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Anti-apoptosis effect of recombinant human interleukin-11 in neonatal hypoxic-ischemic rats through activating the IL-11Rα/STAT3 signaling pathway

Open AccessPublished:December 13, 2022DOI:https://doi.org/10.1016/j.jstrokecerebrovasdis.2022.106923

      Highlights

      • In this study, we focused on effect of recombinant human interleukin-11(rhIL-11) on Neonatal hypoxic ischemic encephalopathy (HIE).
      • And also,we try to explore the mechanism of the rhIL-11.
      • We found that rhIL-11 reduces neuronal apoptosis by activating the brain IL-11Rα/STAT3 pathway.
      • Our results may provide a basis for other researchers to find new synergistic treatments for HIE.

      Abstract

      Hypoxia-ischemia (HI) is one of the most common causes of death and disability in neonates. Apoptosis contributes to HI development. Interleukin-11(IL-11) has been shown to protect mice from cerebral ischemia/reperfusion injury. However, whether IL-11 exerts the anti-apoptotic effect on HI injury is unclear. In this study, we demonstrated that recombinant human IL-11 (rhIL-11) prevented apoptosis of rat neonates with HI through activating IL-11Rα/STAT3 signaling. Sprague-Dawley rat pups on the 7th day after birth were used to establish an HI injury model. The expression levels of IL-11Rα and GP130 were increased first and then decreased after HI. In contrast, IL-11 expression was first decreased and then increased. Immunofluorescence staining showed that IL-11Rα was localized in neurons and oligodendrocytes. RhIL-11 treatment alleviated hippocampal and cortical damages, significantly reduced cerebral infarction volumes, cerebral edema, and loss of the Nissl body and nerve cells, and also ameliorated the outcomes of HI injury and long-term neurological deficits. In addition, rhIL-11 treatment upregulated the expressions levels of Bcl-2 and p-STAT3/STAT3, and downregulated the protein concentrations of the lytic protease, and cleaved-caspase-3. Furthermore, GP130 inhibitor and JAK1 inhibitor reversed the protective effects of rhIL-11. Overall, rhIL-11 showed an anti-apoptosis effect on the brain after HI injury. Our results indicated that rhIL-11 reduced neuronal apoptosis by activating the brain IL-11Rα/STAT3 pathway.

      Key Words

      Introduction

      Hypoxic ischemia (HI) leads to severe brain injury during the neonatal period, which mainly causes neuron and white matter damage.
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      Interleukin-11 (IL-11) belongs to the IL-6 family, also called GP130 cytokine.
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      IL-11 is an anti-inflammatory factor.

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      In vitro studies showed that rhIL-11 inhibited the production of tumor necrosis factor α(TNF-α) and IL-1β. All ligands of the IL-6 family cytokines have cytokine-specific receptors, usually called α receptors that are expressed in the brain.
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      However, the protective mechanism of IL-11 remains unclear. IL-11 has been shown to promote chemotherapy resistance of lung adenocarcinoma cells by activating the IL-11R/JAK/STAT3 anti-apoptotic signaling pathway.
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      JAK1 is necessary for IL-11 and its receptor complex to activate STAT3.

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      Another study showed that IL-11 initiated anti-apoptotic signals by activating the JAK1-STAT3 pathway. Tyrosine phosphorylation (Tyr1022/Tyr1023) of JAK1 and phosphorylation (Tyr705) of STAT3 are the activation markers of the JAK1/STAT3 signaling pathway.
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      STAT3 activation has been shown to upregulate Bcl-2 expression, an anti-apoptotic member of the Bcl family,

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      and reduce neuronal apoptosis.
      In the present study, we hypothesized that rhIL-11 activated the IL-11Rα/STAT3 pathway to produce anti-apoptotic proteins and reduce activation of the lytic protein, caspase-3, thereby reducing the apoptosis of neurons, which could provide a neuroprotective effect after HI.

      Results

      Expression of IL-11Rα, GP130, and IL-11 in the brain after HI

      IL-11Rα expression was decreased shortly after HI, then began to rise at 12 h, and reached a peak at 24 h. There was a significant difference compared with the sham group at 24 h. GP130 protein and IL-11Rα exhibited a similar pattern. Compared with the sham group, endogenous IL-11 protein levels were increased at 6 h after HI, then began to decline and reach its lowest point at 12 h, but gradually increased from 24 h to 72 h. These results are shown in Table 1 and Fig. 1.
      Table 1Expression of IL-11Rα, GP130, and IL-11 in the brain after HI.
      GroupsIL-11RαGP130IL-11
      Sham0.64±0.100.75±0.060.53±0.03
      6h0.57±0.140.64±0.050.81±0.04
      p< 0.01 compared with the Sham group.
      12h0.77±0.100.80±0.050.63±0.08
      24h1.01±0.07
      p< 0.05.
      0.91±0.04
      p< 0.05.
      0.71±0.08
      48h0.80±0.090.78±0.030.72±0.05
      72h0.74±0.100.66±0.050.83±0.10
      p< 0.01 compared with the Sham group.
      Note: Data are presented as the mean ±SEM(n=6).
      low asterisk p< 0.05.
      low asterisklow asterisk p< 0.01 compared with the Sham group.
      Fig 1
      Fig. 1Endogenous expression of interleukin-11 receptor alpha (IL-11Rα), glycoprotein 130(GP130) and interleukin-11(IL-11) in the neonatal brain after hypoxic-ischemic (HI) injure. (A) Representative western blot bands of IL-11Rα; IL-11Rα increased from 12h to 24h post HI then dropped, and significantly different at 24h (p< 0.01); (B) Representative western blot bands of GP130 that exhibited a similar pattern with IL-11Rα; (C) Representative western blot bands of IL-11; After HI, IL-11 protein tended to decrease first and then increase, with the lowest point at 12h. Data are mean ± SEM. *p < 0.05, **p < 0.01 vs sham. n = 6, one-way ANOVA followed by the LSD test.

