Korean J Physiol Pharmacol 2021; 25(2): 97-109
Published online March 1, 2021 https://doi.org/10.4196/kjpp.2021.25.2.97
Copyright © Korean J Physiol Pharmacol.
Fan Yang1,#, Ya Li2,3,4,#, Xun Sheng5, and Yu Liu6,*
1Department of Clinical Nutrition, The First People’s Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology, 2Department of Clinical Laboratory, The First Affiliated Hospital of Kunming Medical University, 3Yunnan Institute of Laboratory Diagnosis, 4Yunnan Key Laboratory of Laboratory Medicine, 5School of Stomatology, Kunming Medical University, 6Department of Pharmacy, The First People’s Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology, Kunming 650032, Yunnan, China
Correspondence to:Yu Liu
E-mail: chubashi4@163.com
#These authors contributed equally to this work
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Neonatal hypoxia/ischemia (H/I), injures white matter, results in neuronal loss, disturbs myelin formation, and neural network development. Neuroinflammation and oxidative stress have been reported in neonatal hypoxic brain injuries. We investigated whether Paeoniflorin treatment reduced H/I-induced inflammation and oxidative stress and improved white matter integrity in a neonatal rodent model. Seven-day old Sprague–Dawley pups were exposed to H/I. Paeoniflorin (6.25, 12.5, or 25 mg/kg body weight) was administered every day via oral gavage from postpartum day 3 (P3) to P14, and an hour before induction of H/I. Pups were sacrificed 24 h (P8) and 72 h (P10) following H/I. Paeoniflorin reduced the apoptosis of neurons and attenuated cerebral infarct volume. Elevated expression of cleaved caspase-3 and Bad were regulated. Paeoniflorin decreased oxidative stress by lowering levels of malondialdehyde and reactive oxygen species generation and while, and it enhanced glutathione content. Microglial activation and the TLR4/NF-κB signaling were significantly down-regulated. The degree of inflammatory mediators (interleukin 1β and tumor necrosis factor-α) were reduced. Paeoniflorin markedly prevented white matter injury via improving expression of myelin binding protein and increasing O1-positive olidgodendrocyte and O4-positive oligodendrocyte counts. The present investigation demonstrates the potent protective efficiency of paeoniflorin supplementation against H/I-induced brain injury by effectually preventing neuronal loss, microglial activation, and white matter injury via reducing oxidative stress and inflammatory pathways.
Keywords: Brain injury, Hypoxia, Neuroinflammation, Paeoniflorin, TLR4/NF-κB signaling
Hypoxia/ischemia (H/I) induced brain injury due to lack or insufficient supply of glucose and oxygen to the brain is leading causes of neonatal morbidity and mortality with a higher occurrence rate of 2–6 per 1,000 births [1]. Neonatal H/I results in white matter injury, neuronal cell loss, impaired neuronal function due to failure of neural network subsequently leading to neurological impairments [2,3] as motor and cognitive disabilities, epilepsy, and autonomic dysfunction [4-6]. White matter is found to be highly vulnerable to injury following H/I insult. H/I-induced loss of oligodendroglia, axonal damage, and impaired myelin formation have been reported in the pathogenesis of cerebral palsy and neurobehavioral deficits [7-10].
OPCs of the developing brain are predominantly liable to H/I insults [11,12]. Loss of early OPCs and failure to differentiate to mature OPCs disrupt myelination in the immature brain [12]. Developing brain is extremely susceptible to oxidative stress due to increased rate of oxygen utilization and low levels of endogenous antioxidant defense and high amounts of fatty acids [13,14]. Studies have suggested oxidative stress significantly contributes to the loss of early OPCs [12,15].
