Chinese Medicine in Diabetic Peripheral Neuropathy: Experimental Research on Nerve Repair and Regeneration

Diabetic peripheral neuropathy (DPN) is one of the most common complications of chronic diabetes mellitus. Pathological characteristics of DPN include axonal atrophy, nerve demyelination, and delayed regeneration of peripheral sensory nerve fibers. The goal of treatment in DPN is not only to ameliorate neurological symptoms but also to slow or reverse the underlying neurodegenerative process. Schwann cells and neurotrophic factors play important roles in the repair and regeneration of peripheral nerves. The present paper reviews current studies and evidence regarding the neurological effects of traditional Chinese medicine, with an emphasis on recent developments in the area of nerve repair and regeneration in DPN.


Introduction
Diabetic peripheral neuropathy (DPN) is a common complication of chronic diabetes. Pathological characteristics of DPN include axonal atrophy, nerve demyelination, and delayed regeneration of peripheral sensory nerve fibers. To our knowledge, the pathophysiological mechanism of DPN in dysfunctional peripheral nerve repair and regeneration is not well understood.
The symptoms associated with DPN have been mentioned in various traditional Chinese medicine (TCM) references. Pujifang (Prescriptions for Universal Relief), an ancient Chinese medicine book written in the Ming dynasty, described the following constellation of symptoms: "The kidney pattern of diabetes consists of symptoms of thirst, dry eye, impotence, and annoying pain in the hands and feet." Moreover, in Wangxugaoyian (Medical Records of Wangxugao) from the Qing dynasty, there was a case of a patient with diabetes noted to have "numbness of hands and feet" and "limbs as cold as ice." The differentiation of DPN implicates the domains of "sinew impediment," "blood impediment," and "leg flaccidity" in Chinese medicine [1].
From the viewpoint of TCM [1], the etiology and pathogenesis of DPN are as follows: (1) with an increased duration of disease in diabetes, a deficiency of yin burns body fluid and blood, resulting in empty heat. This increases blood viscosity, resulting in blood stasis, as well as blockage of sinews and channels; (2) excessive intake of foods high in fat and sugar content results in the deficiency of spleen and stomach, resulting in the accumulation of dampness and phlegm, which has a synergistic effect with stasis; (3) sinew and channels demonstrate poor nourishment because of the deficiency of liver and kidney; (4) the deficiency of yin results in a deficiency of yang, which generates an inner cold that results in microvascular coagulation. These four aspects result in a decreased peripheral flow of qi and blood to muscles, sinew, and channels. With regard to visceral organ systems, DPN is related to the liver, spleen, and kidney. The nature of DPN is deficiency secondarily complicated by excess; the deficiency is the root, and the excess is a subsequent manifestation. The root cause is deficiency in qi, yin, and yang; the subsequent complication is blood stasis and phlegm accumulation. Common patterns and treatments of DPN are summarized in Table 1.
Recently the effects of Schwann cells and neurotrophic factors on the repair and regeneration of peripheral nerve have been of research interest. Recent studies have shown that TCM medications may affect neuronal repair and 2 Evidence-Based Complementary and Alternative Medicine   Table 2). The literature search was conducted in the following database: China Journals Full-Text Database (2002-2012) (http://dlib.cnki.net/kns50/index.aspx). The keywords used were: nerve repair, nerve regeneration, Chinese medicine, acupuncture, sciatic nerve, diabetic rats, Schwann cell, neurotrophic factors, and diabetic neuropathy. The authors read full articles and reached consensus after discussion. The effects and mechanisms of Chinese medicine on nerve repair and regeneration were reviewed. Articles included in the study covered the following domains of TCM: (1) Chinese herbal medicine therapy and (2) acupuncture and moxibustion. Research of monomers, review articles, and abstracts were excluded. A total of 21 peer-reviewed papers written in Chinese were included in this paper.

Schwann Cells
Schwann cells are glial cells of the peripheral nerve system. They are important for maintaining the microenvironment for regeneration of peripheral nerves. Schwann cells not only support the repair of peripheral nerves, but they also induce, stimulate, and modulate axonal regeneration and myelin formation via expression and secretion of multiple proteins, peptides, and other bioactive substances. Thus, Schwann cells play an important role in promoting repair and regeneration after peripheral nerve injury. In hyperglycemia, a series of changes, including abnormal expression of proteins and enzymes, result in increased apoptosis and decreased cell proliferation and repair signals [23,24]. Therefore, inhibiting apoptosis and promoting growth of Schwann cells may be crucial in the prevention and treatment of DPN.

