Regulatory Effect of Electroacupuncture on Ceramide Galactosyltransferase Expression in Sciatic Nerve of Experimental Diabetic Rats

Jiang Chao1,, Liu Ping, Zhang Yun-yun3, Zhang Qiu-juan3, Huang Gao-min

1 Department of Geriatrics, Shaanxi Provincial Hospital of Traditional Chinese Medicine, Xi’an 710003, China

2 Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200030, China

3 Yueyang Hospital of Integrated Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China

BASIC STUDY

Regulatory Effect of Electroacupuncture on Ceramide Galactosyltransferase Expression in Sciatic Nerve of Experimental Diabetic Rats

Jiang Chao1,2, Liu Ping2, Zhang Yun-yun3, Zhang Qiu-juan3, Huang Gao-min2

1 Department of Geriatrics, Shaanxi Provincial Hospital of Traditional Chinese Medicine, Xi’an 710003, China

2 Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200030, China

3 Yueyang Hospital of Integrated Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China

Author: Jiang Chao, M.D., attending physician.

E-mail: 280165056@qq.com

Objective: To observe the change of ceramide galactosyltransferase (CGT) expression in sciatic nerve of experimental diabetic rats after electroacupuncture (EA) treatment.

Methods: The rat model of diabetic peripheral neuropathy (DPN) was established with Streptozotocin (STZ). Twenty-eight rats were randomly divided into a normal group, a model group, an EA acupoint group, and an EA non-acupoint group, with 7 rats in each group. Acupuncture on points Shenshu (BL 23) and Zusanli (ST 36) was performed in the EA group using EA electronic device with continuous wave, 2 Hz, 20 min every time, once every other day for 12 times. Acupuncture (on the tip of rat tail) in EA non-acupoint group was performed with the same electronic parameters and time. The model group and the normal group were not given any treatment, except the same fixation as that in the EA acupoint group. After 12 treatments, the albumen and mRNA expressions of CGT of sciatic nerve in rats from each group were measured with real-time polymerase chain reaction (RT-PCR), fluorescence quantitative PCR (FQ-PCR) and immunohistochemical methods.

Results: After modeling, the CGT expression was significantly lower than that in the normal group (P＜0.01); however, after 12 EA treatments, the CGT positive cells in the EA acupoint group were up-regulated and significantly higher than those in the EA non-acupoint group and the model group (P＜0.01). The CGT mRNA expression of the rats after modeling was also higher than that of the normal rats (P＜0.01), and the expression in the EA acupoint group was significantly lower than those in the model group and the EA non-acupoint group (P＜0.01,P＜0.05).

Conclusion: EA treatment with acupoints displays some therapeutic or preventive effect in the recovery of DPN via reversing the abnormal expression of CGT mRNA in sciatic nerves of experimental diabetic neuropathy rats, and it is worthy of further study.

Acupuncture Therapy; Electroacupuncture; Sciatic Nerve; Diabetic Neuropathies; N-Acylsphingosine Galactosyltransferase

Diabetic neuropathy (DN), a microvascular complication of diabetes, comprising disorders of peripheral nerve in people with diabetes but ruling out other causes, is a generally accepted disabling disease for all of us. As one of the most common diseases, diabetic peripheral neuropathy (DPN) is closely related to considerable mortality, morbidity, and diminished quality of life. According to the clinical epidemiological data, the prevalence ofneuropathy in diabetic patients is about 30%, whereas up to 50% of patients will certainly develop serious neuropathy in their disease. In addition, it's estimated that universal prevalence of diabetes will be about 472 million people by 2030, and DPN will affect 236 million persons worldwide, causing lots of medical bills[1-5]. Currently, medications are often used to help minimize pain and other uncomfortable sensations. Alternative or complementary treatments including electroacupuncture (EA) may be helpful for some symptoms. In China, EA has been regarded as an alternative treatment for inflammatory pain and paresthesia for several decades. Moreover, more than 40 disorders have been endorsed by the World Health Organization (WHO) as conditions that can benefit from acupuncture treatment, in which peripheral neuropathy was included. Meanwhile, many reports have declared the effectiveness of EA treatment for DPN. According to the latest reports in the American journal ofNature Neuroscience, acupuncture has been found to cause the human body to release some natural painkillers[6]. In addition, our previous studies have shown that acupuncture can significantly improve the DPN patient’s clinical symptoms. EA can not only up-regulate the nerve growth factor (NGF) mRNA of sciatic nerve, but also significantly accelerate the motor nerve conduction velocity and sensory nerve conduction velocity in DPN rats. Also, it can significantly promote the structure of myelin. Preventing or delaying the damages of the structure and function of peripheral nerves is helpful for peripheral neural repair[7-9]. However, the molecular mechanisms underlying the therapeutic effect of EA have not been thoroughly clarified. Therefore, it is necessary to find out a much more reliable scientific method to reveal the exact mechanism of EA. Ceramide galactosyltransferase (CGT) is the ratelimiting enzyme in the biosynthesis of galactocerebroside (GalC), which is a major glycolipid constituent in the myelin sheath. Approximately 25%-30% of all lipids consist of GalC and sulfatides (3-sulfate ester of GalC) in both central nervous system (CNS) and peripheral nervous system (PNS)[10]. It’s reported that the defects in CGT mRNA can cause neural morphological abnormalities and slow down the conduction velocity. Our previous study also found that the neural morphological and functional abnormalities in DPN rats had become more serious, but CGT mRNA expression had been increased significantly after 8 weeks[11]. Obviously, we can understand the changes of the structure or function of myelin via the changes of CGT expression. In this paper, we will explore the evidence linking DPN, EA acupoint treatment with CGT expression of sciatic nerve in rats.

