected on days 3,7,14,and 21,respectively.Alveolar development was evaluated by radical alveolar counts(RAC),including thickness of alveolar septum,ratio of alveolar and septa.Real-time PCR and Western-blot were used to detect levels of markers of epithelial cells (SPC,E-cad) and interstitial cells (N-cad,α-SMA) in AT2 and protein expression.Results On day 7,14,and 21,compared with the control group,RAC (7.38 ± 0.92,9.25 ± 0.70,9.88 ± 0.99) and alveolar area/pulmonary septal area ratios (A/S) (2.53 ± 0.02,3.34 ± 0.09,3.96 ± 0.13) were all higher in BPD group [RAC (5.88 ± 0.83,5.14 ± 0.83,4.38 ± 0.52) and A/S (1.88 ± 0.03,1.95 ± 0.03,1.89 ± 0.02)] (all P ＜ 0.05) ; the alveolar septum (8.53 ± 0.04,10.75 ± 0.46,13.55 ± 0.84) in BPD group were thicker than those (5.77 ± 0.09,4.40 ± 0.12,3.67 ± 0.18) in the control group (all P ＜ 0.01).The expressions of SPC and α-SMA in BPD group were significantly higher than those in the control group on day 14 and 21 (all P ＜ 0.05).The level of E-cad mRNA (2.43 ± 0.60,2.59 ± 0.48,3.37 ± 0.53) and protein (18.39 ± 1.77,18.29 ± 1.52,11.48 ± 1.72) for E-cad were higher in the control group than those (mRNA:1.48 ± 0.55,1.57 ± 0.48,1.12 ± 0.45 ;protein:9.50 ± 1.38,8.57 ± 1.06,8.22 ± 1.31) in BPD group was lower(P ＜ 0.05),while the level of N-cad was significantly higher(P ＜ 0.05) on day 7,14,and 21.Conclusions In the development of BPD,the markers of lung epithelial cells were down regulation,while the markers of interstitial cells were up-regulation,and these findings suggest that EMT from lung epithelial cells contributes to the occurrence of BPD.
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Objective To explore the effects of bilirubin on myeloid differentiation factor phospho-p38 mitogen-activated protein kinase (p-p38MAPK) and apoptosis in splenocytes of neonatal rats.Methods Seven-day-old Sprague Dawley rats (clean grade),male or female,weighting 12.0-15.0 g,were randomly assigned to 6 groups.There were blank control group (Ⅰ),lipopolysaccharide (LPS) control group (Ⅱ),15 mg/kg bilirubin control (free-LPS) group (Ⅲ),15 mg/kg group (Ⅳa),30 mg/kg group (Ⅳb) and 50 mg/kg group (Ⅳc),and then subsequently divided into 2 h,5 h and 24 h subgroups in each groups.Some of the 200 newborn rats died amid the experiment,tinally,a total of 144 cases were involved in the analysis of results,and 8 rats in each subgroups.Newborn Sprague Dawley rats were administered at various doses of bilirubin (15 mg/kg,30 mg/kg and 50 mg/kg,respectively) intravenously; 1 h after injection,the rats were administered LPS intraperitoneally at a dose of 1 mg/kg;p-p38MAPK were detected by immunohistochemistry;Apoptosis in splenocytes was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling methods at 2 h 5 h and 24 h after the injection of bilirubin.Results 1.Expression of p-p38MAPK in each group:bilirubin in low-mid concentrations of range inhibited LPS-induced p38MAPK activation (qⅣa =20.93,10.37,respectively at 2 h,and 5 h,all P ＜ 0.01 ;qⅣ b =79.97,14.79,all P ＜ 0.01).The inhibition strengthened with increasing concentration of bilirubin.The effect was observed at 2 h,strengthened at 5 h,disappeared at 24 h.Bilirubin in the high concentrations of range stimulated the expression of p-p38MAPK (qⅣc =32.55,19.23,27.72,respectively at 2 h,5 h and 24 h,all P ＜0.01),observed at 5 h,reduced at 24 h.2.Effects of bilirubin on apoptosis in splenocytes:LPS could increased the apoptosis index (AI) of splenocytes(q =54.62,P ＜ 0.01);The AI of splenocytes had no significant change in low concentrations of range of bilirubin (q =43.92,P ＞ 0.05).Low-mid concentration of bilirubin with LPS reduced the AI of splenocytes (q Ⅳ a =4.48,P ＜ 0.01 ;q Ⅳ b =2.07,P ＜ 0.05),while high concentration of bilirubin with LPS increased the AI of splenocytes (q =5.08,P ＜ 0.01).Conclusions Bilirubin in low-mid concentrations of range could inhibit the expression of LPS-induced p38MAPK,while bilirubin in high concentrations of range stimulated the expression.As the concentration of bilirubin elevated,its inhibition was prolonged.Bilirubin in high concentrations of range bilirubin could induce apoptosis in splenocytes.The immune dysfunction in neonatal hyperbilirubinemia may have something to do with the regulation of phosphorylation of p38MAPK and activation of apoptotic pathways.