      Location and distribution of IL-11Rα

      Immunofluorescent (IF) staining showed that IL-11Rα was expressed on neurons and oligodendrocytes in the sham and HI 24 h groups in different areas of the brain. Additionally, IL-11Rα was abundantly expressed in the cell membranes and cytoplasm (Fig. 2A). The immunofluorescence intensities of IL-11Rα in neurons and oligodendrocytes were increased after HI (Fig. 2B).
      Fig 2
      Fig. 2(A) Location and distribution of interleukin-11 receptor alpha(IL-11Rα) on neurons and oligodendrocytes at 24 h post-hypoxic ischemic encephalopathy using double immunofluorescence staining. In the sham group and the HI 24h group, IL-11Rα was localized in neurons and oliodendrocytes in different brain regions, and was mainly expressed in the cell membrane and cytoplasm. Blue light is nucleus; IL-11Rα (red)+NeuN (green), IL-11Rα (red)+MBP (green) in the figure. Red arrow: IL-11Rα; n=3. Scale bar = 20μm. (B) Quantifying of IL-11Rα fluorescence intensity in neurons and oligodendrocytes per group using ImageJ software. *p< 0.05, **p < 0.01 vs Sham.

      Intraventricular administration of rhIL-11 improved short-term neurobehavior and decreased infarct volumes and brain water content 48 h after hypoxic ischemic encephalopathy

      Exogenous rhIL-11 (0.45 µg/kg, 0.9 µg/kg, and 1.8 µg/kg) was administered by intraventricular injection at 1 h after HI. The medium and high doses of rhIL-11 improved the neurological deficits of animals at 48 h after HI (Fig. 3A–C). Furthermore, infarct volumes of the medium and high dose groups were smaller than those of the HI group (P<0.05). However, there was no significant difference between the HI group and the low dose group (P>0.05). High doses of rhIL-11 reduced brain water content relative to the HI group, but the low and medium dose groups showed no significant effect (Fig. 3D–F). Table 2 shows the effect of rhIL-11 on short-term neurological deficits in HI neonatal rats, and Table 3 shows the effect of rhIL-11 on infarct volume and brain water content in HI neonatal rats.
      Fig 3
      Fig. 3Effects of recombinant human interleukin-11 on infarct volume, brain water content, and short-term neurobehavior at 48 h after hypoxic ischemic encephalopathy. Illustrates that rhIL-11 improves short-term neurobehavior: (A)Righting reflex, (B)Negative Geotaxis test and (C)Neurological scores; (D) Brain water content; (E) Representative sections of TTC staining in the hypoxic-ischemic brain in sham, HI, and HI treated with rhIL-11 0.45 µg/kg, 0.9 µg/kg and 1.8 µg/kg;(F) Quantification of infarct volume showed rhIL-11 treatment significantly reduce infarct volume; Data are mean ± SEM. *p< 0.05, **p < 0.01 vs Sham, #p< 0.05, ##p< 0.01 vs HI, n = 6, Neurological scores are analyzed by Kruskal-Wallis test; the other four indicators are used for one-way ANOVA followed by the LSD test.
      Table 2Effects of rhIL-11 on short term neurological deficits in HI neonatal rats.
      GroupsRighting reflex (s)Negative geotaxis (s)Neurological score
      Sham1.56±0.107.36±0.500.70±0.21
      HI4.32±0.66
      p < 0.01 compared with the Sham group.
      13.74±0.84
      p < 0.01 compared with the Sham group.
      2.90±0.28
      p < 0.01 compared with the Sham group.
      rhIL-11(0.45 μg/kg)4.86±0.3914.32±1.262.70±0.26
      rhIL-11(0.9 μg/kg)2.20±0.37
      p < 0.01 compared with the HI group.
      11.11±0.78
      p < 0.05 or.
      1.70±0.26
      p < 0.05 or.
      rhIL-11(1.8 μg/kg)2.11±0.23
      p < 0.01 compared with the HI group.
      10.57±0.82
      p < 0.05 or.
      1.70±0.21
      p < 0.05 or.
      Note: Data are presented as the mean ±SEM(n=6).
      low asterisklow asterisk p < 0.01 compared with the Sham group.
      # p < 0.05 or.
      ## p < 0.01 compared with the HI group.
      Table 3Effects of rhIL-11 on infarct volume and brain water content in HI neonatal rats.
      GroupsInfarct volume (%)Brain water content (%)
      Sham0.00±0.0087.69±0.07
      HI34.51±1.71
      p < 0.01 compared with the Sham group.
      88.62±0.08
      p < 0.01 compared with the Sham group.
      rhIL-11(0.45 μg/kg)32.74±2.5488.68±0.14
      rhIL-11(0.9 μg/kg)25.87±0.97
      p < 0.01 compared with the HI group.
      88.42±0.11
      rhIL-11(1.8 μg/kg)26.26±2.16
      p < 0.01 compared with the HI group.
      88.18±0.05
      p < 0.01 compared with the HI group.
      Note: Data are presented as the mean ±SEM (n=6).
      low asterisklow asterisk p < 0.01 compared with the Sham group.
      ## p < 0.01 compared with the HI group.

