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Neuroprotective effects of andrographolide on chronic cerebral hypoperfusion-induced hippocampal neuronal damage in rats possibly via PTEN/AKT signaling pathway Da-Peng Wanga,*,1, Shu-Hui Chenb,1, Di Wangc, Kai Kangd, Yi-Fang Wua, Shao-Hua Sua, Ying-Ying Zhange, Jian Haia,* a
Department of Neurosurgery, Tong Ji Hospital, Tong Ji University School of Medicine, Shanghai, 200065, China Department of Radiation Oncology and Head and Neck Surgery, Jiangxi Cancer Hospital, Nanchang, Jiangxi, 330029, China Undergraduate of Grade 2018, School of Integrated Traditional Chinese and Western Medicine, Jining Medical University, Jining, Shandong, 272067, China d Department of Research and Surveillance Evaluation, Shanghai Center for Health Promotion, Shanghai, 200040, China e Department of Oncology, Xiangya Hospital, Central South University, Changsha, 410008, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Andrographolide Chronic cerebral hypoperfusion Neuronal damage PTEN/AKT signaling Mechanism
To explore the potential effects of andrographolide on chronic cerebral hypoperfusion (CCH)-induced neuronal damage as well as the underlying mechanisms. Rat CCH model was established by 2-vessel occlusion (2VO). The CCH rats received andrographolide treatment for 4 weeks. The neuron loss was detected by using neuronal nuclei (NeuN) immunofluorescent staining. The expression levels of phospho-phosphatase and tensin homolog deleted on chromosome ten (p-PTEN), protein kinase B (AKT), p-AKT, and cysteinyl aspartate specific proteinase-3 (Caspase-3) proteins were accessed by Western blotting. Moreover, the neuronal apoptosis of hippocampus tissues was detected via terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) staining. CCH reduced the number of NeuN-positive cells, while the number was significant increased after andrographolide treatment. CCH increased the proteins expression level of p-PTEN, Caspase-3, and decreased the p-AKT, which were reversed by andrographolide treatment. Furthermore, andrographolide treatment also down-regulated CCH-induced TUNEL-apoptosis rate. Our results suggest that the PTEN/AKT pathway may be modulated by andrographolide and the damaging effects of CCH on hippocampus may be ameliorated by andrographolide treatment. Andrographolide may act as a potential therapeutic approach for chronic ischemic insults.
1. Introduction The global number of people living with dementia is expected to increase to 130 million in 2050 (Eggink et al., 2019). Increasing evidence suggests that chronic cerebral hypoperfusion (CCH) serves as a key vascular risk factor associated with Alzheimer’s disease, vascular dementia (VaD), and cognitive impairment (Lim et al., 2019; Silva et al., 2019). CCH is a sustained reduction of cerebral blood flow, which may trigger a cascade of pathophysiological processes, such as neurodegeneration, neuroinflammatory response, apoptosis, and excessive neuronal autophagy (Wang et al., 2018). Those neuropathological events may lead to neuronal injury in vulnerable regions of the brain, such as the hippocampus and cerebral cortex, showing learning and memory decline (Damodaran et al., 2019). However, there are
currently no effective therapeutic targets for the treatment of CCH-related cerebrovascular diseases. Andrographolide (C20H30O5, Fig. 1) is a major active compound extracted from Andrographis paniculata (A. paniculata), a traditional herb that is used to treat various human diseases, including knee osteoarthritis, multiple sclerosis, fever, diarrhea, and cancer chemotherapy-induced cognitive impairment (Lu et al., 2019; Seo et al., 2019). This compound possesses potent anti-inflammation, anti-depressant, anti-bacteria/virus, and anti-neoplasm pharmacological effects, but the underlying mechanisms remain unclear (Lu et al., 2019; Zhang et al., 2019b). Pharmacodynamic studies have revealed that andrographolide can cross the blood-brain barrier (BBB) and protect against cerebral ischemia/reperfusion injury (Yen et al., 2013). In a rat model of permanent middle cerebral artery occlusion, andrographolide
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Correspondence authors at: 389 Xincun Road, Putuo District, Shanghai, 200065, China. E-mail addresses: [email protected] (D.-P. Wang), [email protected] (J. Hai). 1 Da-Peng Wang and Shu-Hui Chen contributed equally to this work. https://doi.org/10.1016/j.acthis.2020.151514 Received 20 August 2019; Received in revised form 2 January 2020; Accepted 23 January 2020 0065-1281/ © 2020 Elsevier GmbH. All rights reserved.
Please cite this article as: Da-Peng Wang, et al., Acta Histochemica, https://doi.org/10.1016/j.acthis.2020.151514
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2.3. Detection of neuron loss via NeuN immunofluorescent staining Brain sections (4 μm thick) pretreated to reduce endogenous peroxidase activity after blocked with 10 % normal goat serum at room temperature for 1 h. The slices were incubated with NeuN antibody (1: 100, Cell Signaling Technology, MA, USA) overnight at 4 °C. After washed in PBS, the sections were incubated with the Alexa 488/594conjugated goat anti-rabbit antibody (1:200; Invitrogen, Carlsbad, CA, USA) at 37 °C for 30 min. Sections were then mounted with 4ꞌ,6-diamidino-2-phenylindole (DAPI). The immunostaining was visualized by an experimenter blinded to the groups using fluorescence microscopy (Zeiss, Thuringia, Germany) at 200× magnification. For the quantitative analysis, the average cells number of four non-overlapping hippocampal CA1 areas (three slides for each brain) was calculated using National Institutes of Health (NIH) Image J software (Bethesda, MD, USA) as previously reported(Su et al., 2018).