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Objective To monitor the function of infection on myelination in white matter damage,neonatal Wistar rats of postnatal day 2 (P2) and postnatal day 7 (P7) were injected intraperitoneally with the same doses of lipopolysaccharides (LPS),and 2',3 '-cyclic nucleotide phosphodiesterase (CNPase) and myelin basic protein (MBP) were labeled in immature oligodendrocytes and mature oligodendrocytes.To investigate the function of tumor necrosis factor(TNF)-α according to test the change of TNF-α expression in the brain.Methods Ninty-six neonatal Wistar rats were randomly divided into four groups (each group 24 rats):group A:LPS (5.0 mg/kg) was injected intraperitoneally on P2 ; group B:LPS (5.0 mg/kg) was injected intraperitoneally on P7 ;group C1 and C2 were control groups in which equal amount of normal saline was injected intraperitoneally on P2 or P7.The expression of CNPase at 24 h after injection and MBP at P14 in brain tissue of each group were measured by immunohistochemistry and express of TNF-α mRNA at 4 h after the injection was measured by RT-PCR.Results Punctate hemorrhage in the corpus callosum,external capsule and intraventricular hemorrhage were seen in group A.Periventricular leukomalacia appeared in the corpus callosum and glial cells hyperplasia could be seen periventricular in P14 rat brains,but not found in the group B and any of the saline-injected rat brains.Compared with group C1 and C2 respectively,CNPase-positive cells showed obvious decrease in the area of white matter in periventricular in group A(106.93 ± 2.62 vs 113.67 ± 2.69,P ＜ 0.01) and group B (96.37 ± 1.82 vs 101.65 ± 2.01,P ＜ 0.01).Following LPS treatment in group A,the protein expression of MBP in neonatal brain decreased evidently compared with group C1 at P14 (128.21 ± 2.99 v s 134.81 ± 2.98,P ＜ 0.01),while no significant change was found between group B and group C2(134.77 ±3.68 vs 134.81 ±2.98,P ＞0.05).After 4h of the LPS treatment,the level of TNF-α mRNA was greatly increased in group A,it was significantly higher than that in group B (1.79 ± 0.04 vs 1.18 ± 0.04,P ＜ 0.01).Conclusion Intraperitoneal injection of LPS to the development neonatal rats can lead to dysmyelination and white matter damage.The expression of TNF-oα mRNA increased significantly in these immature neonatal rats,while only myelination delay occurred in those of mature neonatal rats without dysmyelination.
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Objective To observe the expression of hypoxia-inducible factor 1 α (HIF-1α) in rat brain after hypoxia-ischemia(HI),and to explore the possible mechanism of L-thyroxine (L-T4) on HIF-1α expression.Methods Sixty-four postnatal 7-day Sprague-Dawley rats were randomly divided into 4 groups:the sham operation group,HI group,menstruum-treated group and L-T4-treated group.HIBD models were generated according to Rice model method.The rats in menstruum-treated group and L-T4-treated group were respectively administrated of intraperitoneal injection of menstruum with the equal volume and 2 μg/100g L-T4,once a day,for 5 days.The expressions of HIF-1α and phospho-protein kinase B(p-Akt) protein were detected by means of immunohistochemistry.Reverse transcription-polymerase chain reaction was used to detect the level of HIF-1α mRNA.Results The levels of p-Akt protein(50.168 ±4.259),HIF-1α protein (72.795 ±6.121) and HIF-1α mRNA (0.448 ± 0.035) were upregulated compared with those in the sham operation group (8.080 ±0.369,38.581 ± 2.846,0.174 ± 0.015),and the differences were significant (all P ＜ 0.05).The levels of p-Akt protein (82.765 ± 6.271),HIF-1 α protein (117.350 ± 9.374) and HIF-1 α mRNA (0.618 ± 0.042) in L-T4-treated group were higher than those in HI group,and the differences were significant (all P ＜ 0.05).The level of HIF-1 α protein was positively correlated with p-Akt protein in HI group and L-T4-treated group [r(HI) =0.635,P=0.048;r(L-T4) =0.694,P=0.026].Conclusions L-T4 can upregulate HIF-1α mRNA and protein expression in neonatal rats with hypoxia-ischemia brain damage.Phosphatidylinositol-3-kinase/protein kinase B signaling pathway may be involved in L-T4 upregulating HIF-1α mRNA and protein expression.