      The rhIL-11 reduced neuropathic damage after HI

      After 48 h of HI, hematoxylin and eosin (H&E) staining showed that the brain injury of the model group was more severe than that of the sham group. After HI, there were multiple damaged cells in the hippocampal dentate gyrus, and the nuclei were pyknotic and condensed. The ischemic frontotemporal cortex showed tissue damage. Medium and high doses of rhIL-11 reduced the numbers of abnormal damaged cells in the dentate gyrus and frontal temporal cortex, reduced the loss of brain tissue and necrosis of nerve cells in the right brain, and overall improved the morphology and arrangement of nerve cells. There was no abnormality in the sham group (Fig. 4A).
      Fig 4
      Fig. 4Effect of recombinant human interleukin-11 on pathological morphology, Nissl substance in brain tissue of newborn rats at 48 h after hypoxic ischemic encephalopathy. (A) Representative images of H&E staining displays that rhIL-11 improves pathological morphology. Red arrow: the dentate gyrus(DG) of the hippocampus. Yellow arrow: irregular atrophy of neurons in neonatal rat cerebral cortex. Scale bar = 1000/200/20μm. (B) Representative pictures of Nissl staining shows high-dose rhIL-11 significantly reduces the loss of Nissl bodies in the CA1, CA3 and DG regions of hippocampus and cerebral cortex, and is dose-dependent; n = 3, Scale bar = 1000/200μm.
      Brain histopathology and morphology of neonatal rats at 48 h after HI are shown in Fig. 4. Nissl bodies in the CA1, CA3, and DG regions of the hippocampus and cortex were almost all lost in the HI group, and only small amounts of Nissl bodies were stained in the low dose group. In the medium dose group, the CA1, CA3, and DG regions and cortex were partially lost; in the high dose group, a small amount of Nissl bodies was lost in the CA3 regions (Fig. 4B). To examine the effect of rhIL-11 on neuronal apoptosis in the brain, TUNEL staining was performed at 48 h after HI. In the TUNEL assay, green fluorescent spots represent apoptotic cells. After HI, apoptotic cells were mainly distributed in the hippocampus and cerebral cortex, and the density of apoptotic cells was greatly increased in the HI group, while rhIL-11 treatment at both medium and high dose significantly reduced apoptotic cells relative to the HI group. Especially, rhIL-11 at a high dose remarkably decreased the numbers of apoptotic cells (Fig. 5).
      Fig 5
      Fig. 5Effect of recombinant human interleukin-11 on nerve cells about TUNEL staining in brain tissue of newborn rats at 48 h after hypoxic ischemic encephalopathy. Representative pictures of TUNEL staining shows rhIL-11 reduce fluorescence intensity of TUNEL positive cells in cerebral cortex and hippocampus. And the high dose is the most significant. n = 3, Scale bar = 1000/200μm.

      The rhIL-11 treatment ameliorated long-term neurological dysfunction at 4 weeks post-HI induction

      The results of a Morris water maze showed that rats in the HI group had spatial memory loss, which was evaluated by measuring the latency and the number of effective wear stages in the probe trials.
      The HI group had a significantly longer escape latency to find a platform than the sham group in the place navigation trial. The rhIL-11 treatment improved memory and learning abilities (Fig. 6A-C). Representative images of brain tissue morphology at 4 weeks after HI showed that there were vacuoles in the HI group, and almost all of the right hippo-campus was largely shrunken, while rhIL-11 treatment partially but obviously restored right hippocampal structures (Fig. 6D). Table 4 shows the effects of rhIL-11 on long-term neurological outcomes of rats at 4 weeks post-HI.
      Fig 6
      Fig. 6Effects of recombinant human interleukin-11 on long-term neurological outcomes and brain pathology in rats 4 weeks after induction of hypoxic-ischemic encephalopathy. Intraventricular administration of rhIL-11 ameliorated long term neurological deficits. (A) Tendency chart of escape latency in rats during place navigation; (B) The number of times the rat crosses the platform and (C)effective area in the probe trail; (D) Representative images of HE staining displays that rhIL-11 improves pathological morphology at 4w post HI (n=3). Data are mean ± SEM, *p < 0.05, **p < 0.01 vs Sham, n = 6, About the Morris water maze is analyzed by Kruskal-Wallis test.
      Table 4Effects of rhIL-11 on long-term neurological outcomes of rat at 4 weeks post HI.
      GroupsLatency(s)Platform crossover numberEffective area crossover number
      Day 2Day 3Day 4Day 5
      Sham72.55±11.6735.88±7.9040.48±5.0218.68±3.713.33±0.565.33±0.76
      HI109.00±5.3389.43±9.8757.35±10.7157.43±5.960.33±0.21
      p < 0.05 compared with the Sham group.
      2.00±0.58
      p < 0.05.
      rhIL-11(1.8 µg/kg)102.45±10.5061.77±13.5459.99±17.7548.83±5.312.33±0.67
      p < 0.05 compared with the HI group.
      4.83±1.05
      p < 0.05 compared with the HI group.
      Note: Data are presented as the mean ±SEM (n=6).
      low asterisk p < 0.05.
      low asterisklow asterisk p < 0.05 compared with the Sham group.
      # p < 0.05 compared with the HI group.

      Effects of bazedoxifene (GP130 inhibitor) and ruxolitinib (JAK1 inhibitor) on the IL-11Rα /STAT3 signaling pathway at 48 h after HI