Fig. 1. The chemical structure of andrographolide.
shows neuroprotective effects by inhibiting microglial activation and the production of proinflammatory cytokine (tumor necrosis factor-α and interleukin-1β) (Chan et al., 2010; Yang et al., 2019). In addition, andrographolide exerts significant anti-cytotoxicity effects via upregulating the mammalian target of rapamycin (mTOR) signaling in SHSY5Y cells (Zhang et al., 2019a). However, the role of andrographolide in CCH and its influence on hippocampal neurons remain poorly understood. In this study, we herein explored its potential neuroprotective effects as well as the underlying mechanisms in rat CCH model.
2.4. Detection of protein expressions of PTEN/AKT signaling via western blotting Proteins were extracted from hippocampus tissues. The BCA method (Beyotime Biotechnology, Shanghai, China) was used to detect the concentration of protein. Subsequently, protein samples were separated by 10 % or 12 % sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After blocking with 5 % non-fat milk for 1 h, the PVDF membranes were incubated with primary antibodies against pPTEN (Ser380) (1:1000), AKT (1:1000), Phospho-AKT (1:1000), cleaved-Caspase-3 (1:1000), and GAPDH (1:5000) (Cell Signaling Technology, MA, USA) at 4 °C overnight. The PVDF membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were detected by enhanced chemiluminescence (Millipore, Watford, UK) with three biological replicates. For the quantitative analysis, the relative band density normalized against the GAPDH level using the Image Pro Plus 6.0 software (Bethesda, MD, USA).
2. Materials and methods 2.1. Antibodies and reagents Andrographolide CAS No. 5508587 was from Sigma-Aldrich Sigma, St. Louis, MO, USA, with purity ≥ 98 %. It was dissolved in 1 % dimethyl sulphoxide, and stored at 4 °C. Rabbit monoclonal antibodies against neuronal nuclei NeuN No. 24307, cleaved cysteinyl aspartatespecific proteinase-3 Caspase-3 No. 9664, and glyceraldehyde 3-phosphate dehydrogenase GAPDH No. 5174 were from Cell Signaling Technology Danvers, MA, USA. Rabbit polyclonal antibodies against phospho-phosphatase and tensin homolog deleted on chromosome ten p-PTEN Ser380 No. 9551, protein kinase B AKT No. 9272, and p-AKT No. 9271 were from Cell Signaling Technology. The Alexa 488/594conjugated goat anti-rabbit antibody is from Invitrogen Carlsbad, CA, USA. A bicinchoninic acid BCA Kit is from Beyotime Biotechnology Shanghai, China. The In Situ Cell Death Detection Kit is from Promega Madison, WI, USA.
2.5. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis assay Brain sections were processed with the In Situ Cell Death Detection Kit (Promega, Madison, WI, USA) to assess the number of apoptotic cells in the hippocampal CA1 area as the manufacturer’s instructions. Negative controls were performed in the reaction buffer without the TdT. TUNEL-positive cells were counted at 200× magnification in four non-overlapping microscope fields (Olympus, Tokyo, Japan) in hippocampal CA1 areas in each sample (three slides for each brain) and expressed as the mean percent of TUNEL-positive cells to the number of total cells by two investigators blinded to the groups(Shan et al., 2018).
2.2. Animal grouping and modeling A total of twenty-five SD male weighing 100 g ± 10 g were purchased from the Laboratory Animal Center of Shanghai. Rats were routinely fed in the Tongji University Animal Center under the temperature of (24 ± 1)°C, the humidity of 60 % and a 12/12 h light/dark cycle. Experimental protocols were performed following the Chinese legislation on the use and care of laboratory animals, and were approved by the Animal Ethics Committee of Animal Center. Rat CCH model was established by 2-vessel occlusion (2VO) as previously reported (Wang et al., 2018). Briefly, the bilateral common carotid arteries were exposed and ligatured by surgical 4–0 silk after anesthesia. The rats received the same treatment without 2VO were set as controls. Survival rate was 24 of 25 rats. All rats were randomly divided into three groups: (1) the Sham group; (2) the 2VO group; (3) the 2VO and andrographolide treatment group (2VO + And). The sample size was based on previous studies (n = 8 per group) (Choi et al., 2018; Das et al., 2017; Wang et al., 2019). Rats in the 2VO + And group received 1 % andrographolide in dimethyl sulfoxide at 1 ml/kg for 4 weeks by intraperitoneal (i.p.) injection. Rats in the 2VO group received an equal mount vehicle per day for 4 weeks.
2.6. Statistical analysis All experimental data were presented as mean ± standard deviation (SD). Tukey's posthoc test was used to validate the one-way ANOVA for comparing results between groups. The analyses were performed using the SPSS 22.0 statistical software (IBM, USA). p < 0.05 was depicted as statistically significant. 3. Results 3.1. Effects of andrographolide on NeuN immunofluorescent staining The immunoreactivity of NeuN, a definitive marker of mature neurons, reduce in various CNS diseases and neurodegenerative states (Duan et al., 2016). As shown in Fig. 2, the number of NeuN-positive cells reduced in the 2VO group (p < 0.05, vs. Sham), while the number 2
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Fig. 2. Effects of andrographolide on hippocampal neuron loss. (A) Representative NeuN- immunofluorescence in hippocampus CA1 region. (B) Statistical analysis of the number of NeuN-positive cells among groups. (n = 8 per group, * p < 0.05 Sham vs. 2VO, # p < 0.05, 2VO vs. 2VO + And).
3.4. Effects of andrographolide on TUNEL apoptosis rate in hippocampus tissues
significantly increased in the 2VO + And group (p < 0.05, vs. 2VO) (Fig. 2B). The results suggest that andrographolide could ameliorate CCH-induced hippocampal neuron loss.