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Objective To explore the changes of tumor necrosis factor-α (TNF-α),nuclear factor kappa B (NF-κB),free radical in brain tissue in newborn rats with hypoxic-ischemic brain damage(HIBD),to clarify the role of TNF-α,NF-κB,free radical in HIBD.Methods The 56 SD neonatal rats were randomly divided into control group and hypoxic-ischemic 6 h,12 h,24 h,48 h,72 h,7 d groups.The conventional method was used to establish HIBD model.The level of TNF-α was measured by enzyme-linked immumosorbent assay,NF-κB was detected by electrophoretic mobility shift assay.The neuron apoptosis was identified by flow cytometry.Superoxide dismutase(SOD),molondialdehyde (MDA),glutathione peroxidase(GSH-Px) were identified by spectrophotography.Results The rate of neuron apoptosis,the contents of TNF-α,NF-κB,MDA in brain tissue were significantly higher in 6 h,12 h,24 h,48 h,72 h groups than those in contral group(all P ＜0.05),and the apoptosis,MDA reached the peak at the 24 h after hypoxia,TNF-α reached the peak at the 12 h after hypoxia,NF-κB reached the peak at the 48 h after hypoxia.But the content of SOD and the activity of GSH-Px in hypoxic-ischemic group were significantly lower than those in control group at 6 h,12 h,24 h,48 h,72 h after hypoxia.And the rate of neuronal apoptosis was positively correlated with TNF-α,NF-κB and MAD (all P ＜ 0.05),negatively correlated with GSH-Px and SOD (all P ＜ 0.05),the contents of TNF-α,NF-κB were positively correlated with MAD (all P ＜ 0.05),negatively correlated with the content of SOD and the activity of GSH-Px (all P ＜ 0.05).Conclusions TNF-α,NF-κB,free radical are early changed after HIBD,So they play an important role in HIBD,inflammation and free radical can promote brain damage.
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Objective To explore the expression of KL-6/MUC1 and the possible impact on alveolar development in lung tissue of newborn rats with hyperoxia-induced bronchopulmonary dysplasia (BPD).Methods Sixty-four newborn rats were randomly divided into 2 groups:hyperoxic group and control group.The rats in hyperoxic group were exposed to high oxygen volume fraction of 900 mL/L,and the rats in control group were exposed to normal oxygen volume fraction of 210 mL/L.The experimental control factors were the same in two groups.Eight rats were randomly selected from each group on day 1,3,7,and 14 after oxygen exposure.The alveolar development was evaluated by the number of radial alveolar count (RAC) and the alveolar area/pulmonary septal area ratio (A/S).The location,distribution,and expression of KL-6/MUC1 in the lung tissue were detected by the fluorescent immunoassay,Western blot,and reverse transcription polymerase chain reaction.Results Compared with the control group,the RAC in hyperoxic group decreased on day 3 and continued to decline on day 14.The A/S in hyperoxic group increased on day 7 and peaked on day 14 (P ＜0.05).KL-6/MUC1 expressed in both bronchial epithelial cells and alveolar epithelial cells of newborn rats.KL-6/MUC1 protein in hyperoxic group peaked on day 1 and decreased later,which was higher than that of the control group (P ＜ 0.05),the level of KL-6/MUC1 was positively correlated with RAC (r =0.707,P ＜ 0.05)and negatively correlated with A/S(r =-0.716,P ＜ 0.05).MUC1 mRNA in hyperoxic group was slightly higher than that in control group,but no significant inter-or intra-group difference was observed (P ＞ 0.05).Conclusions The highest expression of KL-6/MUC1 can be observed in the early phase of hyperoxic exposure,and it decreases to the lowest on the key point of pulmonary development.KL-6/MUC1 may play an important role in the alveolar development.