Neuroinflammatory responses, characterized by activation of microglial cells, have been observed in neonatal hypoxic brain injuries [16,17]. Accumulating experimental data reveals that neuroinflammatory processes are critically involved in H/I-induced loss of early OPCs in developing brain [18-20]. The microglial cells are involved in the regulation of proliferation of neurons and oligodendrocytes [21,22]. Toll-like receptor 4 (TLR4) is expressed on microglia and mediates neuroinflammatory diseases [23]. Increased expression of TLR4 in microglia following hypoxic insult has also been reported
Paeoniflorin, monoterpene glucoside, is one of the primary bioactive compounds of the plant,
Isoflurane and paeoniflorin was obtained from Sigma-Aldrich, St. Louis, MO, USA. Antibodies against cleaved caspase-3, TLR4, TNF-α, Bad, Bcl-2, β-actin and hypoxia-inducible factor-1 alpha (HIF-1α) (Cell Signaling Technology, Beverly, MA, USA). For expression analysis NF-κBp65, IκBα, p-IκBα, myelin binding protein (MBP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), were utilized. Most other reagents and chemicals were purchased from Sigma-Aldrich, St. Louis, MO, USA indicated otherwise.
Female, pregnant Sprague Dawley rats (n = 25) were obtained from the Institution Animal breeding center and were kept separately in sterile polypropylene rat cages under controlled animal house conditions (temperature 22°C–23°C; relative humidity 55%–60%; 12 h/12 h day-night cycle). The rats were strictly observed for pups to be born and the day of birth was noted as postnatal day 0 (P0). The experimental design and the procedures were endorsed by the Institutional ethical committee, and care and handling of the animals were in strict adherence to the guidelines issued by the National Institutes of Health on the care and use of animals [31].
P3 pups were randomly assigned to study groups (6 groups; n = 24/group). Group I - control; Group II - H/I control; Group III to V - H/I induced and treated with paeoniflorin at 6.25 mg/kg body weight (b.wt), 12.5 mg/kg b.wt and 25 mg/kg b.wt respectively. Group VI pups were not exposed to H/I insult but were treated with 25 mg/kg b.wt paeoniflorin.
H/I was induced in P7 pups as described earlier by Rice
P3 pups were administered paeoniflorin (6.25, 12.5, 25 mg/kg b.wt), orally
The rat pups were sacrificed on P8 (n = 6/group), P10 (n = 6/group) and on P14 (n = 6/group) under isoflurane anaesthesia by transcardial perfusion of saline-heparin tailed by ice-cold paraformaldehyde solution in 0.1 M PBS. Brains were excised immediately and used for analysis.
Neurobehavioral deficits were assessed in H/I induced rats on P8, P14 and P21. The extent of the neurological deficit was graded based on parameters as - symmetry of movements, symmetry of the forelimbs, spontaneous activity, climbing, touch response, and vibrissae touch response. The behaviour of animals was graded on a scale between 0–4. The animals were scored as: 0 - no deficit (normal); 1 - mild deficit; 2 - moderate deficit; 3 - severe deficit; and 4 - very severe deficit [33,34].
Tissue viability and infarct size was measured following 24 h after H/I on P8 and P14 day. Brain tissue sections of 5 µm thickness (n = 6/group; n = 4/animal) were cut at the level of interaural distance 5.40 mm and bregma 3.60 mm. The sections were embedded in paraffin were incubated with cresyl violet stain. Tissue sections were visualised (Carl Zeiss Stemi 2000-C stereomicroscope; Zeiss, Oberkochen, Germany) and the infarcted location was measured with NIH ImageJ software (Version1.42; National Institutes of Health, Bethesda, MD, USA). The infarcted region was unstained, while normal areas appeared stained with cresyl violet. The extent of infarction was determined as follows infarct area (%) = ([C – I] / C) × 100; C - mean of the contralateral area and I - mean value of the ipsilateral area.
Staining with terminal transferase-mediated dUTP nickend-labeling (TUNEL) was done to evaluate neuronal apoptosis following H/I. Sections of tissues measuring about 5 µm thickness (n = 6/group) were treated as per the instructions specified (DeadEnd fluorometric TUNEL system kit; Promega, Madision, WI, USA) and were analysed for apoptotic cells by NIS-Elements BR imaging processing and analysis software (Nikon Corporation, Tokyo, Japan).