As shown in Fig. 4, the number of TUNEL-positive cells (brownyellow cells) in the 2VO group was markedly larger than that in the control group (p < 0.05), while andrographolide treatment significantly decreased the positive cells. Combined with the above Western Bolt findings of cleaved Caspase-3, the results suggest that andrographolide could suppress CCH-induced neuronal apoptosis.
3.2. Effects of andrographolide on expressions of pathway proteins AKT signaling mediates many aspects of cellular processes, including nutrient uptake, cell growth, proliferation, and survival (Sanchez-Alegria et al., 2018). We further detected the expression of PTEN/AKT signaling elements using western blotting (Fig. 3). Phosphorylated PTEN significantly increased in the 2VO group compared with the Sham group (p < 0.05) (Fig. 3B). Inversely, CCH significantly decreased the phosphorylation of AKT protein (p < 0.05, vs. Sham) (Fig. 3C). However, those changes were reversed by andrographolide treatment (p < 0.05, respectively) (Fig. 3). The data indicate that the effects of andrographolide involves in mediating PTEN/AKT pathway.
4. Discussion Chronic cerebral ischemia is a complex neurological process with a series of pathological mechanisms, in which CCH plays an important role in neuropathological deterioration (Lee et al., 2018). A lot of preclinical works have demonstrated that CCH can cause BBB disruption, inflammatory responses, white matter injury, and neurobehavioral defect (Du et al., 2017). In our previous studies, neuronal degeneration, glial activation, and synaptic plasticity dysfunction were found in hippocampus and cerebral cortex of rats with CCH (Lin et al., 2010; Wang et al., 2017). Although some therapeutic methods have been used in clinic, there is still a translation gap between the pathological mechanism and clinical treatment (Duncombe et al., 2017). A perspective for the development of neuroprotectants from Andrographis paniculata is
3.3. Effects of andrographolide on Caspase-3 level in hippocampus tissues As shown in Fig. 3, the level of cleaved Caspase 3 protein obviously enhanced in CCH rat hippocampus tissue compared with control (p < 0.05). Compared with the 2VO group, andrographolide markedly reduced cleaved Caspase-3 protein expression in rat hippocampus tissue (p < 0.05, vs. 2VO) (Fig. 3A, D).
Fig. 3. Effects of andrographolide on expressions of pathway proteins and apoptosis protein. (A) Representative Western blotting bands of p-PTEN, p-AKT, AKT, and Caspase-3 (cleaved form). (B) Fold change of p-PTEN/GAPDH. (C) Fold change of p-AKT/AKT. (D) Fold change of Caspase-3/GAPDH. (n = 8 per group, * p < 0.05 Sham vs. 2VO, # p < 0.05, 2VO vs. 2VO + And). 3
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Fig. 4. Effects of andrographolide on hippocampus neuronal apoptosis. (A) Representative TUNEL staining in hippocampus CA1 region. (B) Statistical analysis of the apoptosis rate (n = 8 per group, * p < 0.05 Sham vs. 2VO, # p < 0.05, 2VO vs. 2VO + And).
neurons against hypoxia-induced injury (Wang et al., 2012), consistent with our findings. Recently, many studies have reported that andrographolide has anti-inflammatory and neuroprotective effects in both experimental and clinical trials (Bertoglio et al., 2016; Pearngam et al., 2019; Wang et al., 2019; Xu et al., 2019). Andrographolide can attenuate chronic unpredictable mild stress-induced depressive-like behavior in mice (Geng et al., 2019). In patients with glioma, andrographolide shows a good safety profile against cancer chemotherapy-induced cognitive impairment (Bertoglio et al., 2016).We previously reported that andrographolide ameliorates CCH-induced cognitive impairment due to inhibition of astrocyte activation and improvement of neuroprotectant brain-derived neurotrophic factor signaling (Wang et al., 2019). In this study, the level of cleaved Caspase-3 protein expression and the TUNELapoptotic rate were increased in the 2VO group, while those enhancements were partly reversed by andrographolide, suggesting its effects in rats with CCH associated with anti-apoptosis. An analysis of hippocampal synaptosomes by electron microscopy also reveals that the length of synapses is restored with andrographolide treatment in a mice model of early-onset Alzheimer's disease (Cisternas et al., 2019). There are some limitations to this study. Firstly, only histological morphologic changes, cell survival signals, and apoptosis-related protein in the hippocampus were accessed and further pharmacodynamic tests, especially screening and optimization of drug concentration, can provide more definitive evidence of the mechanism of neuroprotective effect of andrographolide in this cerebral ischemia model. Inspiringly, Bilia AR et al has demonstrated that andrographolide encapsulated in human albumin nanoparticles improves locomotor activity and cognitive function of TgCRND8 mice (an Alzheimer's disease model) with extraordinary versatility, nontoxicity, nonimmunogenicity, strong biocompatibility, and high biodegradability (Bilia et al., 2019). Although, andrographolide shows beneficial potentiality against neuropathy in various regions of brain namely hypothalamus, cerebellum, hippocampus, and cerebral cortex in diabetic rats (Naik et al., 2017). In this study, it would be more helpful to elucidate the role of andrographolide in CCH-induced neural damage if the assessment was also performed in other brain regions, such as the cortex and cerebellum.
