†These authors contributed equally.
“Tianma” (Gastrodia) and “gouteng” (Uncaria) are both widely used to treat cerebral ischemia. At the same time, “ezhu” (Curcuma longa) or turmeric, is derived from the dried roots of C. longa. It is a polyphenol known for
its anti-inflammatory effects and its promotion of blood vessel endothelial
function. This study explored the neuroprotective effects of a water extract of
“tianma”, “gouteng”, and “ezhu” against ischemic
injury. Flow cytometry analysis showed that
Gastrodia, Uncaria, and Curcuma reduced the
proportion of apoptotic cells in CoCl
Stroke is characterized by interrupted blood flow in the brain leading to the acute neurological deficit, and it represents a significant contributor to global morbidity and mortality (Benjamin et al., 2018). Herein, we focus on ischemic stroke, the pathogenesis of which is usually due to cerebral artery occlusion or severe stenosis, although cerebral sinus or cortical vein thrombosis are also contributors. Risk factors strongly associated with ischemic stroke include advanced age, smoking, hypertension, diabetes, combined heart disease, carotid artery stenosis, and dyslipidemia (Boehme et al., 2017; Poorthuis et al., 2017; Shindo and Tomimoto, 2014; Tsai et al., 2015). The core problem of stroke is the death of neurons, and mechanisms responsible for neuronal death include mitochondrial related deaths, free radical injury, excitotoxicity, apoptosis, autophagy, and aseptic inflammation (Sekerdag et al., 2018). Intravenous recombinant tissue plasminogen activator (rtPA) is currently recommended for acute treatment options by clinical guidelines as a therapy for stroke in eligible patients. Although it is efficacious, it carries a risk of hemorrhagic complications, and its administration is limited due to the effective time window.
Traditional Chinese medicine (TCM), the most widely used form of herbalism around the world, is a sophisticated system of medical theory and practice that is quite different from modern medicine. The decoction “tianma gouteng yin”, which includes the herbs “tianma” (Gastrodia) and “gouteng” (Uncaria), is deemed in TCM as a treatment for wind disorder, calming liver wind, as well as activating blood and clearing heat. It is also used in the treatment of ischemic stroke and brain-related disease due to the mechanisms of antioxidant and anti-apoptotic (Liu et al., 2015; Xian et al., 2016; Zhang et al., 2004). “Ezhu” (Curcuma) is used to the treatment of stagnation of “qi” (the body’s vital energy), blood stasis induced chest and abdominal pain, dysmenorrhea, and limb pain. Previous studies have demonstrated its neuroprotective effects against cerebral ischemia-reperfusion injury (Huang et al., 2018; Li et al., 2017). Since “tianma”, “gouteng” and “ezhu” may have a synergistic therapeutic effect on cerebral ischemic injury, in the present study, we want to verify it through in vivo and in vitro experiments.
Crude Gastrodia (product ID. ECN000234), Uncaria (product ID. ECN000233) and Curcuma (product ID. ECN000195) (GUC) were purchased from Bencaopu online Chinese medicine shop, Laoruilin Pharmacy Chain Co., Ltd. (Zhangzhou, P. R. China). The herbs were authenticated according to the methods recorded in The Pharmacopoeia of P. R. China 2015 Edition. Voucher specimens of Gastrodia, Uncaria, and Curcuma were collected at the Shanghai Museum of Traditional Chinese Medicine. The GUC extract was prepared as follows: Gastrodia (105.0 g) was immersed in 2.5 L distilled water for one hour and then boiled for 40 min. Uncaria (140.9 g) and curcumin (105.5 g) were then added. Heat the mixture at reflux for 15 minutes and then cooled to room temperature and filtered. The resulting filtrate was rotary evaporated and lyophilized for further study (Fig. 1).
Extraction protocol of “tianma gouteng ezhu” (Gastrodia, Uncaria, and Curcuma) water extracts (GUC).
“Tianma gouteng ezhu” protects neuronal survival under hypoxia.
GUC reduced the percentage of apoptotic B35 and SH-SY5Y cells after CoCl
“Tianma gouteng ezhu” reduced apoptotic proteins under hypoxia.
(A) Protein bands of Bcl-2, Bax, Caspase-3, and Actin of CoCl
The rat neuroblastoma B35 and the human neuroblastoma
cell lines (purchased from
Cell Resource Center, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences) were grown in Dulbecco’s
modified Eagle’s medium (Hyclone; GE Healthcare) supplemented with 10% FBS
(Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin
and 0.1 mg/ml streptomycin in a humidified atmosphere at 37
B35 and SH-SY5Y cells treated with CoCl
Male Sprague-Dawley (SD) rats (Animal experiment center, School of Medicine,
Shanghai Jiao Tong University), weighting 220-250 g, were housed in cages of
The 18 rats were divided into three groups arbitrarily: sham, control (MCAO-CK), and GUC treatment (MCAO-GUC). Rats in the MCAO-CK and sham groups were administered with distilled water after middle cerebral artery occlusion (MCAO) or a sham operation. In contrast, rats of the MCAO-GUC group were fed with 250 mg/kg GUC once daily for 7 days. The MCAO operation was executed as follows: SD rats were anesthetized intraperitoneally with 350 mg/kg of chloral hydrate (VWR International Ltd), then fixed to a surgical plate. We isolated and ligated the external carotid artery (ECA) and its branch vessels. Then, a 4-0 nylon suture was inserted into the internal carotid artery (ICA) through the ECA and forward to the anterior cerebral artery to block the middle cerebral artery (MCA). After 2 hours, reperfusion was achieved by visual removal of the nylon filament. In sham rats, operations were conducted analogously without the usage of nylon filament.