      The expression levels of p-STAT3/β-actin (Fig. 7B), p-STAT3/STAT3 (Fig. 7C), cleaved-caspase3/β-actin (Fig. 7F), and cleaved-caspase3/procaspase3 (Fig. 7G) were up-regulated, while Bcl-2 (Fig. 7E) expression was decreased in the HI group. The rhIL-11 treatment upregulated the expression levels of p-STAT3/STAT3 and anti-apoptotic protein Bcl-2 were further increased; in contrast, it downregulated the expression of cleaved-caspase3/procaspase3. Both bazedoxifene (GP130 inhibitor) and ruxolitinib (JAK1 inhibitor) impaired rhIL-11 upregulated p-STAT3/STAT3 and Bcl-2(Fig. 7E), and downregulated cleaved-caspase3/procaspase3 (Fig. 7G). Table 5 shows corresponding protein expression changes in the IL-11R/STAT3 pathway.
      Fig 7
      Fig. 7Effects of bazedoxifene and ruxolitinib on the IL-11Rα /STAT3 pathway at 48 hours after hypoxic-ischemic encephalopathy in-duction. (A)∼(C) Representative western blots bands, p-STAT3/β-Actin and p-STAT3/STAT3 in the IL-11Rα /STAT3 signaling pathway. The data was normalized to the expression level of β-actin, which was used as a loading control. (D)∼(G). Representative western blots bands apoptotic proteins, Bcl-2/β-Actin, cleaved-caspase3/β-Actin and cleaved-caspase3/procaspase3. Data are mean ± SEM, *p < 0.05, **p < 0.01 vs Sham, #p < 0.05, ##p < 0.01 vs HI, $p < 0.05, $$p < 0.01 vs rhIL-11, n = 6, one-way ANOVA followed by the LSD test.
      Table 5Corresponding protein expression changes in IL-11Rα/STAT3 pathway.
      GroupsP-STAT3 / β-actinP-STAT3 / STAT3Bcl-2 / β-actincleaved-caspase3/β-actincleaved-caspase3/pro-caspase3
      Sham0.38±0.050.71±0.080.75±0.040.60±0.100.56±0.05
      HI0.73±0.09
      p< 0.01 vs Sham group.
      1.02±0.06
      p< 0.05.
      0.59±0.041.07±0.131.10±0.17
      rhIL-110.99±0.09
      p< 0.01 vs Sham group.
      ,
      p< 0.05,##p< 0.01 vs HI group.
      1.39±0.17
      p< 0.01 vs Sham group.
      ,
      p< 0.05,##p< 0.01 vs HI group.
      0.96±0.10
      p< 0.05,##p< 0.01 vs HI group.
      0.74±0.120.76±0.15
      rhIL-11+Bazedoxifene0.68±0.06
      p< 0.01 vs rhIL-11 group.
      0.90±0.07
      p< 0.01 vs rhIL-11 group.
      0.76±0.151.47±0.35
      p < 0.05
      1.23±0.26
      rhIL-11+Ruxolitinib0.50±0.04
      p< 0.01 vs rhIL-11 group.
      0.77±0.08
      p< 0.01 vs rhIL-11 group.
      0.71±0.050.79±0.151.31±0.33
      Note: Data are mean ± SEM (n=6).
      low asterisk p< 0.05.
      low asterisklow asterisk p< 0.01 vs Sham group.
      # p< 0.05,##p< 0.01 vs HI group.
      $ p < 0.05
      $$ p< 0.01 vs rhIL-11 group.

      Discussion

      In this study, we reported that rhIL-11 could improve short-term outcomes and long-term outcomes of neonatal rats with HI. The protein expression levels of endogenous IL-11Rα and GP130 expression were decreased shortly after HI, then began to rise at 12 h, and reached a peak at 24 h. After rhIL-11 treatment, the expression levels of p-STAT3/STAT3 and anti-apoptotic protein Bcl-2 were further increased. Inhibiting JAK1/STAT3 signaling by bazedoxifene or ruxolitinib abrogated rhIL-11 upregulated p-STAT3/STAT3 and Bcl-2 expression.
      In the early stage after HI, diffuse swelling of brain tissue was found. Over time from 0 h to72 h, brain edema gradually reached a peak and was gradually absorbed. Cytotoxic cerebral edema was the most important pathological change in the early stage of HI injury. In the acute phase, the decreased cerebral blood flow reduced the delivery of oxygen and glucose to the brain, leading to anaerobic metabolism. As a result, production of adenosine triphosphate was decreased, while the production of lactic acid was increased, leading to brain edema. Therefore, evaluating cerebral edema was very important for the treatment of the disease[24, 25]. Six different time points contributed to our understanding the process of brain edema.
      In this study, the level of endogenous IL-11 protein was increased at 6 h and then began to be decreased. It reached its lowest point at 12 h. This is similar to the results of previous studies.

      Genet GF, Bentzer P, Ostrowski SR. et al., Resuscitation with Pooled and Pathogen-Reduced Plasma Attenuates the Increase in Brain Water Content following Traumatic Brain Injury and Hemorrhagic Shock in Rats. J Neurotrauma, 2017.34(5):1054–1062.

      ,

      Douglas-Escobar M. and Weiss MD., Hypoxic-ischemic encephalopathy: a review for the clinician. JAMA Pediatr, 2015.169(4):397–403.

      Therefore, we speculated that cerebral HI injury caused the release of inflammatory factors, and also produced anti-inflammatory factors for self-defense.

      Priyadarshini S. and Aich P., Effects of psychological stress on innate immunity and metabolism in humans: a systematic analysis. PLoS One, 2012.7(9):e43232.

      Studies have shown that GP130 mRNA was expressed in neurons at the pyramidal cell layer and granulosa cell layer in animals, and was upregulated after transient forebrain ischemia.

      Choi JS, Kim SY, Cha JH. et al., Upregulation of gp130 and STAT3 activation in the rat hippocampus following transient forebrain ischemia. Glia, 2003.41(3):237–46.

      To determine the presence and expression of the two receptors targeted by IL-11 in the brain, we examined the time course expression of endogenous IL-11Rα and GP130 after HI. Our results showed that both of them were gradually increased after 6 h, and began to decrease after reaching a peak at 24 h. This was consistent with previous research.

      Choi JS, Kim SY, Cha JH. et al., Upregulation of gp130 and STAT3 activation in the rat hippocampus following transient forebrain ischemia. Glia, 2003.41(3):237–46.

      We confirmed localization and expression of IL-11Rα on neurons in HI injury models. IL-11Rα was indeed expressed in the oligodendrocytes of neonatal rats and located in the cell membrane and cytoplasm, which was consistent with previous studies[28]. Tao et al. confirmed that IL-11Rα was primarily expressed in the cell membrane and cytoplasm of cancer cells.
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      • Song H.
      • Chen L.
      Cancer-associated fibroblasts treated with cisplatin facilitates chemoresistance of lung adenocarcinoma through IL-11/IL-11R/STAT3 signaling pathway.
      Some studies showed that IL-11Rα was highly expressed in brain tissues of adult C57BL/6J mice,

      Davidson AJ, Freeman SA, Crosier KE. et al., Expression of murine interleukin 11 and its receptor alpha-chain in adult and embryonic tissues. Stem Cells, 1997.15(2):119–24.

      and in oligoendrocytes with various morphologies in the brain.
      • Zhang Y.
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      • John G.R.
      Interleukin-11 potentiates oligodendrocyte survival and maturation, and myelin formation.
      In addition, our IF staining showed that 24 h after HI, the numbers of neurons and oligodendrocytes were significantly decreased.
      A dose-dependent experiment was performed to identify the optimal dosage of exogenous IL-11. Studies have shown that intracerebroventricular injection of rhIL-11 into rat brains caused significant changes in biochemical indicators at doses ranging from 13.5 to 250 ng,

      Lopez–Valpuesta FJ. and Myers RD. Fever produced by interleukin-11 (IL-11) injected into the anterior hypothalamic preoptic area of the rat is antagonized by indomethacin. Neuropharmacology, 1994.33(8):989–94.