a promising treatment for this disease (Yang et al., 2017). In some rodent experiments, andrographolide has been shown to improve middle cerebral artery occlusion-induced brain infarction and behavioral deficits (Chan et al., 2010; Yang et al., 2017; Yen et al., 2016). A new molecule entity derived from andrographolide protects neurons against ischemic stroke by inhibiting the Toll-like receptor 4-NF-κB signaling (Yang et al., 2019). Here, we have demonstrated that andrographolide could increase the NeuN-positive cell number in hippocampus of CCH rats, indicating that andrographolide can improve cerebral ischemiainduced neuron loss, neurodegeneration. Consistent with previous reports (Varela-Nallar et al., 2015), andrographolide shows a favorable role in promoting neurogenesis in adult hippocampus. Correspondingly, andrographolide has been reported to accelerate the proliferation of rat Schwann cells following peripheral nerve injury (Xu et al., 2016). Furthermore, andrographolide protects PC12 neurons against inflammation-mediated neurodegeneration by inhibiting oxidative stress (Xu et al., 2019). Those studies contribute to further exploration of the therapeutic potential of andrographolide in relation to CCH-induced neurodegeneration. PTEN, one tumor suppressor genes, regulates multiple intracellular processes, such as protein interactions, nuclear translation, membrane localization (Malaney et al., 2017). The activity of PTEN is regulated by its phosphorylation at Ser380, abnormal activation of PTEN signaling may result in tumorigenesis, immunological and neurological diseases (Chen and Guo, 2017; Papa and Pandolfi, 2019). Multiple studies have confirmed that cerebral ischemia can upregulate its phosphorylated level (Li et al., 2017; Ueno et al., 2015). In the current study, we also found that the level of p-PTEN was increased in CCH rats compared to that in sham rats. PTEN activation frequently correlates with an inactive status of p-AKT level, which also suggests that AKT may be an important PTEN functional target (Malaney et al., 2017). As shown in western blotting, p-AKT was decreased following upregulated p-PTEN in CCH rats. It is reported that silencing PTEN as well as subsequent activating AKT enhances axonal plasticity and neuron survival (Ueno et al., 2015). After chronic treat with andrographolide for 4 weeks, the levels of p-PTEN and p-AKT were reversed, indicating that andrographolide treatment restored the aberrant PTEN/AKT signaling. Meanwhile, the apoptosis rate in the 2VO group was markedly larger than that in the Sham group, while andrographolide treatment significantly decreased the TUNEL-positive cells number. These results suggest that andrographolide against CCH-induced hippocampal neuronal damage may be mediated by PTEN/AKT signaling. In addition, andrographolide treatment at 1 h after cerebral ischemia is able to ameliorate brain infarction and neurological deficits in a mice model of cerebral ischemic/reperfusion injury through the regulation of PI3K/ Akt signaling (Chern et al., 2011). Moreover, p-PTEN activation is found to aggravate white matter injuries after CCH (Li et al., 2017), and AKT/mTOR pathway activation is found to protect hippocampal
5. Conclusions Our results suggest that the PTEN/AKT pathway could be modulated by andrographolide, as well as the damaging effects of CCH on hippocampus could be ameliorated by andrographolide treatment, proposing a potential neuroprotective effect of andrographolide against chronic ischemic cerebral damage associated with cerebrovascular diseases.
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Da-Peng Wang: Investigation, Formal analysis, Resources, Writing review & editing. Shu-Hui Chen: Investigation, Formal analysis. Di Wang: Conceptualization, Methodology, Writing - original draft. Kai Kang: Conceptualization, Methodology, Writing - original draft. YiFang Wu: Validation, Formal analysis. Shao-Hua Su: Validation, Formal analysis. Ying-Ying Zhang: Investigation, Formal analysis. Jian Hai: Supervision. Declaration of Competing Interest The authors declare that there is no conflict of interest. Acknowledgments The work was supported by the National Nature Science Foundation of China (81974207, 81771410), the Priority of Shanghai key discipline of medicine (2017ZZ02020) and the Key Research and Development Program of Hunan Province (2016JC2073). References Bertoglio, J.C., Baumgartner, M., Palma, R., Ciampi, E., Carcamo, C., Caceres, D.D., Acosta-Jamett, G., Hancke, J.L., Burgos, R.A., 2016. Andrographis paniculata decreases fatigue in patients with relapsing-remitting multiple sclerosis: a 12-month double-blind placebo-controlled pilot study. BMC Neurol. 16, 77. Bilia, A.R., Nardiello, P., Piazzini, V., Leri, M., Bergonzi, M.C., Bucciantini, M., Casamenti, F., 2019. Successful brain delivery of andrographolide loaded in human albumin nanoparticles to TgCRND8 mice, an Alzheimer’s disease mouse model. Front. Pharmacol. 10, 910. Chan, S.J., Wong, W.S., Wong, P.T., Bian, J.S., 2010. Neuroprotective effects of andrographolide in a rat model of permanent cerebral ischaemia. Br. J. Pharmacol. 161 (3), 668–679. Chen, L., Guo, D., 2017. The functions of tumor suppressor PTEN in innate and adaptive immunity. Cell. Mol. Immunol. 