The regulation of method of tonifying Qi and activating blood circulation in the related gene expressions after spinal cord injury

Xiao Fan, Li Zhang *Orthopaedics and Traumatology College of Fujian University of Traditional Chinese Medicine, Fuzhou 35022.

Introduction

Spinal Cord Injury (SCI) is an injury to the spinal cord and cauda equine resulting in dyskinesia, sensory disturbance, dysreflexia and sphincter dysfunction of limbs below lesion level. Common cause is trauma to the spine which leads to spine fracture and dislocation.Spinal cord injury includes primary spinal cord injury and secondary spinal cord injury [1], both of which could lead to paraplegia or even death. Without ideal treatment, spinal cord injury will be a heavy burden on the patients, the families as well as the society [2,3].The pathological reaction of spinal cord injury is related to the post-injury abnormal expressions of related genes, including Aquaporin4 (AQP4) gene expression, B cell lymphoma leukemia 2 (Bcl-2) gene expression, Cysteine aspartate-specific protease(Caspase) gene expression, Heat shock protein (HSP)gene expression, Interleukin (IL) gene expression,Neurotropic factor (NF) gene expression, Nuclear Factor Kappa B (NF-κB) gene expression, Tumor necrosis factor (TNF) gene expression and so on. As an important treatment to spinal cord injury, the method of tonifying Qi and activating blood circulation has shown its prominent effects in clinic [4-7]. The regulating effects of some herbs and prescriptions often used in the method of tonifying Qi and activating blood circulation in the above mentioned gene expressions, including ginseng, salvia miltiorrhiza,astragalus, pseudo-ginseng, ligusticum wallichii,Buyang Huanwu Decoction, Huoxue Tongdu Decoction, and so on. It may be an important mechanism in treating spinal cord injury. The essay briefly summarizes the research progress of the regulation of method of tonifying Qi and activating blood circulation in the related gene expression after spinal cord injury.

The Pathological Mechanisms of SCI and Related Pathways Regulated by Method of Tonifying Qi and Activating Blood Circulation in SCI

Up to now, many pathological mechanisms have been found to be involved in SCI including spinal cord vascular injury, spinal cord edema, lipid peroxidation,intracellular ion imbalance especially Ca2+overload,inflammation, toxicity of excitatory amino acid, and apoptosis of neurons [8]. SCI is undoubtedly of complex pathological mechanisms, but method of tonifying Qi and activating blood circulation is not of"simple" functional mechanisms either which can regulate various pathways instead of a specific pathway to inhibit the abovementioned pathological mechanisms so as to treat SCI, such as EAAS-NMDA-Ca2+pathway [9], excitatory amino acids-glutamate/cystine transporter pathway,mitochondrial pro-apoptotic pathway, and so on.

The Limitations of Current Treatments of SCI

In modern medicine, there are a lot of therapies to treat spinal cord injury including surgery, drugs, cellular transplantation and gene therapy and so on. However,none of these is an ideal method to cure spinal cord injury because of the lack of security, indefinite curative efficacy, controversy in ethics and presence of complications. Surgery, in primary stage of spinal cord injury, can relieve pressure, fix fracture and reconstruct spinal stability and is beneficial to create conditions for restoration of spinal nerves and early rehabilitation.But it is with high surgical risks, high expense and indefinite curative efficacy. Methylprednisolone (MP)is a typical drug used extensively to treat SCI in clinic.However, there is debate about its curative efficacy.Some scholars [10] advocate that administration of MP within 8 hours of spinal cord injury is beneficial to improve the recovery of neurological function, motor function and sensory function, while others [11-13]conducted some randomized clinical trials finding that no evidence supporting the opinion that high-dose MP administration facilitates neurologic improvement in patients with spinal cord injury. But instead the incidence of complications like infection,hyperglycaemia and other metabolic diseases are increased by MP. Cellular transplantation and gene therapy have become hotter and hotter to be applied in treating SCI in some studies recently. However, the application of these approaches is restricted by some problems like how to regulate the differentiation of stem cells and expression of specific gene, how to inhibit immune reaction, and how to address ethical and moral issues.