Chinese Medicine Promotes Schwann Cell Proliferation.
Multiple studies have demonstrated the presence of axonal degeneration and peripheral nerve demyelination in DPN. Characteristic histopathological findings include lipid droplets, Reich granules, and glycogen granules in the cytoplasm of Schwann cells, mitochondrial swelling, and disappearance of mitochondrial cristae, which are indicative of a proapoptotic state. Schwann cell proliferation and migration promote nerve regeneration and thus are likely to mitigate in DPN. Sun et al. [2] applied the serum pharmacological method (Figure 1) to investigate the effect of serum containing Jinmaitong on the proliferation of Schwann cells cultured under hyperglycemic conditions. Compared with a control group treated with neurotrophin, there was no significant difference between the two groups in their effect on enhancing the proliferation of Schwann cells. Furthermore, both groups also increased the expression of nerve growth factor (NGF) in the same cultured Schwann cells. Wu et al. [3] used the XTT method and the 3H2TdR incorporation assay to assess the activity and proliferation of Schwann cells isolated from sciatic nerve tissues of newborn Wistar rats. They confirmed that allyl glycosides significantly reversed the inhibition of proliferation of Schwann cells induced by hyperglycemia.

Chinese Medicine Inhibits Apoptosis of Schwann Cells.
Apoptosis is a manifestation of cell damage. The typical histopathological pattern of mitochondrial swelling and dissolved mitochondrial cristae occur in response to streptozotocin (STZ) administered to diabetic rats [25]. In vitro experiments showed that hyperglycemia reproduces this pattern of apoptosis in Schwann cells [26]. Ji et al. [4] reported that application of Jiangtangshuluofang normalized levels of serum insulin and glycosylated hemoglobin and inhibited the apoptosis of Schwann cells surrounding sciatic nerves in diabetic rats. The mechanism is thought to be related to the inhibition of proapoptotic factors caspase-3 and Bax and promoting of expression of Bcl-2, an antiapoptotic factor. Liu et al. [5] reported that the Chinese medicines astragalus, salvia, and yam have antiapoptotic actions on Schwann cells cultured under hyperglycemic conditions. These medicines increased levels of Bcl-2 expression, while inhibiting expression of caspase-3. Furthermore, the combination of those three herbs was synergistic. In vitro studies showed that the medicated serum containing Jinmaitong decreased the expression of inducible nitric oxide synthase (iNOS), NADPH oxidase p22-phox, 8-OHdG, and active caspase-3 (17 kDA) in Schwann cells, suggesting that Jinmaitong can reduce oxidative injury and apoptosis associated with hyperglycemic conditions (Table 3)