14 (7), 581–589. Chern, C.M., Liou, K.T., Wang, Y.H., Liao, J.F., Yen, J.C., Shen, Y.C., 2011. Andrographolide inhibits PI3K/AKT-dependent NOX2 and iNOS expression protecting mice against hypoxia/ischemia-induced oxidative brain injury. Planta Med. 77 (15), 1669–1679. Choi, S.A., Chong, S., Kwak, P.A., Moon, Y.J., Jangra, A., Phi, J.H., Lee, J.Y., Park, S.H., Kim, S.K., 2018. Impaired functional recovery of endothelial colony-forming cells from moyamoya disease in a chronic cerebral hypoperfusion rat model. J. Neurosurg. Pediatr. 23 (2), 204–213. Cisternas, P., Oliva, C.A., Torres, V.I., Barrera, D.P., Inestrosa, N.C., 2019. Presymptomatic treatment with andrographolide improves brain metabolic markers and cognitive behavior in a model of early-onset Alzheimer’s disease. Front. Cell. Neurosci. 13, 295. Damodaran, T., Muller, C.P., Hassan, Z., 2019. Chronic cerebral hypoperfusion-induced memory impairment and hippocampal long-term potentiation deficits are improved by cholinergic stimulation in rats. Pharmacol. Rep. 71 (3), 443–448. Das, S., Mishra, K.P., Ganju, L., Singh, S.B., 2017. Andrographolide - a promising therapeutic agent, negatively regulates glial cell derived neurodegeneration of prefrontal cortex, hippocampus and working memory impairment. J. Neuroimmunol. 313, 161–175. Du, S.Q., Wang, X.R., Xiao, L.Y., Tu, J.F., Zhu, W., He, T., Liu, C.Z., 2017. Molecular mechanisms of vascular dementia: what can be learned from animal models of chronic cerebral hypoperfusion? Mol. Neurobiol. 54 (5), 3670–3682. Duan, W., Zhang, Y.P., Hou, Z., Huang, C., Zhu, H., Zhang, C.Q., Yin, Q., 2016. Novel insights into NeuN: from neuronal marker to splicing regulator. Mol. Neurobiol. 53 (3), 1637–1647. Duncombe, J., Kitamura, A., Hase, Y., Ihara, M., Kalaria, R.N., Horsburgh, K., 2017. Chronic cerebral hypoperfusion: a key mechanism leading to vascular cognitive impairment and dementia. Closing the translational gap between rodent models and human vascular cognitive impairment and dementia. Clin. Sci. (Lond.) 131 (19), 2451–2468. Eggink, E., Moll van Charante, E.P., van Gool, W.A., Richard, E., 2019. A population perspective on prevention of dementia. J. Clin. Med. 8 (6). Geng, J., Liu, J., Yuan, X., Liu, W., Guo, W., 2019. Andrographolide triggers autophagymediated inflammation inhibition and attenuates chronic unpredictable mild stress (CUMS)-induced depressive-like behavior in mice. Toxicol. Appl. Pharmacol. 379, 114688. Lee, R.H.C., Lee, M.H.H., Wu, C.Y.C., Couto, E.S.A., Possoit, H.E., Hsieh, T.H., Minagar, A., Lin, H.W., 2018. Cerebral ischemia and neuroregeneration. Neural Regen. Res. 13 (3), 373–385. Li, X., Ren, C., Li, S., Han, R., Gao, J., Huang, Q., Jin, K., Luo, Y., Ji, X., 2017. Limb remote ischemic conditioning promotes myelination by upregulating PTEN/Akt/mTOR
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Neuroprotective effects of andrographolide on chronic cerebral hypoperfusion-induced hippocampal neuronal damage in rats possibly via PTEN/AKT signaling pathway Da-Peng Wanga,*,1, Shu-Hui Chenb,1, Di Wangc, Kai Kangd, Yi-Fang Wua, Shao-Hua Sua, Ying-Ying Zhange, Jian Haia,* a
Department of Neurosurgery, Tong Ji Hospital, Tong Ji University School of Medicine, Shanghai, 200065, China Department of Radiation Oncology and Head and Neck Surgery, Jiangxi Cancer Hospital, Nanchang, Jiangxi, 330029, China Undergraduate of Grade 2018, School of Integrated Traditional Chinese and Western Medicine, Jining Medical University, Jining, Shandong, 272067, China d Department of Research and Surveillance Evaluation, Shanghai Center for Health Promotion, Shanghai, 200040, China e Department of Oncology, Xiangya Hospital, Central South University, Changsha, 410008, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Andrographolide Chronic cerebral hypoperfusion Neuronal damage PTEN/AKT signaling Mechanism
To explore the potential effects of andrographolide on chronic cerebral hypoperfusion (CCH)-induced neuronal damage as well as the underlying mechanisms. Rat CCH model was established by 2-vessel occlusion (2VO). The CCH rats received andrographolide treatment for 4 weeks. The neuron loss was detected by using neuronal nuclei (NeuN) immunofluorescent staining. The expression levels of phospho-phosphatase and tensin homolog deleted on chromosome ten (p-PTEN), protein kinase B (AKT), p-AKT, and cysteinyl aspartate specific proteinase-3 (Caspase-3) proteins were accessed by Western blotting. Moreover, the neuronal apoptosis of hippocampus tissues was detected via terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) staining. CCH reduced the number of NeuN-positive cells, while the number was significant increased after andrographolide treatment. CCH increased the proteins expression level of p-PTEN, Caspase-3, and decreased the p-AKT, which were reversed by andrographolide treatment. Furthermore, andrographolide treatment also down-regulated CCH-induced TUNEL-apoptosis rate. Our results suggest that the PTEN/AKT pathway may be modulated by andrographolide and the damaging effects of CCH on hippocampus may be ameliorated by andrographolide treatment. Andrographolide may act as a potential therapeutic approach for chronic ischemic insults.