      ,

      Gourine, A.; Rudolph, K.; Leon, L.; Kluger, M. J. N., Effect of interleukin-11 on body temperature in afebrile and febrile rats. 2000, 8, (1), 8-12.

      therefore we selected low, medium, and high doses of rhIL-11.We confirmed that the medium and high doses alleviated the damage of the brain. Previous studies have shown that IL-11 reduced the infarct volume of MCAO
      • Zhang B.
      • Zhang H.X.
      • Shi S.T.
      • Bai Y.L.
      • Zhe X.
      • Zhang S.J.
      • Li Y.J.
      Interleukin-11 treatment protected against cerebral ischemia/reperfusion injury.
      and ischemia/reperfusion injury models.
      • Obana M.
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      • Fujio Y.
      Therapeutic administration of IL-11 exhibits the postconditioning effects against ischemia-reperfusion injury via STAT3 in the heart.
      Therefore, we think that rhIL-11 is a good choice to treat HI injury.
      We found the pathological changes of the HI injury of newborn rats using H&E staining, Nissl staining, and the TUNEL staining assay. After neurons suffered hypoxic-ischemic damage, the Nissl bodies were significantly reduced. However, rhIL-11 alleviated the decrease in the number of neurons after HI. Previous studies also confirmed that the hippocampus was the most sensitive to HI injury. The hippocampus and related cortices are brain regions closely related to cognitive and memory functions[19].
      Our results showed after rhIL-11 stimulation, STAT3 phosphorylation and its downstream target Bcl-2 were increased, while the expression of the activated cleavage protease, caspase3 was reduced. In addition, to determine whether the increase in p-STAT3/STAT3 was due to rhIL-11, we used the inhibitor, bazedoxifene, which is a specific inhibitor of the membrane receptor, GP130.
      • Wei J.
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      • Zhang R.
      • Li H.
      • Li C.
      • Lin J.
      Bazedoxifene as a novel GP130 inhibitor for Colon Cancer therapy.
      The results showed that rhIL-11 significantly upregulated the levels of p-STAT3/STAT3, which was blunted by the addition of ruxolitinib (JAK1 inhibitor).
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      • Varon C.
      Leukaemia Inhibitory Factor (LIF) inhibits cancer stem cells tumorigenic properties through hippo kinases activation in gastric cancer.
      Our results indicated that rhIL-11 might induce phosphorylation of STAT3 and activate anti-apoptotic signals, thereby reducing neuronal apoptosis. Bazedoxifene as a specific inhibitor has been shown to cross the blood-brain barrier.
      • Hill R.A.
      • Kouremenos K.
      • Tull D.
      • Maggi A.
      • Schroeder A.
      • Gibbons A.
      • Kulkarni J.
      • Sundram S.
      • Du X.
      Bazedoxifene - a promising brain active SERM that crosses the blood brain barrier and enhances spatial memory.
      A study showed that ruxolitinib as a JAK1 inhibitor was used to regulate the JAK-STAT pathway during early brain injury after subarachnoid hemorrhage.
      • Yu L.
      • Liu Z.
      • He W.
      • Chen H.
      • Lai Z.
      • Duan Y.
      • Cao X.
      • Tao J.
      • Xu C.
      • Zhang Q.
      • Zhao Z.
      • Zhang J.
      Hydroxysafflor Yellow A confers neuroprotection from focal cerebral ischemia by modulating the crosstalk between JAK2/STAT3 and SOCS3 signaling pathways.
      Therefore, we think two inhibitors could be injected intraperitoneally. They could enter the brain to exert their effects after crossing the blood-brain barrier. Our results are consistent with studies showing that activation of STAT3 phosphorylation had neuroprotective effects.

      Zhou H, Zhang Z, Wei H. et al., Activation of STAT3 is involved in neuroprotection by electroacupuncture pretreatment via cannabinoid CB1 receptors in rats. Brain Res, 2013.1529:154–64.

      However, several studies have reported that inhibiting the STAT3 signaling pathway could protect the immature brain from HI injury.
      • D'Angelo B.
      • Ek C.J.
      • Sun Y.
      • Zhu C.
      • Sandberg M.
      • Mallard C.
      GSK3beta inhibition protects the immature brain from hypoxic-ischaemic insult via reduced STAT3 signalling.
      ,
      • Hristova M.
      • Rocha-Ferreira E.
      • Fontana X.
      • Thei L.
      • Buckle R.
      • Christou M.
      • Hompoonsup S.
      • Gostelow N.
      • Raivich G.
      • Peebles D.
      Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) reduces neonatal hypoxic-ischaemic brain damage.
      These contradictory results may be associated with different cell types, and the choice of intervention and drug treatments. As for our study, we found that inducing STAT3 to initiate the anti-apoptotic program might have a protective effect on the brain during the early stages of massive neuronal apoptosis during the HI.
      Our study was different from a previous study on the model and mechanism of interleukin-11 on cerebral ischemia reperfusion injury.
      • Zhang B.
      • Zhang H.X.
      • Shi S.T.
      • Bai Y.L.
      • Zhe X.
      • Zhang S.J.
      • Li Y.J.
      Interleukin-11 treatment protected against cerebral ischemia/reperfusion injury.
      In the present study, only localization of IL-11Rα in oligodendrocytes was verified, but the relevant biochemical indicators of the effect of IL-11 on the loss of oligodendrocytes were not detected, which will be confirmed in future experiments.
      There were some limitations in our study. First, we only focused on pathological changes of neurons after HI. We did not characterize changes in the number and morphology of oligodendrocytes and in white matter damage after administration. Second, we reported the relationship between the IL-11Rα/STAT3 signaling pathway and IL-11. However, there might be other signaling pathways involved.
      In summary, our study showed that rhIL-11 had a protective effect on the rat HI model via the IL-11Rα/STAT3 signaling pathway. The rhIL-11 could be a good choice to treatment HI injury of brain in the future.