Chinese Medicine's Effects on Neurotrophic Factors
Neurotrophic factors are essential for the maintenance and survival of neurons. When peripheral nerve are injured, neurotrophic factors can bond to specific tyrosine kinase receptors on the surface of target cells, preventing neuronal cell death and promoting the repair of neurons and axon regeneration. Neurotrophic factors can be classified into: neurotrophins (including NGF, brain-derived neurotrophic factor, and neurotrophin); neuropoietic cytokines (including Evidence-Based Complementary and Alternative Medicine 5  "Can elevate the mRNA expression of NGF and IGF-1 in sciatic nerve of diabetic rat, which may be one of the mechanisms of acupuncture on diabetic neuropathy"   ciliary neurotrophic factor [CNTF] and interleukins); and the transforming growth factor-beta (TGF-β) superfamily (which can be subdivided into acidic fibroblast growth factors and basic fibroblast growth factors). In addition, there are other neurotrophic factors, such as insulinlike growth factor (IGF) and glial-derived neurotrophic factor [27]. Present studies suggest that diabetes-induced dysfunction of nerve regeneration results partially from decreased levels of some neurotrophic factors or their receptors.
3.1. NGF. NGF was the first discovered and most typical neurotrophic factor. It plays an important role in neuronal development, differentiation, and the maintenance of normal functions. NGF not only protects neurons and reduces their degeneration and death, but it also promotes nerve regeneration after nerve injury. There is a deficiency of NGF in diabetes, and reduced levels or activity of NGF plays a significant role in the pathogenesis of diabetic neuropathy [28]. Qu et al. [8] observed that 12 weeks after the success of a STZ-induced diabetic neuropathy rat model, compared with the normal rats, the tail-flick latency was significantly prolonged, the pain threshold was significantly lower, and NGF protein and mRNA expression in the sciatic nerve were significantly reduced in the model rats. Moreover, NGF-mRNA expression level in the sciatic nerve was negatively correlated with the tail-flick latency and was positively correlated with the mechanical pain threshold in the model rats.
The model rats were orally administered with the Chinese medicine compound Jinmaitong in three different dosage groups: large, medium, and small. After the intervention, the medium-dosage group of Jinmaitong showed that the tailflick latency was significantly reduced, the pain threshold significantly increased, and sciatic NGF-mRNA and protein expression were significantly increased, compared with the model control group. Furthermore, in vitro study proved that the serum containing Jinmaitong promotes secretion of NGF in high-glucose cultured Schwann cells [2]. Deng and Zhang [9] investigated the effects of Qitengtongluoyin on protein expression of NGF and neuropeptide substance P (SP) in sciatic nerves of STZ-induced diabetic multiple neuropathy rats, and proved that Qitengtongluoyin can prevent and treat sciatic neuropathy in STZ-induced diabetic multiple neuropathy rats, probably via promoting the expression of NGF and SP protein. Yu et al. [10] reported that a 6-week intervention of the Chinese medicine compound Tangmoning in STZ-induced diabetic rats resulted in a significant increase of NGF mRNA and that the effect was similar to that of methycobal. Xu and Yang [11] reported that Yishentongluofazufang can increase the NGF content in the serum of STZ-induced rats. Wang and Liu [12] proved that Tangbikang can increase serum NGF level and increase the expression of NGF mRNA of sciatic nerve in diabetic rats. Ma et al. [13] testified in their experimental study that certain extracts of morus alba can improve diabetic peripheral neuropathy in alloxan-induced diabetic rats, via promoting expression of NGF and myelin protein in sciatic nerves. Besides the above-mentioned Chinese herbal medicine studies, recent studies have shown that acupuncture and moxibustion can improve DPN, possibly via their effects on NGF. Dong et al. [14] conducted electroacupuncture (EA) on the points of Shenshu (BL 23) and Zusanli (ST 36) in STZinduced diabetic rats. After the intervention was done 12 times, the EA group showed increased NGF-positive cells and increased NGF mRNA expression in sciatic nerve, compared with the model group, suggesting EA upregulates expression of protein and mRNA of NGF and improves nerve repair in DPN. Huang et al. [15] found that EA treatment of diabetic rats resulted in increased expression of NGF mRNA in the sciatic nerve. Yin et al. [16] applied moxibustion on STZinduced diabetic rats, moxaed at the points of Yishu (Ex-B3) and Zusanli (ST 36), 15 min each point, once daily for 56 consecutive days, found that blood glucose significantly decreased, nerve conduction velocity significantly increased, and NGF content significantly increased in treatment group, compared with those in model group, suggesting that moxibustion has functions of peripheral nerve protection which may be related to its promotion of NGF expression of nerve.

CNTF.
CNTF has multiple biological activities: promoting survival of neurons and protecting motor neurons; inhibiting degeneration of axons of motor nerves; enhancing growth speed of axon; preventing muscle atrophy. In addition, administration of CNTF results in promoting regeneration of peripheral nerves [29]. CNTF protein and bioactivity are reduced in the peripheral nerve of diabetic rats, and CNTF treatment improved nerve regeneration and prevented nerve-conduction slowing in diabetic rats, suggesting CNTF plays an important role in nerve regeneration in DPN [30]. Wang et al. [17] observed the Chinese medicine compound Jinmaitong's effects on CNTF expression in diabetic neuropathy rats, and confirmed that Jinmaitong can upregulate the expression of the protein and mRNA of CNTF in the sciatic nerves of diabetic neuropathy rats. For in vitro study, Wang et al. [18] prepared drug-containing serums with the application of 15 times the adult dosage of both Jinmaitong and neurotrophin and grouped as the blank control group (no cells added), normal control group (added with normal rat serum), high-glucose group (added with glucose), Jinmaitong group (added with serum containing Jinmaitong and normal rat serum), and neurotropin group (added with serum containing neurotropin). Except for the blank control group and normal control group, the 50 mmol/L glucose was added to all the groups to achieve high-glucose Schwann cell models. The expression of CNTF and CNTF mRNA was detected by SABC immunohistochemistry method and real-time fluorogenetic quantitative PCR, respectively. Results showed that compared with the normal control group, the CNTF and CNTF mRNA expression in the high-glucose group, Jinmaitong group, and neurotropin group were lowered. Compared with the highglucose group, the CNTF and CNTF mRNA expression of the Jinmaitong group and neurotropin group were increased, and the CNTF mRNA expression in the Jinmaitong group was higher than that in the neurotropin group. This suggests that Jinmaitong upregulates the expression of CNTF and CNTF mRNA of Schwann cells cultured in high-glucose medium.  3.3. IGF-1. IGF-1 promotes cell growth and proliferation, and it promotes the growth of axons. Recent studies have shown that IGF-1 nourishes and supports motor and autonomic nerves. Decreased serum IGF-1 level and IGF-1 mRNA expression are shown in experimental diabetic rats, and administration of IGF-1 results in improvement of diabetic neuropathy [31]. Zeng et al. [19] applied reverse transcription polymerase chain reaction assay and confirmed that the expression of IGF-1 mRNA was decreased in sciatic nerves in STZ-induced diabetic rats; the expression level of IGF-1 mRNA and glucose was negatively correlated; treatment with Xiaokelingnongsuoye resulted in increased expression of IGF-1 mRNA in sciatic nerves. Xu et al. [20] set up a rat model of diabetic peripheral neuropathy and investigated Qingyingtang's effects on sciatic nerve conduction velocity, histopathological changes, and the expression of IGF-1 in tissue. They found that Qingyingtang enhanced sciatic nerve conduction velocity, improved histopathological changes, and increased the expression of IGF-1 in serum and tissue, confirming that Qingyingtang nourishes the sciatic nerve and promotes the sciatic nerve repair, possibly via increasing expression of IGF-1. Another study reported that Tangmoning can improve pathological changes in the sciatic nerve in rats, and it has protective effects on DPN, which might be related to its upregulation of the expression of IGF-1 protein [21]. Yin et al. [16] proved that moxibustion can upregulate IGF-1 mRNA expression of sciatic nerve in diabetic rats.