1. Introduction The global number of people living with dementia is expected to increase to 130 million in 2050 (Eggink et al., 2019). Increasing evidence suggests that chronic cerebral hypoperfusion (CCH) serves as a key vascular risk factor associated with Alzheimer’s disease, vascular dementia (VaD), and cognitive impairment (Lim et al., 2019; Silva et al., 2019). CCH is a sustained reduction of cerebral blood flow, which may trigger a cascade of pathophysiological processes, such as neurodegeneration, neuroinflammatory response, apoptosis, and excessive neuronal autophagy (Wang et al., 2018). Those neuropathological events may lead to neuronal injury in vulnerable regions of the brain, such as the hippocampus and cerebral cortex, showing learning and memory decline (Damodaran et al., 2019). However, there are
currently no effective therapeutic targets for the treatment of CCH-related cerebrovascular diseases. Andrographolide (C20H30O5, Fig. 1) is a major active compound extracted from Andrographis paniculata (A. paniculata), a traditional herb that is used to treat various human diseases, including knee osteoarthritis, multiple sclerosis, fever, diarrhea, and cancer chemotherapy-induced cognitive impairment (Lu et al., 2019; Seo et al., 2019). This compound possesses potent anti-inflammation, anti-depressant, anti-bacteria/virus, and anti-neoplasm pharmacological effects, but the underlying mechanisms remain unclear (Lu et al., 2019; Zhang et al., 2019b). Pharmacodynamic studies have revealed that andrographolide can cross the blood-brain barrier (BBB) and protect against cerebral ischemia/reperfusion injury (Yen et al., 2013). In a rat model of permanent middle cerebral artery occlusion, andrographolide
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Correspondence authors at: 389 Xincun Road, Putuo District, Shanghai, 200065, China. E-mail addresses: [email protected] (D.-P. Wang), [email protected] (J. Hai). 1 Da-Peng Wang and Shu-Hui Chen contributed equally to this work. https://doi.org/10.1016/j.acthis.2020.151514 Received 20 August 2019; Received in revised form 2 January 2020; Accepted 23 January 2020 0065-1281/ © 2020 Elsevier GmbH. All rights reserved.
Please cite this article as: Da-Peng Wang, et al., Acta Histochemica, https://doi.org/10.1016/j.acthis.2020.151514
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2.3. Detection of neuron loss via NeuN immunofluorescent staining Brain sections (4 μm thick) pretreated to reduce endogenous peroxidase activity after blocked with 10 % normal goat serum at room temperature for 1 h. The slices were incubated with NeuN antibody (1: 100, Cell Signaling Technology, MA, USA) overnight at 4 °C. After washed in PBS, the sections were incubated with the Alexa 488/594conjugated goat anti-rabbit antibody (1:200; Invitrogen, Carlsbad, CA, USA) at 37 °C for 30 min. Sections were then mounted with 4ꞌ,6-diamidino-2-phenylindole (DAPI). The immunostaining was visualized by an experimenter blinded to the groups using fluorescence microscopy (Zeiss, Thuringia, Germany) at 200× magnification. For the quantitative analysis, the average cells number of four non-overlapping hippocampal CA1 areas (three slides for each brain) was calculated using National Institutes of Health (NIH) Image J software (Bethesda, MD, USA) as previously reported(Su et al., 2018).
Fig. 1. The chemical structure of andrographolide.
shows neuroprotective effects by inhibiting microglial activation and the production of proinflammatory cytokine (tumor necrosis factor-α and interleukin-1β) (Chan et al., 2010; Yang et al., 2019). In addition, andrographolide exerts significant anti-cytotoxicity effects via upregulating the mammalian target of rapamycin (mTOR) signaling in SHSY5Y cells (Zhang et al., 2019a). However, the role of andrographolide in CCH and its influence on hippocampal neurons remain poorly understood. In this study, we herein explored its potential neuroprotective effects as well as the underlying mechanisms in rat CCH model.
2.4. Detection of protein expressions of PTEN/AKT signaling via western blotting Proteins were extracted from hippocampus tissues. The BCA method (Beyotime Biotechnology, Shanghai, China) was used to detect the concentration of protein. Subsequently, protein samples were separated by 10 % or 12 % sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After blocking with 5 % non-fat milk for 1 h, the PVDF membranes were incubated with primary antibodies against pPTEN (Ser380) (1:1000), AKT (1:1000), Phospho-AKT (1:1000), cleaved-Caspase-3 (1:1000), and GAPDH (1:5000) (Cell Signaling Technology, MA, USA) at 4 °C overnight. The PVDF membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were detected by enhanced chemiluminescence (Millipore, Watford, UK) with three biological replicates. For the quantitative analysis, the relative band density normalized against the GAPDH level using the Image Pro Plus 6.0 software (Bethesda, MD, USA).
2. Materials and methods 2.1. Antibodies and reagents Andrographolide CAS No. 5508587 was from Sigma-Aldrich Sigma, St. Louis, MO, USA, with purity ≥ 98 %. It was dissolved in 1 % dimethyl sulphoxide, and stored at 4 °C. Rabbit monoclonal antibodies against neuronal nuclei NeuN No. 24307, cleaved cysteinyl aspartatespecific proteinase-3 Caspase-3 No. 9664, and glyceraldehyde 3-phosphate dehydrogenase GAPDH No. 5174 were from Cell Signaling Technology Danvers, MA, USA. Rabbit polyclonal antibodies against phospho-phosphatase and tensin homolog deleted on chromosome ten p-PTEN Ser380 No. 9551, protein kinase B AKT No. 9272, and p-AKT No. 9271 were from Cell Signaling Technology. The Alexa 488/594conjugated goat anti-rabbit antibody is from Invitrogen Carlsbad, CA, USA. A bicinchoninic acid BCA Kit is from Beyotime Biotechnology Shanghai, China. The In Situ Cell Death Detection Kit is from Promega Madison, WI, USA.
2.5. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis assay Brain sections were processed with the In Situ Cell Death Detection Kit (Promega, Madison, WI, USA) to assess the number of apoptotic cells in the hippocampal CA1 area as the manufacturer’s instructions. Negative controls were performed in the reaction buffer without the TdT. TUNEL-positive cells were counted at 200× magnification in four non-overlapping microscope fields (Olympus, Tokyo, Japan) in hippocampal CA1 areas in each sample (three slides for each brain) and expressed as the mean percent of TUNEL-positive cells to the number of total cells by two investigators blinded to the groups(Shan et al., 2018).