The Advantages of Method of Tonifying Qi and Activating Blood Circulation of Traditional Chinese Medicine

The method of tonifying Qi and activating blood circulation of traditional Chinese medicine has distinct advantages in its low cost and effective clinical results,having become a novel approach to treat SCI. Besides,the method exerts its effect on SCI in multi channels and multi targets, by alleviation of spinal hydrops,reduction of toxicity of excitatory amino acid,suppression of lipid peroxidation, Ca2+overload and inflammation [8].

Theoretical Basis of the Application of Method of Tonifying Qi and Activating Blood Circulation in Spinal Cord Injury

Spinal cord injury is categorized as “flaccidity syndrome” and “bodily indolence” in traditional Chinese medicine. Spine houses the spinal cord and governor vessel. Running through the spinal cord,governor vessel, as the sea of yang meridians and the junction of yang energies, controls the yang energies all over the body. Injuries due to tumbling will damage the spine, and further damage the spinal cord and governor vessel. It will damage the three yang meridians of hand and foot, kidney-yang and Qi and blood circulation, leading to pathological reactions like retrograding of Qi and blood, Qi stagnation and blood stasis, Qi and blood deficiency and blockage of governor vessel by congestion. Limb function will thus be affected, experiencing limb flaccidity, acratia or even paraplegia. Hence, the pathogenesis of spinal cord injury can be summarized as ineffectiveness of governor vessel’s control in meridians [14]. Based on the above pathogenesis, treatment of spinal cord injury should be guided with the principles of tonifying Qi,activating blood circulation, promoting blood circulation, removing stasis, dredging meridians and recovering spinal cord. Tonifying Qi and activating blood circulation refers to that by using herbs and prescriptions with the effects of tonifying Qi and activating blood circulation like salvia miltiorrhiza,ginseng, astragalus, ligusticum wallichii,pseudo-ginseng, Buyang Huanwu Decoction, Huoxue Tongdu Decoction to attain aims of tonifying Qi,activating blood circulation, promoting blood circulation and dredging meridians. Qi is in control of blood circulation and blood is the origin of Qi that only when Qi is plentiful will blood circulation be activated which will further nourish spinal cord and dredge meridians. Thus, applying method of tonifying Qi and activating blood circulation in treating of spinal cord injury has sufficient theoretical bases.

Researches of Clinical Effects

With sufficient theoretical bases, applying method of tonifying Qi and activating blood circulation in treating spinal cord injury shows its significant therapeutic effect in clinical practice.

Astragalus, an important herb in method of tonifying Qi and activating blood circulation, is widely used in clinic for the treatment of spinal cord injury and clinical studies have found that Astragalus Injection can promote neurological function recovery of patients with thoracolumbar vertebral fracture and spinal cord injury [15].

Buyang Huanwu decoction, which is a representative prescription of method of tonifying Qi and activating blood circulation is used extensively and has a notable effect. In clinical study, Buyang Huanwu decoction can enhance neurological function score of SCI patients via promoting recovery of spinal cord,proving to be effective to treat acute spinal cord injury[5]. Tang Yong el al. [6] carried out a study on adjuvant therapy of spinal cord injury by Buyang Huanwu decoction plus musk, treating the patients in control group with only conventional treatment and those in experimental group with Buyang Huanwu decoction besides conventional treatment. A notable difference was found between control group and experimental group in the score of sensory and motor function(P<0.05) after 4 weeks, which indicated that Buyang Huanwu decoction plus musk is effective in SCI treatment. Besides, in some randomized clinical trials,Buyang Huanwu decoction has been proved not only to be effective to the post-surgery rehabilitation of burst fracture of thoracic-lumbar spine concomitant with incomplete paralysis, similar effect as that of monosialotetera-hexosyl ganglioside, but also to be beneficial to improve the motor function and the motor evoked potential prominently in patients with thoracic-lumbar vertebrae fracture accompanied with spinal cord injury [16,17].

Huoxue Tongdu decoction, an empirical prescription of ZHANG An-zhen professor, has been proved to be able to tonify Qi, activate blood circulation and dredge governor meridian, recover spinal cord in clinic and has a therapeutic effect on spinal cord injury and spinal cord ischemic-reperfusion injury [4,7].