3.4.
Interleukin. Interleukin 1 (IL-1) plays a central role in the regulation of immune and inflammatory responses. It promotes cell proliferation and generation of other cytokines, and regulates metabolism. Recent studies found that IL-1 and IL-6 have functions of promoting regeneration of peripheral nerves; Schwann cell can secret IL-1 which promotes Schwann cells' secretion of NGF; IL-6 can promote nerve regeneration of via up-regulating the expression of CNTF mRNA [32][33][34]. It has not been reported that Chinese medicine promotes nerve repair and regeneration via interleukin. Only a few studies reported that Chinese medicine inhibited inflammation factors in diabetes, so that improved the nerve impairment in DPN. Zhang et al. [22] administered different dosages of Xiaoketongbi to STZ-induced diabetic rats for 2 months and found that Xiaoketongbi improved the peripheral neuropathy and decreased the levels of IL-1β, TNF-α, and CD54 in diabetic peripheral neuropathy rats, suggesting Xiaoketongbi relieves and improves diabetic neuropathy by means of inhibition of inflammation in diabetes.

Conclusion
Diabetic peripheral neuropathy is the result of multiple factors, and the repair and regeneration of peripheral nerves are very complicated procedures that are regulated by multiple factors; furthermore, the micro-environment that is needed for nerve repair and regeneration is not of single factor but composed of multiple related factors. Recent studies have shown that Chinese medicine inhibits apoptosis, promotes proliferation in Schwann cells, and increases expression of multiple neurotrophic factors; therefore, Chinese medicine can improve nerve repair and regeneration in DPN (Figure 2). Chinese medicine has the advantage of providing multiple therapeutic effects on multiple targets, compared with Western medicine, which uses conventional chemical agents and focuses on a single target. Therefore, to a certain extent, the effective single herb or compound of Chinese medicine might offer a more suitable micro-environment, one that is neurologically and physically needed for promoting repair and regeneration of nerves.
As we discussed in this paper, some studies investigated a single factor, thus, limited in explanations of the mechanisms of Chinese medicine's effects on nerve repair and regeneration. Although some Chinese medicine showed effectiveness in vitro studies, it might not be effective clinically, because in vitro cultured cells independently survive in an artificial environment, which is very different from the environment in vivo. Because of the diversity of patterns of Chinese medicine, uncertainty about activities of various ingredients, difficulty of quality control, and unknown interactions between components in the same Chinese medicine compound, research on the mechanisms is very difficult to get further. In short, further research is needed to clarify Chinese medicine's clinical value and the mechanisms of Chinese medicine's functions of nerve repair and regeneration in DPN. Future studies should be carried out with emphasis on both prevention and treatments to clarify the mechanisms by which Chinese medicine promotes nerve repair and regeneration; in the meantime, we need to explore and block the factors that inhibit nerve repair and regeneration.