Rhodiola crenulata attenuates apoptosis and mitochondrial energy metabolism disorder in rats with hypobaric hypoxia-induced brain injury by regulating the HIF-1α/microRNA 210/ISCU1/2(COX10) signaling pathway
Graphical abstract
Introduction
High-altitude conditions, which are characterized by a nonlinear decrease in barometric pressure accompanied by an increase in ultraviolet intensity, as well as a decrease in humidity and ambient temperature, induce an array of hypobaric hypoxic physiological changes in humans and animals (Woods et al., 2017; Girard et al., 2017; Davis and Hackett, 2017; Basnyat and Murdoch, 2003). Such conditions induce a decrease in the inspired partial pressure of oxygen (PO2) in the alveoli, starving arteriovenous blood of oxygen, which triggers excessive ventilation, hypoxic pulmonary vasoconstriction, and elevated cardiac output, resulting in acute mountain sickness (Lawley et al., 2014; Bartsch and Swenson, 2013), high-altitude pulmonary edema (Wang et al., 2016), and high-altitude cerebral edema (Wang et al., 2016; Fayed et al., 2006; Guo et al., 2013; Gong et al., 2018; Hackett et al., 1998). The treatment or improvement of hypobaric hypoxia-induced central nervous system (CNS) damage has recently attracted increasing attention in the field of high-altitude medicine (Hu et al., 2017; X.Y. Zhang et al., 2018). Of the various physiopathologic mechanisms involved, enhanced blood-brain barrier permeability and hypoxic cerebral vasodilation are thought to be the two main pathways (Chryssanthou et al., 1987; Lafuente et al., 2016). In addition, deficient mitochondrial energy production and excessive mitochondrial reactive oxygen species induce an increase in mitochondrial membrane permeability and potential, in turn aggravating the disruption of mitochondrial membrane integrity. Furthermore, elevated hypoxia inducible factor-1 alpha (HIF-1α) mRNA and protein levels stimulate downstream vascular endothelial cell growth factor and erythropoietin generation, enhancing tolerance to hypoxic insult. Under hypoxic challenge, overexpression of microRNA 210 (miR-210) induced by upregulation of HIF-1α was found to reduce expression of iron-sulfur cluster scaffold (ISCU1/2) and cytochrome c oxidase assembly protein (COX10) (Luan et al., 2017). Consequently, mitochondrial electron transfer pathways are terminated and apoptotic pathways are initiated, resulting in caspase cascade reactions and apoptosis (Xu et al., 2013).
Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba (R. crenulata), “Jingtian (景天)” or “Hong Jingtian (红景天),” which belongs to the family Crassulaceae, has long been used as a tonic for physical fitness, for increasing blood circulation, and for enhancing bronchodilation to alleviate asthma. It has been taken as a long-term supplement by permanent lowlanders and highlanders, as well as countless soldiers, travelers, railway workers, and miners in the high-altitude regions of Tibet, Yunnan, and Sichuan in China (Fu et al., 2017; Y.Z. Zhang et al., 2018). R. crenulata has been documented in the three most recent editions of the Chinese Pharmacopoeia (2005, 2010, 2015), as well as the literature on traditional Chinese herbal medicine or Tibetan medicine, such as Shennong's Classic of Materia Medica and Yuewang Yaozhen (Wu, 1985; Mao et al., 2012). From a broader perspective, R. crenulata is known as Suoluomabao (索罗玛保), Segeda (涩疙疸), Ligaduer (力嘎都尔), and other mystical names in the traditional Tibetan medicine of China, and has proven beneficial for people who live in the plateaus, partially because of its plant-derived adaptive traits (Yu et al., 1996; YuTuo, 1983).