Researches on mechanisms of the regulation of gene expressions

Promotion of AQP4 gene expression

AQP4 is a specific protein which can transport hydrone bi-directionally, maintaining the internal and external osmotic pressure of cells. Due to the specific transportation function, Verkman et al. [18] regarded it as a new target to inhibit hydrops post spinal cord injury. As the effective composition of giseng,ginsenoside Rb1 has been proved to boost the AQP4 and mRNA expression remarkably to reduce nerve hydrops and apoptosis in experimental study [19].Besides, experimental results show that the mechanism of Buyang Huanwu decoction to alleviate hydrops is by restraining the AQP4 expression after spinal cord injury and thereby mitigate the secondary damage and promote the limbs function of SCI experimental rats[20].

Promotion of Bcl-2 gene expression

Bcl-2 plays a critical role in the apoptosis process by co-acting with Bax [21]. Bcl-2 inhibits apoptosis while Bax can promote it. The ratio of Bcl-2 and Bax reflects the apoptosis level that increase in the ratio indicates cell survival and decrease indicates cell death.Tetramethylpyrazine (TMP) is extracted from ligusticum wallichii. SUN Hai-yan et al. [22]conducted an experiment by intraperitoneal injection of TMP into SCI experimental rats with the findings that TMP is able to promote Bcl-2 gene expression and inhibit Bax gene expression. It can be inferred that TMP probably functions to protect nerve via regulation of Bcl-2 and Bax gene expression. In the study about the influence of joint use of sodium aescinate (SE)extracted from ash bark and basic fibroblast growth factor (bFGF) on SCI experimental rats modelling by Allen WD method, ZHAO Peng et al. [23] found that SE possibly decreases apoptosis to protect nerve by elevating Bcl-2 gene expression. Compared with the control group, the experimental groups respectively applied with SE, bFGF and SE plus bFGF all experience an increase in Bcl-2 gene expression and the difference is statistically significant. In the TUNEL test of apoptosis, positive cells in all the experimental groups obviously reduced and the difference was statistically significant. In addition, it was found by SHANG Jian-wei et al. [24] through experiment that ginsenoside can also help neurological function recovery through promoting Bcl-2 gene expression and decreasing post-SCI apoptosis.

Promotion of NF gene expression

Neurotrophic factor (NF) is a class of polypeptide factors with the function of maintaining cell survival,promoting cell proliferation and differentiation and sustaining cell normal functions, mainly including nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), bFGF, ciliary neurotrophic factor (CNF) and neurotrophic factor 3 (NT3).Numerous studies have demonstrated that these neurotrophic factors play an important role in spinal cord restoration, neuron survival and synapse regeneration after spinal cord injury [25]. Total panax notoginseng saponins (tPNS) is the effective ingredient of pseudo-ginseng. To study the influence of tPNS to BDNF expression after spial cord injury, LI Hua ea al.[26] respectively injects tPNS and physiological saline intraperitoneally to the hemisected SCI experimental rats and detects BDNF expression with immunofluorescence method at 1d, 3d, 7d, 14d and 28d after injection, finding that BDNF expression at lesion side increases gradually, and at the same time,BDNF expression of tPNS group increased prominently compared with control group, which indicated that tNPS may improve the post spinal cord injury microenvironment, protect neuron, accelerate spinal cord replacement and enhance limbs function through promoting BDNF expression. Besides,compound salvia miltiorrhiza can protect spinal cord from spinal cord secondary injury via enhancement of NGF and BDNF expressions [27].

Inhibition of Caspase gene expression

Caspase gene family is in a critical part of the mechanisms of cell apoptosis, regulating the apoptosis signal by mediating different signal pathways. Caspase 3 in particular, plays a leading role in apoptosis, known as the death protease [28,29]. WANG Jian et al. [30]conducted an experiment to observe the effect of TMP on neuron apoptosis and expression of Caspase-3 mRNA in rats with spinal cord injury. 120 rats were divided randomly into 4 groups including normal group, model control group, MP treatment group and TMP treatment group. The result is that there is no prominent difference between TMP treatment group and MP treatment group at each time point in apoptosis and Caspase-3mRNA expression, but compared with model control group, the difference is notable,indicating that TMP may inhibit Caspase-3mRNA expression to reduce neuron apoptosis as MP. JIANG Jian-feng et al. [31] found that extracts of ginkgo leaves can down-regulate Caspase-3 expression during the primary phases of spinal cord injury to reduce apoptosis and thereby protect neurons.

