In this study, we investigated the mechanism by which Yiqi Dingxuan Yin improves cerebral ischaemic injury in a rat model. Our results showed that Yiqi Dingxuan Yin can improve neural function and morphology after cerebral ischaemia injury in rats, with a more significant effect at 14 days. The mechanism may involve the upregulation of EPO/EPOR, VEGF, and BDNF/TrkB protein expression, which promotes angiogenesis and improves cerebral blood flow and oxygen supply, thereby protecting the form and function of neurons and ultimately promoting the restoration of damaged neural function.
AIS belongs to the category of ‘stroke disease’ in traditional Chinese medicine, and ‘qi deficiency and blood stasis’ is considered the main pathological mechanism. Buyang Huanwu decoction is the representative prescription in Wang Qingren’s ‘Yilin Gaicuo’, a manuscript from the Qing Dynasty. This decoction has the effect of invigorating qi, promoting blood circulation, and dredging collaterals. With the development of traditional Chinese medicine and continuous study of stroke disease, it has gradually been recognised that the disease has a complex aetiology and multiple pathogeneses. Common causes include obstruction of collaterals by wind and phlegm, disturbance of wind and fire, yin deficiency and wind movement, qi deficiency and blood stasis, and deficiency of the liver and kidney [12]. Yiqi Dingxuan Yin is modified based on the Buyang Huanwu decoction, which has three effects: invigorating qi and activating blood circulation, tonifying the kidney and marrow, and dispelling wind/resolving phlegm (these effects are traditionally considered in unison).
A cascade reaction after cerebral ischaemia can cause changes in free radicals, oxidative stress, and inflammatory reactions. This series of pathophysiological changes eventually leads to the apoptosis of nerve cells [13, 14].
Angiogenesis is an important factor in the process of recovery after ischaemic stroke. Following cerebral ischaemia, neovascularisation increases the blood flow supply in the cerebral ischaemia area, promotes the formation of the neovascular network, provides energy for the ischaemic and hypoxic brain tissue, and promotes the repair of neurological damage [15, 16]. The angiogenic factors released in this process include EPO, VEGF, and BDNF [17, 18].
EPO is mainly secreted by the kidney and liver, and its basic function is to promote erythropoiesis. The expression of EPO protein in the normal human brain tissue is low but increases under hypoxia, which indicates that EPO has a protective effect and repairs nerve injury. As an endogenous cytokine in the central nervous system, EPO can act on neuronal EPOR through the paracrine action of neurons and glial cells, thereby activating the downstream signal transduction pathway and participating in brain protection after ischaemia [19–22]. EPO can also reduce the expression of matrix metalloproteinase-9 and increase the expression of transforming growth factor-β1 to play a neuroprotective role by activating AMP-activated protein kinase, upregulating Kruppel-like factor 2 expression, increasing nitric oxide production, and improving angiogenesis.
As the strongest vascular permeability factor, VEGF is a highly specific vascular endothelial growth factor that promotes angiogenesis and plays an important role in the recovery of cells after MCAO [23]. After cerebral ischaemic injury, the expression of VEGF in the brain tissue increases significantly compared with that before injury [24]. After binding to its receptor, VEGF promotes the proliferation and migration of microvascular endothelial cells, induces neovascularisation, improves local blood supply, and protects endothelial cells from programmed death. Conversely, VEGF can promote microvascular regeneration to maintain a stable brain microenvironment and improve the microenvironment in a pathological state to protect neurons, which is one of the biological bases for the venation of qi and blood by which brain neurons are nutritionally supported [25].
BDNF is one of the primary members of the neurotrophic factor family that promotes neurogenesis and angiogenesis in rats with cerebral ischaemia [26]. Neuroprotective factors in the hippocampus of rats are sensitive to the ischaemic injury of neurons, and BDNF can activate intracellular signal transduction pathways to play a neuroprotective role by binding to TrkB [27, 28]. This results in the regeneration, repair, and functional recovery of nerve cells by antagonising the toxicity of intracellular Ca2+ overload-related excitatory amino acids; enhancing the activity of antioxidant enzymes; reducing the damage by oxygen free radicals; inhibiting apoptosis, necrosis, and caspase-3 activity; and regulating the expression of Bax and Bcl-2 [29].
Butylphthalide, a drug developed and used in China, can promote the formation of collateral vessels after ischaemic stroke, improve energy metabolism of the brain tissue, scavenge free radicals, inhibit neuronal apoptosis and pyroptosis, and protect neurons [9].
In this study, the protein expression of EPO/EPOR, VEGF, and BDNF/TrkB in rat hippocampi increased to varying degrees following cerebral ischaemia. Yiqi Dingxuan Yin markedly upregulated the expression of these proteins, accompanied by improvements in the defects of nerve function, changes in cell morphology, and degree of apoptosis; the effect was more significant over the course of the intervention, which may be attributed to the upregulation of the endogenous EPO/EPOR, VEGF, and BDNF/TrkB proteins.
This study has some limitations. We were unable to conduct quantitative immunohistochemical analysis and investigation of more specific mechanisms of action. Furthermore, we did not evaluate the effects of the combination of Yiqi Dingxuan Yin and butylphthalide.