Stroke, a cerebrovascular accident, is a clinical event associated with focal or diffuse brain function deficits caused by acute cerebral circulation disturbance. Stroke is also the second leading cause of death in urban and rural residents diagnosed with malignant tumors.1 There are two main categories of stroke according to their pathological properties: ischemic stroke and hemorrhagic stroke. Intracerebral hemorrhage (ICH) refers to a hemorrhage in the brain parenchyma and is characterized by rapid onset, neurological deterioration, and poor outcome.2 Acute intracerebral hemorrhage (AICH) was observed in 23.4% of patients who had a stroke in China, 46% of whom have died or suffered a severe disability within one year,3 and 36% of survivors remained moderately to severely disabled at discharge.4 Many modifiable risk factors, including arterial hypertension, excessive consumption, decreased low-density lipoprotein cholesterol, and low serum triglyceride levels, can cause AICH.5 Conventional therapies for AICH include hematoma removal, edema attenuation, and intracranial pressure reduction. However, contrary to expectations, the effectiveness of therapy is unsatisfactory.6 Therefore, it is necessary to develop new strategies to treat ICH.
Traditional Chinese Medicine (TCM) has been shown positively effect on stroke.6 Professor Ren Jixue, who has made remarkable contributions in TCM encephalopathy, especially in emergency cases, established the remarkably effective method of “removing blood stasis” to treat hemorrhagic stroke. In particular, Di Dang decoction (DDD) is a classical TCM prescription for acute hemorrhagic stroke and is representative prescription to “remove blood stasis,” which comes from the “Treatise on febrile diseases”. Specifically, DDD promotes blood circulation and removes blood stasis, effectively purging and clearing the viscera. DDD includes rhubarb (Latin name: Rheum palmatum L), peach kernel (Latin name: Prunus persica L. Batsch), leech (Latin name: Whitmania pigra Whitman), and gadfly (Latin name: Tabanus mandarinus Schiner). Using DDD to treat AICH has a long history of demonstrated effective clinical therapeutic effects. Our previous study showed that DDD significantly reduced brain water content and intracerebral hematoma volume in rats with ICH by up-regulating the expression of brain-derived neurotrophic factor, tyrosine kinase B and vascular endothelial growth factor (VEGF).7 Previous study also showed that Di Dang decoction inhibits endoplasmic reticulum stress-mediated apoptosis induced by oxygen glucose deprivation and intracerebral hemorrhage through blockade of the GRP78-IRE1/PERK pathways.8 We also found that the protective effect of Di Dang decoction against AlCl 3-induced oxidative stress and apoptosis in PC12 cells through the activation of SIRT1-Mediated Akt/Nrf2/HO-1pathway.9 In addition, previous reports showed that leech alcohol extract and rhubarb water extract alleviated the inflammation of peripheral tissue and cerebral edema in rats with ICH.10,11 Peach seed water extract up-regulated VEGF and VEGF receptor 2 after ICH in a mouse model6 and inhibited glucose deprivation injury in PC12 cells.12 However, its specific molecular mechanism of protecting against acute intracerebral hemorrhage stroke in rats remains unclear. This study aimed to investigate the effect of DDD on AICH stroke rats and to clarify its mechanism of action to guide its clinical use.
The experimental animal protocol was approved by the Animal Ethics Committee of Changchun University of Traditional Chinese Medicine (Approval No.: 20180008). All animal experimental procedures were performed in accordance with the guidelines of the National Institutes of Health on the care and use of animals. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved animal quarters in our hospital. Studies were carried out on adult Sprague Dawley (SD) rats (180–220 g, 8–10 weeks of age) obtained from Changchun Yi si Laboratory Animal Technology Co. Ltd. All animals were housed under identical conditions (room temperature at 25 °C and 12/12 h light/dark cycle), and allowed ad libitum access to food and water. Before inducing the AICH model, all rats were fed for one week.
DDD Preparation and HPLC Fingerprint Analysis
According to the original prescription from the “TCM prescriptions dictionary,” DDD is composed of four Chinese components: rhubarb (Chinese name: Dahuang, Latin name: Rheum palmatum L, Family: Polygonaceae, Batch number: 170916, Part used: root and rhizome), peach kernel (Chinese name: Taoren, Latin name: Prunus persica L. Batsch, Family: Rosaceae, Batch number: 171011, Part used: seed), leech (Chinese name: Shuizhi, Latin name: Whitmania pigra Whitman, Family: Hirudinidae, Batch number: 170824, Part used: whole animal), and gadfly (Chinese name: Mengchong, Latin name: Tabanus mandarinus Schiner, Family: Tabanidae, Batch number: 171015, Part used: whole animal). All dried components were purchased from the Affiliated Hospital of the Changchun University of Chinese Medicine and identified through the department of Medicine. First, the herbs were minced, mixed, soaked for 30 min in distilled water, and then decocted in distilled water at 100 °C for 30 min. The above procedure was repeated three times, merging decoctions, and increasing the concentration. The concentrated solution was freeze-dried under a vacuum and grounded into a powder. According to the ratio of equivalent dose converted by body surface area between human and rat, the powder was dissolved in distilled water to final concentrations of 0.625 g/mL (high-dose), 0.3125 g/mL (medium-dose), and 0.15625 g/mL (low-dose) for later use.
As our team reported,8,9 we have established a method for the detection of active ingredients from DDD via high-performance liquid chromatography (HPLC, Agilent, Santa Clara, CA, US). Eighteen major peaks of DDD extract were identified using HPLC. Gallic acid, amygdalin, sennoside B, rhein-8-glucoside, sennoside A, emodin, chrysophanol, aloe-emodin, and rhein in DDD were identified by comparing the retention time from high-performance liquid chromatography with good reproducibility.
Experimental Animal Grouping
A total of 135 healthy SD rats were randomly assigned to five groups: control, model, DDD low-dose, DDD medium-dose, and DDD high-dose groups. Twenty-seven healthy SD rats were used in each group. Each group was further divided into three subgroups, each of which contained at least nine rats.
AICH Stroke Model Establishment
The model, low-, medium-, and high-dose groups were intraperitoneally injected using a posterior pituitary injection of 2 U/kg once daily for 14 days. After 14 days, the modified Nath method was used to replicate the AICH model.8 Rats were intraperitoneally injected with 1% sodium pentobarbital, fixed in the supine position. The skin was prepared, the femoral artery was separated, and its distal end was ligated. Furthermore, 50 µL of femoral arterial blood was extracted, the artery was then ligated and surgically sutured. Subsequently, rats were fixed in a stereoscopic brain locator; the skin was prepared and routinely disinfected, the anterior fontanelle was separated, the caudate nuclear was localized, and 50 µL of autologous blood were slowly injected. After the operation, the scalp was sutured and coated with penicillin powder. The control group underwent the same procedure excluding the injection of autologous blood into the caudate nucleus.
Determination of model success: after the rats were fully alert following surgery, the modified Neurological Severity Score (mNSS)13 was used to evaluate the neurological deficit of ICH model rats; an mNSS score 6, and hematoma formation could be seen in the perfused brain tissue, indicating that the model was successfully established. If the symptoms of neurological impairment were too mild, absent, or too great, rats with impaired consciousness, difficulty in moving, or that died, they were discarded. Gavage was given immediately after successful modeling according to their respective dose concentrations; while DDD in the control and model groups was replaced with an equivalent dose of normal saline (each group was administered 1 mL/100 g of body weight gavage intragastrically once a day at the same time).
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