Immunohistochemical examination was done to determine the effects of H/I on myelination in H/I induced pups. The assessments were done on P8 and P14. The excised brains that were post-fixed and equilibrated in 30% sucrose in 0.1 M PBS were used for analysis. Coronal sections (5 µm thickness) sliced at interaural distance 5.40 mm and bregma −3.60 mm level using microtome (Leica 1325; Leica Biosystems, Nussloch, Germany). The sections were cleaned with PBS and then treated with 1% H2O2 in PBS and 0.25% Triton X-100 in PBS (blocking solution) to exclude any activity of endogenous peroxidase. The sections were cleaned with PBS and incubated with specific primary antibodies against MBP, pre-myelinating oligodendrocytes (anti-O4; clone 81; Millipore, Billerica, MA, USA), immature oligodendrocytes (anti-O1; clone 59; Millipore) and incubated at 4°C overnight. Following incubation with primary antibodies, the sections were cleaned thrice with PBS and treated with horse-radish peroxidase tagged secondary antibodies for 1 h, treated with diaminobenzidine (DAB) and hematoxylin counterstained. The tissue sections were then dehydrated in ethanol and treated with xylene. The immunolabeled tissue sections were visualised and analysed using a light microscope (Olympus BX 50; Olympus, Tokyo, Japan).
Microglial activation was determined by assessing the expression of ionized calcium binding adaptor molecule 1 (Iba-1) as previously mentioned by Arteaga
Excised brain tissues (n = 6/group) were blended using cell lysis buffer from Cell Signaling Technology, USA (20 mM Tris-HCl [pH 7.5], 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 150 mM NaCl, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM Na2EDTA, 1 mM beta-glycerophosphate). The whole cell extracts obtained were subjected to centrifugation (3,000 rpm; 15 min) at 4°C. Supernatant collected was used for analysis. Cytosol and nuclear fractions were separated from the whole cell homogenate using ReadyPrep Protein Extraction Kit (Cytoplasmic/Nuclear) from Bio-Rad (Hercules, CA, USA). Total protein content of the whole cell extracts and in cytosol and nuclear fractions were quantified using kits from Thermo Fischer Scientific (Waltham, MA, USA). For Western blot analysis 60–80 µg amount of protein sample (n = 6/group) were loaded on SDS-PAGE (10%–15%) gels and were electrophoresed. The separated protein bands were blot transferred on to polyvinylidinene fluoride (PVDF; Thermo Fischer Scientific) and the membranes were blocked in 5% non-fat dry milk in 1x TBST to exclude any endogenous peroxidase activity. After washing with TBST, the PVDF membranes were incubated nightlong at 4°C with the primary antibodies. The membranes were cleaned using TBST and treated with secondary antibody (Horse radish peroxidase-conjugated; 1:2,000; Santa Cruz Biotechnology) at room temperature for 60 min. After incubating with secondary antibodies, the membranes were cleaned again with TBST and the bands were observed using enhanced chemiluminescence system (Millipore). The positive bands obtained were scanned and analysed using Image J software (Supersignal, Pierce, IL, USA).
Concentrations of pro-inflammatory mediators - IL-1β and TNF-α in the brain tissues post H/I insult were determined by ELISA. The fore brain tissues were homogenised in 0.1 M ice-cold Phosphate buffer and centrifuged. The supernatant was used for analysis of total protein content and for TNF-α and IL-1β levels using kits from RD systems (Minneapolis, MN, USA).
Brain tissue samples (n = 6) were homogenized from each treatment group with 1:10 (w/v) PBS on ice and centrifuged (3,000 rpm; 15 min, 4°C). Accumulated supernatant was used for determination of ROS generation, lipid peroxidation and glutathione (GSH) levels. Total quantity of protein in supernatant was found using Bradford protein assay kit (Bio-Rad). Levels of ROS in the brain tissues on P14 was detected using
Lipid peroxidation levels of the H/I-induced brain was determined as malondialdehyde (MDA) content. MDA and GSH levels were detected by means of assay kits from Sigma-Aldrich, according to the manufacturer's instructions.
The results of the investigation were statistically analysed using SPSS software (Version 22.0; IBM Co., Armonk, NY, USA). The obtained data were subjected to One-way analysis of variance (ANOVA) and Duncan’s Multiple Range Test (DMRT). p < 0.05 was regarded significant.