Inhibition of IL gene expression

A large amount of inflammatory mediators like IL-1,IL-6 will be produced at lesion after spinal cord injury.As important inflammatory mediators inside the body,IL-1 and IL-6 promote vascular endothelial cell to express ICAM-1 to activate inflammation and destroy microenvironment of spinal cord. HAO Yang-quan et al. [32] injected Huangqi Injection to spinal cord injury model rats, finding it can reduce IL-1 expression to inhibit ICAM1 and inflammation to improve limbs function, diminish pathological changes of tissues and protect spinal cord. Furthermore, the inhibition of IL-1β and TNF-α expression and up-regulation of IL-10 expression were regulated by TMP to protect spinal cord neuron and inhibit inflammation caused by spinal cord ischemia reperfusion in a study [33].

Inhibition of NF-κB gene expression

NF-κB was found by Sen on 1986 from the extractive of B lymphocytes. It is an important moderator of inflammatory factor which can combine with κB fragment, the enhancer of immune globulin light-chain gene, to regulate the expression and transcription of immune factor, chemotactic factor，growth factor and other cell factors to regulate inflammation [34,35].NF-κB activated abnormally by CNS injury may promote the expression of cell factors to active apoptosis of neurons. ZHANG Li et al. [36]investigated the effect of Tanshinon IIA sulfonate on expression of NF-κB and VCAM-1 during spinal cord ischemia reperfusion injury indicating Tanshinon IIA could inhibit the expression of NF-κB to reduce spinal cord ischemia reperfusion injury, which may be the mechanism of protection of spinal cord. Similar to the effect of Tanshinone IIA, Compound Salvia miltiorrhiza can inhibit the expression of NF-κB to reduce inflammation to diminish spinal cord secondary injury to protect spinal cord [37]. As a representative prescription of method of tonifying Qi and activating blood circulation, Huoxue Tongdu decoction is a classic prescription to treat spinal cord injury [4,7]. It is proved to be effective to spinal cord injury in clinic by inhibition of NF-κB and VCAM-1 expression to reduce inflammation and spinal cord injury ischemia injury [38].

Inhibition of TNF expression

TNF is a cell factor producedby activated mononuclear macrophages. It is an important inflammatory mediator which can activate and promote expressions of many cell factors to aggravate inflammation, playing a significant role in spinal cord injury [39]. Tanshinone, the effective ingredient of salvia miltiorrhiza had been proved to inhibit the expression of TNF-α and IL-1β and up-regulate IL-1Ra expression to protect nerves [40].

Figure 1: The pattern diagram of method of tonifying Qi and activating blood circulation in the treatment of SCI

Regulation of HSP gene expression

The mechanism of HSP in spinal cord injury is very complicated due to its dual effect [41]. One sort of HSP like HSP27 and HSP70 protect cells from injuries by ischemia and hypoxia and reduce apoptosis accordingly while another sort of HSP like HSP47 promotes release of inflammatory mediators resulting in inflammation and scar tissue fibrosis after spinal cord injury. ZHANG Li et al. [42] studied the effect of Tanshinone IIA on the expression of HSP70 and Bcl-2 via injection of Tanshinone to experimental rats with spinal cord ischemia reperfusion injury, finding that it can promote expression of HSP70 and Bcl-2 and inhibit Bax expression, which may be its mechanism to reduce apoptosis and pathological injury.

Conclusion

Abnormal gene expression is a post-SCI process during which various kinds of cell factors and harmful substance will be produced. Some researches have found that method of tonifying Qi and activating blood circulation can regulate post-SCI gene expression,attain the goals of reducing hydrops, inhibiting inflammation, regulating apoptosis, improving spinal cord microenvironment and protecting neuron and thereby enhance limbs function after SCI (Figure 1).This provides a new thought and method to spinal cord injury treatment and related research. But mechanisms of many herbs used in method of tonifying Qi and activating blood circulation to treat SCI remain unclear as many researches are laboratory studies with little clinical practice. As molecular biology develops continuously and scientists make progress in researching traditional Chinese medicine constantly,traditional Chinese medicine is expected to bring its advantages into full play and obtain considerable development in treating spinal cord injury, benefiting the whole mankind.

Competing interests

The authors declare that they have no competing interests.

Acknowledgement

This work is financially sponsored by National Nature Science Foundation of China (81273775).

Reference

1. Gao YL, Li Y, Li WL. The progress of drug treatments of spinal cord injury. Zhejiang J Trauma Surg 2013, 18(1): 141-144.