Brain slices of rats were stained by 2,3,5-triphenyl tetrazolium chloride (TTC). Brains were harvested and immersed in ice-cold saline for 20 minutes, then cut into 2 mm-thick slices. After that, brain slices were stained with 2% TTC solution at room temperature without light for 20 minutes, and images were captured subsequently. Infarct regions were analyzed using Adobe Photoshop CC 2018 (Adobe Systems Inc). The infarct area of each slice was multiplied by 2 (mm) to estimate infarct volume per slice. Similarly, the total infarct volume was the summation of infarct volumes of all slices.
Rats were euthanized, and the brains were isolated and cut into
The data for each study group were given as mean
In the B35 cell line, the percentage of apoptotic cells was significantly lower
in the group treated with CoCl
Cerebral infarct caused by MCAO was apparent in both the control group and the GUC group, occurring in the right cerebral hemisphere, the cortex, basal ganglia, and hippocampus. GUC treatment was associated with a dramatic reduction of infarct volume of 40% when compared with the control group (P = 0.0256) (Fig. 4).
“Tianma gouteng ezhu” enhanced neuronal survival in MCAO models. (A) TTC staining of slices of brains. (B) Statistical analysis of sham, MCAO-CK, and MCAO-GUC groups by oneway ANOVA. MCAO: middle cerebral artery occlusion; CK: control check; GUC: Gastrodia, Uncaria, and Curcuma.
Immunofluorescence was applied to examine the expression of Caspase-7. The results showed that the expression of Caspase-7 was evident in the MCAO-CK groups (Fig. 5), while hardly evident in the sham group. Furthermore, the expression of Caspase-7 was much lower in the MCAO-GUC group than that of the control group, which suggested that GUC helped to reduce the expression of Caspase-7 in vivo.
“Tianma gouteng ezhu” reduced Caspase-7 expression in MCAO models. The expression of Caspase-7 was examined by immunofluorescence. The MCAO-GUC group showed a downregulation of the level of Caspase-7 compared to the MCAO-CK group, while in the sham group Caspase-7 was negligible. MCAO: middle cerebral artery occlusion; CK: control check; GUC: Gastrodia, Uncaria, and Curcuma.
The application of traditional Chinese
medicine in cerebral ischemia is often used
in the form of prescriptions. The so-called “Jun Chen Zuo Shi” compatibility
method, which emphasizes the combination of therapeutic herbs, has demonstrated
prominent effects in stroke, although exact mechanisms of action are unclear
(Chen, 2012; Jayakumar et al., 2015). The present study extracted crude
Gastrodia, Uncaria, and Curcuma and explored the
benefits of their combination in the immediate post-stroke period. According to
our results, GUC could protect the B35 and SH-SY5Y cells from CoCl
Furthermore, the apoptosis protein Caspase-7 was significantly downregulated with GUC treatment, indicating a restrained onset of the apoptosis pathway. Our results have confirmed the protective effect of water extract of GUC on cerebral ischemia and cell hypoxia. However, the underlying mechanism needs to be further elucidated. Existing data can only indicate that the therapeutic effect is related to the inhibition of the apoptotic pathway. As is known, the apoptosis regulatory network is complex. Experiments have found that active components of Gastrodia, Uncaria, and Curcuma function by different mechanisms to improve neuro-hypoxia/ischemic injury. The phenolic components of Gastrodia can relieve cerebral ischemia/reperfusion damage and improve prognosis in rats, and the effects seem to occur via activation of a Nrf2-mediated cell protection system (Alfieri et al., 2013; Kam et al., 2011; Kim et al., 2007; Shi et al., 2018; Yu et al., 2010).
Moreover, 4-hydroxy benzyl alcohol (4-HBA), an important
phenolic constituent of Gastrodia confers antioxidative,
anti-inflammatory and anti-apoptotic effects in neurons and microglia, and that
the molecular mechanism underpinning this is anti-Zn
Furthermore, curcumin could shift the microglial phenotype from the pro-inflammatory M1 state to the anti-inflammatory and tissue reparative M2 type, as evidenced in mouse (Liu et al., 2017). Indeed, previous studies have confirmed that Gastrodia, Uncaria, and Curcuma alone have protective effects on ischemic brain damage. The essence of TCMs is compatibility, emphasizing the role of prescriptions. Studies of “Tianma Gouteng Yin” in cerebral ischemia support the effectiveness of TCM treatment of cerebral infarction (Xian et al., 2016).
While our research has experimentally demonstrated that GUC has a therapeutic effect on hypoxic-ischemic brain damage via the reduction of apoptosis. In fact, except for GUC, traditional Chinese medicine with the anti-apoptosis effects is commonly practiced in cerebral ischemia. The TCM preparation “Naoshuantong” capsule, Cerebralcare Granule, and Astragalus injection, three traditional Chinese medicine preparations that are widely used clinically in China, are also thought to inhibit the activation of apoptosis, their mechanisms may be associated with the ERK activation, upregulated AKT phosphorylation or down-regulated JNK phosphorylation (Sun et al., 2015).
GUC protects B35, and SH-SY5Y cell lines treated with CoCl
LR and ZW conceived and designed the research. ZHW and BC conducted most of the experiments. YL and JX contributed new reagents or analytical tools and analyzed data and helped with some of the experiments. BC wrote the manuscript. YL analyzed the data and helped revise the draft. All authors read the manuscript carefully, agree with the manuscript content, and are willing to take responsibility for the research contents.
The animal studies were approved by the Ethics Committee of Shanghai Pudong Hospital, Fudan University.
The authors acknowledge funding support from the Pudong New Area Health and Family Planning Commission (PWZzk2017-16) and the Outstanding Leaders Training Program of Pudong Health Committee of Shanghai (PWRL2017-03).
The authors declare no conflict of interest.