The P8, P14 and P21 rats that were exposed to hypoxic insult on P7 were assessed for behavioural responses and neuromuscular coordination. The H/I induced rats exhibited difficulties to walk and to move around indicating neurological deficits (Fig. 1). The flexibility of the left limbs was found to be reduced and the rats struggled to respond to the test stimulus. The neurological deficits were observed to be substantively (p < 0.05) reduced in animals that were treated with paeoniflorin at 6.25, 12.5 and 50 mg doses. Furthermore, on day P21 rats treated with paeoniflorin noticeably (p < 0.05) exhibited development in the neuro-behavioural responses and in muscular coordination
Infarct area following H/I was determined by cresyl violet staining. Infarcted regions appeared as unstained regions vs. normal regions stained with cresyl violet. The ischemic areas were observed predominantly in the striatum and in the fronto-parietal cortex regions (Fig. 2A, B). Pre- treatment with paeoniflorin at 6.25, 12.5 and 25 mg doses resulted in a substantially (p < 0.05) reduced infarct volume following H/I (Fig. 2A, B). Nevertheless, supplementation of 25 mg dose more expressively (p < 0.05) decreased cerebral infarct size in comparison to lower doses. In P8 rats, the infarct volume decreased from 68.15% to 15.72% and in P10 rats the infarct volume reduced to 10.08% and in P14 rats the infarct volume reduced to 5.79% with paeoniflorin treatment at 25 mg. Further, group 6 rats that were treated with paeoniflorin at 25 mg alone did not exhibit any neuronal injury. Also, interestingly, irrespective of the dosage given, the infarct size was significantly reduced in rats.
Neuroprotective effects of paeoniflorin pre-treatment was further evaluated by TUNEL assay. The data obtained illustrated that administration of paeoniflorin produced a significant (p < 0.05) decline in H/I-induced neuronal cell death (Fig. 3A, B). The apoptotic cell counts decreased from 149 cells/mm2 in H/I control to 52 cells/mm2 on paeoniflorin treatment at 25 mg/kg dose in P8 rats, 19 cells/mm2 in P10 rats and 12 cells/mm2 in P14 rats.
To evaluate further the neuroprotective effects of paeoniflorin treatment prior and post H/I induction, the expression of the proteins associated with cell apoptosis were evaluated by Western blotting in P8 rats exposed to H/I on P7 (Fig. 4A, B). Up-regulated expression of Bad and Cleaved caspase-3 were noticed in the H/I control group
H/I-induced oxidative stress is well documented in H/I-induced brain injury [35]. ROS and lipid peroxidation levels in brain tissues were measured in P8 pups at 24 h following induction of H/I and in P10 rats at 72 h post H/I insult. A multi-fold increase in ROS and MDA levels following H/I was observed (Fig. 5A, B). ROS generation was found to increase to 375.08% in H/I control on P8 and to 407.08% in P10 rats
GSH levels in H/I control rats were found to be reduced (p < 0.05) in H/I-induced P8 rat pups (Fig. 5C). Twenty four hours post H/I induction, GSH content decreased to 13.76 nM/mg protein in H/I control
Activation of microglia were assessed by determining the expression of the macrophage marker Iba-1. H/I-induction resulted in a significant (p < 0.05) rise in Iba-1-postive cells (Fig. 6A). Activated microglial cells were observed in the dentate gyrus and in the hippocampal CA 1 areas in the H/I control animals vs. normal controls. In contrast, Iba-1 expression levels were noticed to be remarkably decreased in P8 and in P10 pups treated with paeoniflorin at all the 3 tested doses compared to H/I control.
TLR4 levels were seen to increase multi-fold post H/I to 205.10% in P8 and to 280.26% in P10. HIF-1α levels were elevated to 190% in P8 and to 289.1% at P10. The up-regulated expression of TLR4 and HIF-1α observed following 24 h and 72 h of H/I induction were down-regulated significantly (p < 0.05) in rats supplemented with paeoniflorin (Fig. 6B). TLR4 and HIF-1α expression reduced to 129% and 115.2% respectively in P8 rats administrated with 25 mg Paeoniflorin. These observations suggest that paeoniflorin effectively reduced microglial activation.