2. Baaj AA, Uribe JS, Nichols TA, et al. Health care burden of cervical spine fractures in the United States: analysis of a nationwide database over a 10-year period. J Neurosurg Spine 2010, 13(1):61-66.

3. Elisabeth P, Shiroma EJ, Wilson DA. Statewide investigation of medically attended adverse health conditions of persons with spinal cord injury. J Spinal Cord Med 2010, 33(3): 221-231.

4. Zhang JH, Zhang L. Clinical experience of professor ZHANG Li on application of Huoxue Tongdu Decoction. Chin J Tradit Chin Med Pharm 2014, 29(6): 1875-1877.

5. Zhang Y. The clinical observation of Buyang Huanwu Decoction for acute spinal cord injury.Fujian University Traditional Chinese Medicine, 2014.

6. Tang Y, Qi W, Ye CL, et al. The clinical study on the adjuvant therapy of spinal cord injury by Buyang Huanwu decoction plus musk. J Chin Med 2013, 28(2): 288-290.

7. Liu Y, Zhang JH, Zhang L. Using the treatment experience of professor ZHANG An-zhen on the treatment of spinal cord injury. Chin J Trad Feb Traum Orthop 2014, 22(2): 63-65.

8. Zhang JH, Zhang L. Study on function and mechanism of traditional Chinese medicine on the microenvironment of spinal cord ischemia-reperfusion injury. Chin J Tradit Chin Med Pharm 2014, 29(1): 208-211.

9. Lin X. Influence of Tanshinone on the EAAs-NMDA-Ca2+ Pathway in a rat models of spinal cord ischemia/reperfusion injury. Fujian University Traditional Chinese Medicine,2010.

10. Bracken MB, Shepard MJ, Collins WF, et al.Methylprednisolone or naloxone treatment after acute spinal cord injury: 1-year follow-up data. J Neuro Surg 2009, 76(1): 23-31.

11. Yasuo I, Yoshihisa S, Masao T, et al. Does high dose methylprednisolone sodium succinate really improve neurological status in patient with acute cervical cord injury?: a prospective study about neurological recovery and early complications.Spine 1976, 34(20): 2121-2124.

12. Matsumoto T, Tamaki T, Kawakami M, et al. Early complications of high-dose methylprednisolone sodium succinate treatment in the follow-up of acute cervical spinal cord injury. Spine, 2001,26(26): 426-430.

13. Zhu ZC, Jiao W, Cai GD, et al. Early complications of high-dose methylprednisolone in acute spinal cord injury patients. Injury 2008,39(7): 748-752.

14. Wang MX, Wang X, Yao GH. Chinese medicine for the understanding of the pathogenesis of spinal cord injury. Guid J Tradit Chin Pharm 2004, 10(6):7-11.

15. Luo JP. Experimental study and clinical observation on the impact of astragalus membranaceus on neural fuction after spinal cord injury. Fujian College of Traditional Chinese Medicine, 2013.

16. Zhang TL. Observation rehabilitation effects on post-surgery of burst fracture thoracic-lumbar spine concomitant with incomplete paralysis using Buyang Huanwu Decoction. Chengdu University of Traditional Chinese Medicine, 2012.

17. Zhong GX. Clinical Study of Buyang Huanwu Decoction on the thoracic and lumbar vertebrae fracture accompanied with spinal cord injury.Fujian College of Traditional Chinese Medicine,2009.

18. Alan S, Verkman, Julien, et al. Aquaporin-4:orthogonal array assembly, CNS functions, and role in neuromyelitis optica. Acta Pharm Sin 2011,32(6): 702-710.

19. Huang F. Experimental study on spinal cord ischemia-reperfusion injury in rats by panaxoside Rb1 adjust AQP4 expression. Jilin University,2012.

20. Wang W, Xie J, Fang J, et al. The effect in the AQP4 expressive of experimental spinal cord injury in rats by Buyang Huanwu decoction. Chin J Rehabil 2005, 20(1): 3-5.

21. Geng WQ, Hou Y, Sun SM, et al. The research progress of proto-oncogene Bcl-6. Prog Vet Med 2013, 34(3): 110-113.

22. Sun HY, Chen XW, Gui BJ, et al. The effect of gene expression after spinal cord injury in rats by ligustrazine. J Cervicod Lumb 2005, 26(6):425-428.