The main constituents extracted from the roots and rhizomes of R. crenulata are salidroside, tyrosol, gallic acid, and ethyl gallate (Han et al., 2016), which enrich energy (Qi) and blood flow, and increase pulse rate to treat antiasthma. Pharmacological evidence further suggests that R. crenulata could also mitigate CNS damage induced by plateau hypoxia such as encephalalgia, dizziness and hypomnesis. The underlying mechanisms are thought to involve antioxidative stress responses (Wu et al., 2018), increased mitochondrial membrane permeability stability and apoptotic inhibitory effects. However, involvement of the HIF-1α/microRNA 210/ISCU1/2 (COX10) signaling pathway in R. crenulata-induced protection of altitude/hypobaric brain injury remains unclear, and details of the underlying mechanisms remain unknown. In this study, a rat model of high-altitude brain injury, successfully established using a hypobaric and hypoxic animal container in our pre-laboratorial study was employed to determine the anti-hypoxic effects of root and rhizome aqueous extract from R. crenulata on hypobaric hypoxia-induced brain injury.
Section snippets
Preparation of R. crenulata
Root and rhizome samples of R. crenulata were collected in 12 villages in Cuola Township, Jiali County, Nagqu, in the Tibet Autonomous Region (Fig. 1A, B, C, and D). Identification was carried out by Zhang Yi, a research scholar in the Ethnopharmacology Department of the Ethnic Medicine College, Chengdu University of Traditional Chinese Medicine.
HPLC analysis of gallic acid, salidroside, tyrosol, and ethyl gallate contents in aqueous extracts of R. crenulata
Fine R. Crenulata powder was obtained using a low temperature pulverizer and a simple reflux extraction was performed using a reflow extractor (Fig. 1
HPLC analysis of RCAE
To determine the content of gallic acid, salidroside, tyrosol, and ethyl gallate, RCAE was analyzed by HPLC. Concentrations were determined using the following curve regression equations: gallic acid (y = 3323.6x + 97.39, r = 0.9999), salidroside (y = 329.81× - 14.72, r = 1), tyrosol (y = 791.54× - 8.64, r = 1), and ethyl gallate (y = 3749.2× - 26.62, r = 0.9992). Fig. 1G and H shows the chromatographic profile and chemical structure of the above four compounds. The retention times of gallic
Discussion
Histopathological examinations have confirmed that a lack of oxygen can cause cell swelling, shrinkage, glial cell proliferation, an unclear nucleus and nucleolus, capillary hyperemia or congestion, a decrease in neuron number, and even degeneration and necrosis in hippocampal and cortical tissue (Krugers et al., 2000; Churilova et al., 2012; Czerniczyniec et al., 2015). In this study, H&E staining was employed to determine the protective effect of RCAE on hypoxia-induced neuronal injury.
Conclusions
In conclusion, our findings suggest that oral treatment with Rhodiola crenulata protected rat neurons from HH-induced apoptosis by regulating the HIF-1α/microRNA 210/ISCU1/2 (COX10) signaling pathway. However, details of the underlying mechanisms and active components involved in the upregulation of HIF-1α, miR-210, ISCU1/2, and COX10 expression remain unknown. Further in vitro and in vivo studies are therefore needed to confirm these results.
Authors’ contributions
Xiaobo Wang, Ya Hou and Xianli Meng constructed the hypobaric hypoxia (HH)-induced brain injury model, performed hematoxylin and eosin staining, western blot and quantitative reverse transcription-quantitative polymerase chain reaction analysis. Qiuyue Li, Xuanhao Li and Wenxiang Wang determined the content of Rhodiola crenulata aqueous extract by HPLC and Glide molecular docking methodology. Xiaobo Wang, Ya Hou and Xiaopeng Ai determined the serum indicators, Nissl staining, TUNEL staining and
Disclosure statement
The authors have no conflicts of interest to declare.
Funding sources
This work was supported by the National Key R&D Program of China (2017YFC1703904), Science & Technology Department of Sichuan Province (2018JY0467) and Sichuan Science and Technology Program (2019YJ0480).
Acknowledgements
We would like express our sincere gratitude to Jinsong Su for providing the Rhodiola crenulata herbs used in this study.
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These authors contributed equally to this work.