NF-κB signaling is known to be activated under hypoxic conditions [36]. H/I induction in P7 rats caused a markedly enhanced (p < 0.05) nuclear NF-κB (p65) expression with substantially (p < 0.05) lower cytosolic levels of NF-κB (p65) (Fig. 7). The observations indicate activation of NF-κB. Furthermore up-regulated expression of TNF-α, and levels of phosphorylation of the regulatory kinase-IκBα were noted both at 24 h and 72 h post H/I. However, we noticed a marked increase in nuclear NF-κB (p65) expression at P10 when compared at 24 h. Paeoniflorin supplementation at all the 3 doses resulted in a significant suppression (p < 0.05) of NF-κB p65 (nuclear fraction) expression
Brain levels of proinflammatory cytokines - IL-1β and TNF-α at 24 h after H/I were observed to be significantly raised (p < 0.05)
White matter integrity post H/I insult was detected by calculating the density of MBP positive cells by immunohistochemical analysis. A significant reduction in MBP positive cell counts were observed at 24 h following H/I. MBP positive counts was noticed to be reduced by 29.12% at 24 h and 18.08% at 72 h post H/I in the in the corpus callosum of H/I control rats in comparison to normal control rats (Fig. 9). Prior treatment with paeoniflorin from P3 significantly (p < 0.05) improved MBP counts from 29.12% to 37.24%, 49.33% and 68.79% with 6.25 mg, 12.5 mg and 25 mg doses respectively at P8. Paeoniflorin supplementation at 25 mg improved MBP counts to 71.23% at P10. However, paeoniflorin alone administered rats exhibited no significant difference in MBP-positive cell counts on P8 and P10.
OPCs proliferation and maturation are critical for myelination. We noticed a significant decrease in the O4-positive oligodendrocytes and O1-positive oligodendrocytes at 24 h and 72 h following H/I insult in P8 and in P10 rat pups. Paeoniflorin treatment was found to effectively reduce the loss of OPCs (Fig. 9). In P8 pups, paeoniflorin at 25 mg significantly (p < 0.05) improved the counts of O4-positive oligodendrocytes to 89.67% from 16.14% in H/I control and O1-positive counts to 93.10%
Perinatal H/I-induced brain injury is the major cause of death and long-term neurological impairments [37]. The pathogenesis of H/I-induced brain injury involves several mechanisms- including, neuroinflammation, oxidative stress and apoptosis [13,18,38]. Lack of effective treatment strategies makes the identification of novel compounds inevitable. The present research was aimed to study the effects of systemic administration of paeoniflorin in animal model of neonatal H/I brain injury.
Paeoniflorin administered from P3 was found to effectively improve neurobehavioral scores and reduce neuronal apoptosis and infarct volume. Immunoblotting analysis revealed that paeoniflorin significantly down-regulated expression of cleaved caspase-3, chief marker of apoptotic cell death and pro-apoptotic protein, Bad. Interestingly paeoniflorin enhanced expression of anti-apoptotic protein, Bcl-2. The balance between anti-apoptotic and pro-apoptotic proteins is crucial in regulating cell existence [39]. The up-regulated Bcl-2 expression by paeoniflorin was noticed to be in line with decreased TUNEL positive cell counts, reflecting the anti-apoptotic effects of paeoniflorin.
Oxidative stress is well documented as a major contributor of H/I-induced brain injury [38]. Raised levels of free radicals including ROS are seen during the 2nd phase of H/I brain injury that occurs between 6–48 h of H/I insult. These elevated ROS levels extent for days post H/I insult contributing to further complications as chronic neuroinflammation, decreased neurogenesis and cell death [35,38]. In line with the previous reports, increased ROS production and MDA levels were observed in our study reflecting oxidative stress following H/I at 24 h and 72 h. Supressed GSH levels suggest utilisation of antioxidant defences in responses to stress. Paeoniflorin supplementation prior to H/I insult and post insult was found to remarkably reduce ROS production and MDA content and as well increased GSH levels. The observations suggest the anti-oxidant efficacy of paeoniflorin.