2.2. Animal grouping and modeling A total of twenty-five SD male weighing 100 g ± 10 g were purchased from the Laboratory Animal Center of Shanghai. Rats were routinely fed in the Tongji University Animal Center under the temperature of (24 ± 1)°C, the humidity of 60 % and a 12/12 h light/dark cycle. Experimental protocols were performed following the Chinese legislation on the use and care of laboratory animals, and were approved by the Animal Ethics Committee of Animal Center. Rat CCH model was established by 2-vessel occlusion (2VO) as previously reported (Wang et al., 2018). Briefly, the bilateral common carotid arteries were exposed and ligatured by surgical 4–0 silk after anesthesia. The rats received the same treatment without 2VO were set as controls. Survival rate was 24 of 25 rats. All rats were randomly divided into three groups: (1) the Sham group; (2) the 2VO group; (3) the 2VO and andrographolide treatment group (2VO + And). The sample size was based on previous studies (n = 8 per group) (Choi et al., 2018; Das et al., 2017; Wang et al., 2019). Rats in the 2VO + And group received 1 % andrographolide in dimethyl sulfoxide at 1 ml/kg for 4 weeks by intraperitoneal (i.p.) injection. Rats in the 2VO group received an equal mount vehicle per day for 4 weeks.
2.6. Statistical analysis All experimental data were presented as mean ± standard deviation (SD). Tukey's posthoc test was used to validate the one-way ANOVA for comparing results between groups. The analyses were performed using the SPSS 22.0 statistical software (IBM, USA). p < 0.05 was depicted as statistically significant. 3. Results 3.1. Effects of andrographolide on NeuN immunofluorescent staining The immunoreactivity of NeuN, a definitive marker of mature neurons, reduce in various CNS diseases and neurodegenerative states (Duan et al., 2016). As shown in Fig. 2, the number of NeuN-positive cells reduced in the 2VO group (p < 0.05, vs. Sham), while the number 2
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Fig. 2. Effects of andrographolide on hippocampal neuron loss. (A) Representative NeuN- immunofluorescence in hippocampus CA1 region. (B) Statistical analysis of the number of NeuN-positive cells among groups. (n = 8 per group, * p < 0.05 Sham vs. 2VO, # p < 0.05, 2VO vs. 2VO + And).
3.4. Effects of andrographolide on TUNEL apoptosis rate in hippocampus tissues
significantly increased in the 2VO + And group (p < 0.05, vs. 2VO) (Fig. 2B). The results suggest that andrographolide could ameliorate CCH-induced hippocampal neuron loss.
As shown in Fig. 4, the number of TUNEL-positive cells (brownyellow cells) in the 2VO group was markedly larger than that in the control group (p < 0.05), while andrographolide treatment significantly decreased the positive cells. Combined with the above Western Bolt findings of cleaved Caspase-3, the results suggest that andrographolide could suppress CCH-induced neuronal apoptosis.
3.2. Effects of andrographolide on expressions of pathway proteins AKT signaling mediates many aspects of cellular processes, including nutrient uptake, cell growth, proliferation, and survival (Sanchez-Alegria et al., 2018). We further detected the expression of PTEN/AKT signaling elements using western blotting (Fig. 3). Phosphorylated PTEN significantly increased in the 2VO group compared with the Sham group (p < 0.05) (Fig. 3B). Inversely, CCH significantly decreased the phosphorylation of AKT protein (p < 0.05, vs. Sham) (Fig. 3C). However, those changes were reversed by andrographolide treatment (p < 0.05, respectively) (Fig. 3). The data indicate that the effects of andrographolide involves in mediating PTEN/AKT pathway.
4. Discussion Chronic cerebral ischemia is a complex neurological process with a series of pathological mechanisms, in which CCH plays an important role in neuropathological deterioration (Lee et al., 2018). A lot of preclinical works have demonstrated that CCH can cause BBB disruption, inflammatory responses, white matter injury, and neurobehavioral defect (Du et al., 2017). In our previous studies, neuronal degeneration, glial activation, and synaptic plasticity dysfunction were found in hippocampus and cerebral cortex of rats with CCH (Lin et al., 2010; Wang et al., 2017). Although some therapeutic methods have been used in clinic, there is still a translation gap between the pathological mechanism and clinical treatment (Duncombe et al., 2017). A perspective for the development of neuroprotectants from Andrographis paniculata is
3.3. Effects of andrographolide on Caspase-3 level in hippocampus tissues As shown in Fig. 3, the level of cleaved Caspase 3 protein obviously enhanced in CCH rat hippocampus tissue compared with control (p < 0.05). Compared with the 2VO group, andrographolide markedly reduced cleaved Caspase-3 protein expression in rat hippocampus tissue (p < 0.05, vs. 2VO) (Fig. 3A, D).