      Materials and methods

      Animals

      In this study, a total of 180 neonatal Sprague-Dawley rat pups (12–18 g, 7-days-old) were collected. The mortality rate after HI injury was 8%. They were provided by the Animal Center of Guizhou Medical University. Female and male pups were evenly divided, and all animal-related procedures were approved by the Animal Care Welfare Committee.

      HI injury

      In this study, an improved Rice-Vannucci rat model was used to study neonatal hypoxic-ischemic encephalopathy.
      • Li B.
      • Dasgupta C.
      • Huang L.
      • Meng X.
      • Zhang L.
      MiRNA-210 induces microglial activation and regulates microglia-mediated neuroinflammation in neonatal hypoxic-ischemic encephalopathy.
      The 7-day-old infant rats were weighed and anesthetized with a R500IP small animal anesthesia machine, and the infant rats were fixed on the surgical heating pad with constant temperature. Deep anesthesia was given with 4−5% isoflurane, followed by 1−2% isoflurane for maintenance. The exposed neck was sterilized with iodine and alcohol. An incision of about 5 mm was made in the middle of the neck. After the right common carotid artery was found and dissociated, it was double ligated with sutures and a cut in the middle, and the incision was sutured and put back to the mother rat, followed by rest. After 1 h, hypoxic treatment was performed with 8% oxygen and 92% nitrogen in a 37°C constant temperature water bath for 2 h. When the hypoxia was over, the pups were returned to their mothers. In the sham operation group, only the right common carotid artery was exposed without ligation and hypoxia, and other steps were the same as in the model group.
      • Knox-Concepcion K.R.
      • Figueroa J.D.
      • Hartman R.E.
      • Li Y.
      • Zhang L.
      Repression of the glucocorticoid receptor increases hypoxic-ischemic brain injury in the male neonatal rat.

      Drug administration

      In the intracerebroventricular injection, the specific agonist of IL-11 receptor used in this study was recombinant human IL-11(rhIL11; Peprotech, Rocky Hill, NJ, USA). RhIL-11 was dissolved in 0.9% saline and administered at doses of 0.45 µg/kg, 0.90 µg/kg and 1.8 µg/kg, respectively. One hour after the HI model was established, rhIL-11 was administered via lateral ventricular injection using a stereotactic brain locator (RWD Life Science, China). The newborn rats were anesthetized and fixed on a stereotaxic instrument. The bregma was exposed by cutting an incision in the surface of the skull. With bregma as the coordinates at 1.5 mm lateral, 2 mm posterior, and 2.7 mm below the skull surface, 3 µL of the drug was administered to the lateral ventricle of the right cerebral hemisphere at 1 µL/min.
      • Feng Z.
      • Ye L.
      • Klebe D.
      • Ding Y.
      • Guo Z.N.
      • Flores J.J.
      • Yin C.
      • Tang J.
      • Zhang J.H.
      Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage.
      ,
      • Li Y.
      • Xiao D.
      • Dasgupta C.
      • Xiong F.
      • Tong W.
      • Yang S.
      • Zhang L.
      Perinatal nicotine exposure increases vulnerability of hypoxic-ischemic brain injury in neonatal rats: role of angiotensin II receptors.
      The HI group was injected with saline. After the drug was injected, the needle was left for 5 min, then removed, and the incision was sutured.
      To identify the mechanism of IL-11, the specific inhibitors of GP130 and JAK1 used in this study were bazedoxifene and ruxolitinib (MCE, San Rafael, CA, USA). Bazedoxifene acetate and ruxotinib phosphate were dissolved in 0.9% normal saline and intraperitoneally injected into neonatal rats at 3 mg/kg and 5.6 mg/kg, respectively, 1 h before treatment with rhIL-11.

      Experimental protocol

      In experiment I, the expression levels of IL-11Rα and GP130 were explored by western blots at six time points after HI. A total of 36 rats were randomly divided into the sham group (n =6) and HI injury group (n =30).
      In experiment II, the co-localization of IL-11Rα with NeuN and MBP were determined by double immunofluorescent staining in the sham (n =3) and HI groups (n =3) at 24 h.
      In experiment III, the effects of administration of exogenous IL-11 after HI injury were evaluated. Rats were divided into five groups: Sham (n = 12), HI (n = 12), HI+ rhIL-11 (0.45 µg/kg) (n = 12), HI + rhIL-11 (0.90 µg/kg) (n = 12), and HI+rhIL-11 (1.80 µg/kg) (n = 12). At 48 h post-HI, short-term neurobehavioral outcomes were examined. Furthermore, infarct volume and brain water content were measured at 48 h. The samples for short term neurobehavioral tests were shared from Experiment III.
      In experiment IV, the effects of rhIL-11 on pathological changes of the brain were studied, using H&E staining, Nissl staining, and TUNEL staining. The sections from the same brain tissues were used in these three types of staining procedures. Rats were divided into five groups: Sham (n = 3), HI (n = 3), HI+ rhIL-11 (0.45 µg/kg) (n = 3), HI+rhIL-11 (0.90 µg/kg) (n = 3), and HI +rhIL-11 (1.80 µg/kg) (n = 3).
      In experiment V, the long-term effects of rhIL-11 after HI, neurobehavioral function were performed at 4 weeks post-HI. Rats were divided into sham (n = 6), HI (n = 6), and HI + rhIL-11 (n = 6) groups. Then, three rats in each group were randomly selected for H&E staining.
      In experiment VI, the effects of bazedoxifene (GP130 inhibitor) and ruxolitinib (JAK1 inhibitor) on the IL-11Rα /STAT3 signaling pathway at 48 h after HI were determined. Rats were divided into Sham (n = 6), HI (n = 6), HI+rhIL-11 (n = 6), HI+ rhIL-11+Bazedoxifene (n = 6), and rhIL-11+Ruxolitinib (n = 6) groups.