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Objective To investigate the expression and significance of Smad4 and Smad7 in newborn rats with hyperoxia-induced chronic lung disease(CLD).Methods Sixty-four newborn Wistar rats 12 h after birth were divided into high-oxygen group (n =32) and air group (n =32,control group) by random number table method.The high-oxygen group was placed in the oxygen glass tank with continuous infusion of oxygen.And 1,3,7,14 d after experiment,tracheal separated,the chest opened to expose heart and lung,slices were Masson staining,undergo dynamic observation of the pulmonary pathological changes under light microscope.Lung fibrosis score was carried out to determine the degree of pulmonary fibrosis,and immunohistochemical technique was used to detect Smad4 and Smad7 protein expression in lung tissue.The expression levels of Smad4 and Smad7 protein in lung tissue were detected with Western blot.Results Compared with the air group,there was statistically significant difference in pulmonary fibrosis score on day 7 (2.67 ± 0.21 vs 0.58 ± 0.17) and day 14 (4.48 ± 0.24 vs 0.63 ± 0.13) in high-oxygen group (P ＜ 0.05) ; Smad4 and Smad7 was main in visible lung epithelial cells and interstitial fibroblasts.Smad4 expression in the high-oxygen group gradually enhanced,compared with the air group (P ＜ 0.05) on day 7 (122.35 ± 10.3 vs 140.08 ±7.77) and day 14(129.7 ± 7.33 vs 144.99 ± 6.49).Smad7 expression in the high-oxygen group first increased and then decreased,expression in the high-oxygen group increased on day 7 (122.35 ± 10.29 vs 130.56 ±9.8),and compared decreased with the air group(P ＜0.05) on day 14(132.16 ±4.38 vs 126.22 ±6.49).Conclusion The newborn rat exposed hyperoxia,the up-regulation of Smad4 protein expression and the down-regulation of Smad7 protein expression are imposible closely related to the happen and development of CLD pulmonary fibrosis.
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ObjectiveTo investigate the effect of different dosage recombinant human erythropoietin(rhEPO),an angiogenesis-like factor,on pulmonary angiogenesis exposed to hyperoxia in newborn rats.MethodsSixty Sprague-Dawley newborn rats were randomly divided into four groups:air group (room air exposure,n =15 ),hyperoxia group ( exposed to 95％ oxygen,n =15 ),hyperoxia + large dosage rhEPO group (received rhEPO 5000 U/kg,intraperitoneally on 1 hour before and 3 days after exposed to hyperoxia,n =15) and hyperoxia + small dosage rhEPO group (received rhEPO 800U/kg,the same time points,n =15 ).The isodose of saline were given intraperitoneally on the same time points in the air group and the hyperoxia group.After 6 d of exposure,the survival rate was compared,CD31 and vascular endothelial growth factor (VEGF) were measured by immunohistochemistry to assess hyperoxia-induced changes in lung morphology.ResultsAfter 6d of exposure,hyperoxia + large dosage rhEPO group prolonged the survival rate in comparison with the hyperoxia group [ 86.7 ％ ( 13/15 ) vs 60.0 ％ ( 9/15 ) ].The expression of lung CD31 [ ( 38.69 ±1.69)％ vs (33.57±4.12)％,P＜0.05] and VEGF (124.4296±7.2823 vs 114.2059 ±-8.345 7,P＜0.05) in newborn rats treated with large dosage of rhEPO was significantly higher than those in hyperoxia group.While there was no significant difference of CD31 [ ( 36.34 ± 1.89 ) ％ ] and VEGF( 115.429 6 ± 6.719 9) in small dosage rhEPO group compared with the hyperoxia group (P＞0.05 ).ConclusionInstead of treatment with small dosage rhEPO (800 U/kg),large dosage rhEPO (5000 U/kg) may have important protective effects on pulmonary angiogenesis in hyperoxia-induced lung injury of newborn rats.