Fig. 3. Effects of andrographolide on expressions of pathway proteins and apoptosis protein. (A) Representative Western blotting bands of p-PTEN, p-AKT, AKT, and Caspase-3 (cleaved form). (B) Fold change of p-PTEN/GAPDH. (C) Fold change of p-AKT/AKT. (D) Fold change of Caspase-3/GAPDH. (n = 8 per group, * p < 0.05 Sham vs. 2VO, # p < 0.05, 2VO vs. 2VO + And). 3
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Fig. 4. Effects of andrographolide on hippocampus neuronal apoptosis. (A) Representative TUNEL staining in hippocampus CA1 region. (B) Statistical analysis of the apoptosis rate (n = 8 per group, * p < 0.05 Sham vs. 2VO, # p < 0.05, 2VO vs. 2VO + And).
neurons against hypoxia-induced injury (Wang et al., 2012), consistent with our findings. Recently, many studies have reported that andrographolide has anti-inflammatory and neuroprotective effects in both experimental and clinical trials (Bertoglio et al., 2016; Pearngam et al., 2019; Wang et al., 2019; Xu et al., 2019). Andrographolide can attenuate chronic unpredictable mild stress-induced depressive-like behavior in mice (Geng et al., 2019). In patients with glioma, andrographolide shows a good safety profile against cancer chemotherapy-induced cognitive impairment (Bertoglio et al., 2016).We previously reported that andrographolide ameliorates CCH-induced cognitive impairment due to inhibition of astrocyte activation and improvement of neuroprotectant brain-derived neurotrophic factor signaling (Wang et al., 2019). In this study, the level of cleaved Caspase-3 protein expression and the TUNELapoptotic rate were increased in the 2VO group, while those enhancements were partly reversed by andrographolide, suggesting its effects in rats with CCH associated with anti-apoptosis. An analysis of hippocampal synaptosomes by electron microscopy also reveals that the length of synapses is restored with andrographolide treatment in a mice model of early-onset Alzheimer's disease (Cisternas et al., 2019). There are some limitations to this study. Firstly, only histological morphologic changes, cell survival signals, and apoptosis-related protein in the hippocampus were accessed and further pharmacodynamic tests, especially screening and optimization of drug concentration, can provide more definitive evidence of the mechanism of neuroprotective effect of andrographolide in this cerebral ischemia model. Inspiringly, Bilia AR et al has demonstrated that andrographolide encapsulated in human albumin nanoparticles improves locomotor activity and cognitive function of TgCRND8 mice (an Alzheimer's disease model) with extraordinary versatility, nontoxicity, nonimmunogenicity, strong biocompatibility, and high biodegradability (Bilia et al., 2019). Although, andrographolide shows beneficial potentiality against neuropathy in various regions of brain namely hypothalamus, cerebellum, hippocampus, and cerebral cortex in diabetic rats (Naik et al., 2017). In this study, it would be more helpful to elucidate the role of andrographolide in CCH-induced neural damage if the assessment was also performed in other brain regions, such as the cortex and cerebellum.
a promising treatment for this disease (Yang et al., 2017). In some rodent experiments, andrographolide has been shown to improve middle cerebral artery occlusion-induced brain infarction and behavioral deficits (Chan et al., 2010; Yang et al., 2017; Yen et al., 2016). A new molecule entity derived from andrographolide protects neurons against ischemic stroke by inhibiting the Toll-like receptor 4-NF-κB signaling (Yang et al., 2019). Here, we have demonstrated that andrographolide could increase the NeuN-positive cell number in hippocampus of CCH rats, indicating that andrographolide can improve cerebral ischemiainduced neuron loss, neurodegeneration. Consistent with previous reports (Varela-Nallar et al., 2015), andrographolide shows a favorable role in promoting neurogenesis in adult hippocampus. Correspondingly, andrographolide has been reported to accelerate the proliferation of rat Schwann cells following peripheral nerve injury (Xu et al., 2016). Furthermore, andrographolide protects PC12 neurons against inflammation-mediated neurodegeneration by inhibiting oxidative stress (Xu et al., 2019). Those studies contribute to further exploration of the therapeutic potential of andrographolide in relation to CCH-induced neurodegeneration. PTEN, one tumor suppressor genes, regulates multiple intracellular processes, such as protein interactions, nuclear translation, membrane localization (Malaney et al., 2017). The activity of PTEN is regulated by its phosphorylation at Ser380, abnormal activation of PTEN signaling may result in tumorigenesis, immunological and neurological diseases (Chen and Guo, 2017; Papa and Pandolfi, 2019). Multiple studies have confirmed that cerebral ischemia can upregulate its phosphorylated level (Li et al., 2017; Ueno et al., 2015). In the current study, we also found that the level of p-PTEN was increased in CCH rats compared to that in sham rats. PTEN activation frequently correlates with an inactive status of p-AKT level, which also suggests that AKT may be an important PTEN functional target (Malaney et al., 2017). As shown in western blotting, p-AKT was decreased following upregulated p-PTEN in CCH rats. It is reported that silencing PTEN as well as subsequent activating AKT enhances axonal plasticity and neuron survival (Ueno et al., 2015). After chronic treat with andrographolide for 4 weeks, the levels of p-PTEN and p-AKT were reversed, indicating that andrographolide treatment restored the aberrant PTEN/AKT signaling. Meanwhile, the apoptosis rate in the 2VO group was markedly larger than that in the Sham group, while andrographolide treatment significantly decreased the TUNEL-positive cells number. These results suggest that andrographolide against CCH-induced hippocampal neuronal damage may be mediated by PTEN/AKT signaling. In addition, andrographolide treatment at 1 h after cerebral ischemia is able to ameliorate brain infarction and neurological deficits in a mice model of cerebral ischemic/reperfusion injury through the regulation of PI3K/ Akt signaling (Chern et al., 2011). Moreover, p-PTEN activation is found to aggravate white matter injuries after CCH (Li et al., 2017), and AKT/mTOR pathway activation is found to protect hippocampal
5. Conclusions Our results suggest that the PTEN/AKT pathway could be modulated by andrographolide, as well as the damaging effects of CCH on hippocampus could be ameliorated by andrographolide treatment, proposing a potential neuroprotective effect of andrographolide against chronic ischemic cerebral damage associated with cerebrovascular diseases.
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