Neuroinflammatory processes are pivotal players in the loss of OPCs, loss of white matter integrity and subsequently causing brain injury [40,41]. Microglial cells are primary immune cells in neuroinflammatory responses and are reported to be critically involved in hypoxia-induced neuronal loss [17,42,43]. The Toll-like receptors (TLRs) are involved in innate defence responses and TLR4 expressed on microglia are reported in several neuroinflammatory diseases [16,23]. TLR4 expression was up-regulated following ethanol induced activation of microglia [44]. In our study we noticed marked activation of microglia at 24 h and at 72 h following H/I insult as evidenced by significantly raised levels of the microglial/macrophage marker, Iba-1. In line with expression levels of Iba-1, TLR4 expression was noticed to be up-regulated.
It is known under that the expression of HIF-1α is strongly upregulated under hypoxia [45,46]. HIF-1α is found to regulate TLR4 expression in microglia and macrophages under hypoxia [21,47]. Here again, we noticed up-regulated expression of HIF-1α following hypoxic insult. The elevated TLR4 expression may be due to enhanced HIF-1α.
Interestingly, we noted NF-κB signaling was significantly up-regulated following H/I induction. The results of our study revealed marked nuclear translocation of NF-κB (p65) post H/I. Murugan
It has been suggested that microglial activation and TLR4/NF-κB-mediated release of inflammatory mediators induce disruption of white matter integrity and subsequently leading to white matter injury [44]. Pro-inflammatory cytokines were found to cause loss of immature OPCs and affect myelination post H/I insult [19,26]. Disruption of myelination occurs due to loss of premyelinating oligodendrocytes and loss of maturation of OPCs [51,52]. OPCs of the immature brain are reported to be specifically vulnerable to H/I insults [11,12]. Low innate antioxidant enzymes in part have reported to contribute to cell death [12,15]. White matter injury is known as a major cause of chronic neurological deficits in H/I survivors [7]. The progenitor cells are pivotal for the replacing the degenerating premyelinated oligodendrocytes and as well in the formation of new myelin. In the present study we noticed significantly reduced MBP expression following H/I insult. Reduced MBP levels are regarded as a marker for white matter injury [53,54]. Similar to the observations of the previous reports, in the present study, marked losses of O4- and O1-positive oligodendrocytes were noticed in P8 and in P10 rat pups that were subjected to H/I insult on P7 [11,18,20].
Paeoniflorin supplementation to the pups prior H/I induction significantly reduced microglial activation as evidenced by decreased Iba1 levels and reduced TLR4 expression. Paeoniflorin down-regulated NF-κB activation in a dose-dependent manner, with 25 mg dose exhibiting higher effects. TNF-α and IL-1β levels were also reduced in the brain tissues suggesting suppression of inflammatory responses. Also, paeoniflorin significantly prevented the loss of O4- and O1-positive oligodendrocytes and improved MBP cell counts in comparison to H/I control group. These observations indicate paeoniflorin prevented white matter injury and improved myelination. Restoration of white matter integrity leads to functional recovery [9]. Thus, paeoniflorin could also aid in neurofunctional improvements post H/I insult. Paeoniflorin significantly reduced oxidative stress and caused down-regulation of HIF-1α. Paeoniflorin-mediated reduction in HIF-1α expression could have partly caused downregulation of TLR4 and thus leading to inhibition of TRL4/NF-κB signaling. Down-regulated NF-κB signaling and inflammatory cytokines by paeoniflorin could have aided in prevention of neuronal loss and white matter injury. The results demonstrate the neuroprotective efficiency of paeoniflorin.
Paeoniflorin pre-treatment significantly attenuated oxidative stress and inhibited up-regulation of TLR4/NF-κB mediated inflammatory responses. Paeoniflorin markedly reduced neuronal loss and white matter injury by improving MBP-positive counts and O4- and O1-positive oligodendrocytes. Our results suggest that paeoniflorin as a potent candidate in therapy of H/I induced neurological impairments.
Supplementary data including one figure can be found with this article online at https://doi.org/10.4196/kjpp.2021.25.2.97.
kjpp-25-2-97-supple.pdfF.Y. and Y.L. had equally contributed to this work and performed all activities relating to this study. F.Y., Y.L., X.S., and Y.L., designed the study and performed experiments and analysed the data and prepared this manuscript.
The authors declare no conflicts of interest.
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