      Western blotting

      The effects of HI on IL-11Rα and GP130 were assessed at sham (0 h, 9 am), 6 h (15 am), 12 h (21 am), 24 h (9 am), 48 h(9 am), and 72 h (9 am) after HI by western blotting as previously described.
      • Ye L.
      • Feng Z.
      • Doycheva D.
      • Malaguit J.
      • Dixon B.
      • Xu N.
      • Zhang J.H.
      • Tang J.
      CpG-ODN exerts a neuroprotective effect via the TLR9/pAMPK signaling pathway by activation of autophagy in a neonatal HIE rat model.
      The effects of rhIL-11 on the IL11Rα/STAT3 signaling pathway were determined using western blotting at 48 h post-HI. Six newborn rats were included at each time point. The brain tissue was removed and stored at -80°C. For western blotting, an appropriate amount of the right brain of these brain tissues was placed in a glass homogenizer, which contained the cerebral cortex, hippocampus, and brain stem, followed by addition of prechilled lysis buffer. After quantitation, an identical amount of protein sample (30 μg) was loaded on 8–15% SDS-polyacrylamide (SDS-PAGE gels for electrophoresis). The SDS-PAGE separated proteins were transferred to PVDF membranes, which were incubated with 5% milk for 1 h, then were incubated with antibodies to IL-11Rα (4D12) (1:200), GP130 (E-8) (1:100), IL-11 (A-9) (1:100), STAT3 (F-2) (1:200), p-STAT3 (B-7) (1:200), caspase-3 (CPP324-1-1) (1:200) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and to cleaved caspase-3 (1:1,000), and primary rabbit antibody to Bcl-2 (1:1,000) (ABclone, Cummings Park, MA, USA) for overnight, washed with washing buffer three times, then incubated with corresponding secondary antibody for 1 h, and washed with washing buffer three times. An enhanced chemiluminescence (ECL) kit (Beyotime, Haimen, China) was used to detect protein signals. Image J (National Institutes of Health, Bethesda, MD, USA) was used to analyze the relative densities of the bands, and the data was normalized to the expression level of β-actin, which was used as a loading control.

      Immunofluorescent (IF) staining

      Double-fluorescence labeling of IL-11Rα with NeuN and double-fluorescence labeling of IL-11Rα with myelin basic protein (MBP) were performed to localize the IL-11Rα protein on neurons and oligodendrocytes as previously described.
      • Wang X.
      • Li T.
      • Liu N.
      • Zhang H.
      • Zhao X.
      • Ma Y.
      Characterization of GLOD4 in leydig cells of tibetan sheep during different stages of maturity.
      At 24 h after HI, the rats were anesthetized, then PBS and paraformaldehyde were infused through the heart. The brains were taken and placed in formalin for fixation, subjected to standard histological processing, embedded in paraffin and cut into 4 μm tissue sections. Paraffin sections were deparaffinized to water: sections were sequentially soaked into xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and distilled water. Antigen repair was then performed. After the section was dried slightly, a circle was drawn around the tissue with a histochemical pen. PBS was removed by shaking and samples were blocked with 3% bovine serum albumin to prevent nonspecific binding, for 30 min. The slides were then incubated first in primary antibodies (diluted with PBS) for IL-11Rα (1:100; Santa Cruz Biotechnology), NeuN (1:200; Servicebio, Wuhan, China), and MBP (1:200; Servicebio) overnight at 4°C, then placed in a wet box containing a small amount of water. After subsequent incubation with Cy3-conjugated goat anti-mouse IgG(H+L) (1:300) and Alexa Fluor® 488-conjugated goat anti-Rabbit IgG (H+L) (1:400, Servicebio), the nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Servicebio). The sections were then sealed, and images were detected using fluorescence microscopy. The nucleus stained by DAPI was blue when excited by ultraviolet light, and positive expression was red or green light labeled with corresponding fluorescein, that is, the Alexa Fluor® 488-labeled cell emitted green light, and the CY3 labeled cells emitted red light.

      Short-term neurobehavioral tests

      At 48 h after HI, the righting reflex, negative geotaxis test results, and neurological severity score were measured. The experiment was divided into five groups: the sham group, HI group, rhIL-11 0.45 µg/kg, 0.9 µg/kg, and 1.8 µg/kg group (n=10 rats/group). For the righting reflex test, rat neonates on their backs were placed on the surface of the table, and the time was recorded when the animal reached the prone position (all four paws were on the ground). For the negative geotaxis test, rat neonates were placed head down on a 45° slope, and the time for the pups to recognize their position on the slope, rotate 180° and face the top of the slope was recorded.
      • Dixon B.J.
      • Chen D.
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      • Nowrangi D.
      • Zhang J.H.
      • Tang J.
      Intranasal administration of interferon beta attenuates neuronal apoptosis via the JAK1/STAT3/BCL-2 pathway in a rat model of neonatal hypoxic-ischemic encephalopathy.
      The neurological severity scoring was as the following: 0 points, normal; 1 point, inability to extend the front paw; 2 points, circled while crawling; 3 points, dumped while crawling; and 4 points, stayed still or lost consciousness.
      • Li C.
      • Mo Z.
      • Lei J.
      • Li H.
      • Fu R.
      • Huang Y.
      • Luo S.
      • Zhang L.
      Edaravone attenuates neuronal apoptosis in hypoxic-ischemic brain damage rat model via suppression of TRAIL signaling pathway.
      The score was positively correlated with the severity of brain damage, and evaluated by two non-investigators in a double-blind manner.