23. Zhao P, Ji ZY, Yang Z, et al. The experimental research of the aescine sodium, basic fibroblast growth factor and the protection of neurons in the two joint use of the synergies after spinal cord injury in rats. Chin Community Dr 2013, 15(3):7-9.

24. Shang JW, Kong KM, Qi WL, et al. Effect of panaxoside on nerve cell apoptosis after spinal cord injury. Guangdong Med J 2006, 27(1): 37-39.

25. Luo W. The changes on the expression of neurotrophic factors and apoptosis genes following hSCI. Kunming Medical college, 2004.

26. Hua L , Chen A, Liang L , et al. Effect of tPNS on Spinal Cord Injury and the Influence on BDNF Expression. Prog Mod Biomed 2012, 12(12):2268-2271.

27. Liu Q. Effect of compound Danshen on acute spinal cord injury in rats NGF, BDNF expression.Liaoning University of Traditional Chinese Medicine, 2011.

28. Guo H, Cai X L. Research progress of Caspase and inducing factors on cell apoptosis. J Henan Med Coll Staff Work 2012, 24(6): 847-850.

29. Zhang X, Gao W J. Study progress of Caspase-3 on nerve cell apoptosis with cerebral ischemia-reperfusion injury. J Chengde Med Coll 2013, 130(1): 55-58.

30. Wang J, Jia YZ, Zhen CW, et al. Effect of tetramethylpyrazine on expression of early apoptosis and Caspase-3 gene in rats with spinal cord injury. Chin Arch Tradit Chin Med 2011,29(12): 2662-2668.

31. Jiang J, Zhang F, Wei A. Experimental study on neuroprotective effect of EGb after spinal cord injury in rats. Mod J Integr Tradit Chin West Med 2012, 21(18): 1958-1960.

32. Hao YQ, Qu Q, Shang RA, et al. Preconditioning of Huangqi injection on the expression of intercellular adhesion Molecule-1 in Spinal Cord Ischemia-reperfusion Injury in Rats. Chin J Orthop Traumatol 2009, 17(6): 10-12.

33. Fan L1, Wang K, Shi Z, et al. Tetramethylpyrazine protects spinal cord and reduces inflammation in a rat model of spinal cord ischemia-reperfusion injury. J Vasc Surg 2011, 54(1): 192-200.

34. Robbins D, Zhao Y. Imaging NF-κB signaling in mice for screening anticancer drugs. Methods Mol Biol 2011, 716(1): 169-177.

35. Zhu BS, Xing CG, Lin F, et al. Blocking NF-κB nuclear translocation leads to p53-related autophagy activation and cell apoptosis. World J Gastroenterol 2011, 17(4): 478-487.

36. Zhang L, An GY, Zhang WG, et al. Effects of tanshinone- II A sulfonate on expression of nuclear factor-kappaB, vascular cell adhesion molecule-1 and hemorrheology during spinal cord ischemia reperfusion injury. Chin J Orthop Traumatol 2012,25(12): 1016-1020.

37. Wang JM. Effect of compound Danshen on inflammatory factor NF-κB after acute spinal cord injury in rats. Shantou College, 2007.

38. Zhang L, Zhang JH, Chen K, et al. Effect of Huoxue Tongdu decoction on inflammatory factor NF-kB and VCAM-1 during spinal cord ischemia-reperfusion injury. Chin J Tradit Chin Med Pharm 2015, 30(4): 1020-1023.

39. Qiu CH, Hou G, Huang DN. Molecular Mechanism of TNF-α Signal Transduction. Chin J Biochem Mol Biol 2007, 23(6): 430-435.

40. Chen XH. Wang HM, Zhang L. Effects of Tanshinone on the Changes of TNF-α, IL-1β and IL-1Ra in Rabbit Model with Spinal Cord Ischemia Reperfusion Injury. Chin J Trad Med Traum Orthop 2012, 20(4): 1-3.

41. Zhou Y, Xu L, Song X, et al. The role of heat shock proteins in spinal cord injury. Eur Spine J 2014, 23(7): 1480-1490.

42. Zhang L, Gan W, An G. Influence of Tanshinone IIa on heat shock protein 70, Bcl-2 and Bax expression in rats with spinal ischemia/reperfusion injury. Neural Regen Res 2012, 7(36): 2882-2888.