      Brain infarct staining and the brain water content assay

      At 48 h post HI, the newborn rats were euthanized. The brain was cut into serial coronal sections (2 mm thick), and soaked in a 2% 2,3,5-triphenyltetrazolium chloride monohydrate (TTC; Solarbio) solution about 5 min, washed with PBS, and photographed after being fixed for 24 h. The percentage of infarct volume of each slice was analyzed using ImageJ software.
      • Li N.
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      • Tang Y.
      • Hou Y.
      • Fan F.
      • Ai X.
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      • Wang X.
      • Zhang Y.
      Tibetan medicine Duoxuekang ameliorates hypobaric hypoxia-induced brain injury in mice by restoration of cerebrovascular function.
      After 48 h HI, the pups were euthanized. The brain tissue was taken, then the wet weight of the brain tissue was immediately determined. The weight of the brain dried at 100°C for 24 h was taken as the dry weight. The formula for the water content of brain tissue is: brain water content (%) = (wet weight-dry weight)/wet weight × 100%.
      • Dixon B.J.
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      • Zhang Y.
      • Flores J.
      • Malaguit J.
      • Nowrangi D.
      • Zhang J.H.
      • Tang J.
      Intranasal administration of interferon beta attenuates neuronal apoptosis via the JAK1/STAT3/BCL-2 pathway in a rat model of neonatal hypoxic-ischemic encephalopathy.

      Pathological evaluation of rat brain tissues

      H&E staining was conducted at 48 h and 4 weeks. The rats were anesthetized, then PBS and paraformaldehyde were infused through the heart. The brain tissues were fixed in 4% formalin, followed by dehydration with gradient ethanol and xylene as follows: 75% ethanol (12 h), 85% ethanol (12 h), 95% ethanol (12 h), anhydrous ethanol (2 h), anhydrous ethanol (2 h), xylene (0.5 h), and xylene for (0.25 h), then embedded after soaking in wax for 3 h. A slicer was then used to slice the coronal brain into 5 µm sections. The tissue was placed in a water bath at 45°C and then baked at 60°C. Next, sections of brain tissue were subjected to sequential dewaxing in xylene (I), xylene (II), 100% ethanol (I), 100% ethanol (II), 75% ethanol, and rinsed with tap water, then stained with H&E (G1003, Servicebio). Finally, the sections were dehydrated, mounted, and examined with a DS-U3 imaging system (Nikon Eclipse E100; Nikon, Tokyo, Japan).

      Nissl staining

      The steps were performed according to the Servicebio Toluidine Blue staining kit instructions. Nissl staining was used to observe the loss or survival of neurons in the cerebral cortex and hippocampus. Paraffin sections of brain tissue were subjected to sequential dewaxing, and rinsed with tap water. The brain slices were then treated with toluidine blue (G1032, Servicebio) for 2–5 min, and rinsed with tap water, followed by treatment with 1% glacial acetic acid. The degree of differentiation was controlled using a microscope. The sections were washed with tap water, then dried in an oven, followed by xylene treatment for 10 min, and sealing with neutral gum. When observed with a microscope, the Nissl bodies of brain tissue were dark blue with a light blue background.

      TUNEL staining

      Paraffin sections of the brain tissue were deparaffinized and rehydrated. Proteinase K working solution was added to incubate the sections at 37°C for antigen retrieval, then the sections were washed with PBS (pH 7.4). To permeabilize cell membranes, 0.1% Triton was used to cover the tissue, followed by incubation at room temperature for 20 min. After equilibrium at room temperature, the appropriate amount of TDT enzyme, dUTP, and buffer from the TUNEL kit (G1501, Servicebio) were used according to the number of slices and tissue sizes, then mixed at a 1:5:50 ratio. This mixture was added to the tissue sections, then the samples were placed in a flat wet box and incubated at 37°C for 2 h. DAPI was used to counterstain the nucleus. The sections were sealed, then observed using a fluorescence microscope.

      Guo J, Cao X, Hu X. et al., The anti-apoptotic, antioxidant and anti-inflammatory effects of curcumin on acrylamideinduced neurotoxicity in rats. BMC Pharmacol Toxicol, 2020.21(1):62.

      Morris water maze test

      To determine the spatial memory and learning ability of rats 4 weeks after HI, we conducted a water maze test with a WMT-100 Morris Water Maze Video Analysis System (TaiMeng, Chengdu, China).

      Morris, R.; Garrud, P.; Rawlins, J.; O'Keefe, J. J. N., Place navigation impaired in rats with hippocampal lesions. 1982, 297, (5868), 681-3.

      According to the four quadrants divided by the video tracking software, we placed the platform in the center of the first quadrant. On the first day of training, the rat was placed on a visible platform to assess its visual ability. For the next 4 days, the platform became invisible to the rat, then animals were placed into water from a different quadrant and allowed to swim freely for 120 s. Rats that found the target platform were allowed to rest on the platform for 10 s. If the rat could not find the platform within 120 s, the experimenter led the rats to the target platform for 10 s, and then carried out the next experiment. The average value of the incubation period of four training sessions was taken as the study result of the rats on a specific day. The platform was removed 24 h after the place navigation test. The rats were allowed to enter from the third quadrant and then swim freely for 120 s. The number of times the rats crossed the target platform and the effective area was recorded by the video tracking system.

      Statistical analysis

      The data were expressed as the mean ± SEM. Data were analyzed by one-way analysis of variance followed by the LSD test. The Kruskal-Wallis test was used if it did not follow a normal distribution. Graphpad Prism 8 software (GraphPad, San Diego, CA, USA) was used to analyze the data. A value of p< 0.05 was considered statistically significant.

      Author contributions

      Ding Zuo conceived, designed the experiments, carried out behavioral tests, western blot, immunohistochemistry, and wrote part of the manuscript. Qian Zheng wrote the manuscript. Mei Xiao, Xiao-ya Wang, Hui-xin Chen and Ying Xiong carried out TTC, HE, Nissl and TUNEL staining, behavioral tests. Jian-wei Xu and Qing Zhang conceived and participated in acquiring and analyzing the presented data. Lan Ye and Zhan-hui Feng conceived, designed, and coordinated the study, and contributed to the critical revision of the manuscript. All authors read and approved the final manuscript.

      Funding

      This research was funded by National Natural Science Foundation of China (No. 81960224, No. 81860248). Institutional Review Board Statement: he animal study protocol was approved by the GuiZhou Medical University the Animal Care Welfare Committee (protocol code NO.1900029, 03/01/2019).

      Conflicts of Interest

      The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

      Acknowledgments

      We thank Professor Qifang Zhang from Guizhou Medical University and International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.

      Appendix. Supplementary materials

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