1 Materials and Methods

1.1 Design

A completely randomized controlled experiment was conducted. The random number was generated by the random number table. The experiment was conducted in the Central Laboratory of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (TCM) between March 2013 and July 2013. This study was carried out in strict accordance with theGuide for the Care and Use of Laboratory Animalsof the National Institutes of Health's recommendations. All procedures were approved and performed in accordance with regulations stipulated by the Committee on Animal Research and Ethics (Institutional Animal Care and Use Committee in Longhua Hospital Affiliated to Shanghai University of TCM).

1.2 Modeling

Twenty-eight male Wistar rats, 4 months old, clean grade, weight 200-220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences, animal license No.: SCXK (Shanghai) 2003-0003. Before modeling, 7 rats were randomly selected to carry out blood sugar, weight and nerve conduction velocity testing, and the remaining 21 rats were used to establish the DPN rat model. Over one night fasting, the rats were administrated with Streptozotocin (STZ, American Sigma company, batch number: S-0130) intraperitoneally 55 mg/(kg·bw), while the rats in the normal group were given the same volume of citric acid-sodium citrate buffer. Four weeks later, the blood sugar was tested after overnight fasting using fast glucometer (Johnson, USA), fasting plasma glucose ≥15 mmol/L. The sensory nerve conduction velocity (SNCV) and motor nerve conduction velocity (MNCV) were significantly slowed down, wagging tail temperature threshold increased, and morphological changes of the sciatic nerve myelinated fibers were revealed, which all reflected the success of modeling. Ultimately, totally 18 rat models were classified randomly into 3 groups, namely, model group (n＝6), EA acupoint group (n＝6), and EA non-acupoint group (n＝6).

1.3 Therapeutic method

According to theRat’s Acupoint Atlas[12], Shenshu (BL 23) and Zusanli (ST 36) were selected. Rats were tied to a wooden platform. The points were punctured by depth of 0.5 cm in the EA acupoint group, by using EA device (type G-6805II), with continuous wave at 2 Hz, 20 min every time, once every other day for 12 times. Acupuncture was performed at the tip of rats’ tails in the EA nonacupoint group, with the same EA device andparameters. The model group and the normal group were not given any treatment but the same fixation as the EA acupoint group.

1.4 Sample collection and preparation

On the second day after the end of treatment, we used 10% chloral hydrate to anaesthetize rats. Bilateral sciatic nerves were taken out under sterile condition. The right sciatic nerve was loaded into vials and quickly placed in liquid nitrogen, and then cryopreserved at －70 ℃, while the left sciatic nerve was fixed in 10% formaldehyde. Finally, all rats were killed by overdose of anesthetic.

1.5 RNA extraction and the CGT mRNA expression

Total RNA was prepared following standard protocols. Briefly, RNAs were extracted from each frozen sample (sciatic nerve tissue 50 mg) using Trizol RNA of general type quick extraction kits (Shanghai Biological Engineering Technology Company). One milliliter Trizol reagent was added, and homogenisation was performed in a tissueLyser (Qiagen, Valencia, CA) at 30 Hz for 3 min twice; adding 50 μL RNase-free water; dissolving RNA. The quality and concentration of the extracted RNAs were detected by UV spectrophotometer, A260/A280 at 1.8-2.0. The samples were stored at －80until use. Total RNA of 1 μg was used to synthesize cDNA using iScript reverse transcriptase (Shanghai Biological Engineering Technology Company) and CGT primer as specified by the manufacturer. PCR was performed in optimized conditions: denatured at 95 ℃ for 2 min, followed by 10 cycles of 45 s at 93and 1 min at 55，and 10 cycles of 30 s at 93and 45 s at 55Primers (Chemicon Company, USA) were as follows: CGT, Pro 5'-AGACTACATTCTTCGCCACGACGGAGC-3', forward 5'-ACCCTGTCAATCGGACTACCTATT-3', reverse 5'-AGCCGAACGGAGGTGATG-3'. Relative quantification was achieved by calculating the ratio between the cycle number (Ct) at which the signal crossed a threshold set within the logarithmic phase of the gene of interest and that of the 18S reference gene. Ct values were the means of duplicates.

1.6 Analysis of CGT protein

Sciatic nerve tissue was fixed for 48 h, embedded in paraffin, and then conducted in serial sections (4 μm)，and dewaxed to water. The tissue sections were then incubated at room temperature in 3% H202in PBS for 10 min to inactivate endogenous peroxidase. The slides were blocked (30 min at 37 ℃) with normal calf serum to reduce non-specific binding, without washing. Diluted Calpainantibody solution (1:100) was dropped at 4 ℃ overnight. Following incubation, the slides were washed 3 times in PBS for 2 min each time. Envision reagent was added at 37 ℃for 30 min. Then, the slides were washed twice in PBS for 3 min each time, in 3% H202in PBS for 8 min plus 0.4 g/L DAB to 0.03% H2O2for color rendering for 8 min. It was then washed and added with Hematoxylin dropwisely for 30 s and then washed again. The samples were blued with Hydrochloric Acid for 2 s and washed; blued with microwave for 30 s, cooled at room temperature. Finally, the samples were added with one or two drops of neutral gum and stored at room temperature for subsequent image analysis. All the operations were carried out in strict accordance with the kit instructions. A microscope (OLYPUS BH2), image acquisition card (Shanghai Shenteng Information Technology Co., Ltd.), and a digital camera or a CCD camera [Panasonic (MV-cp410)] were used to collect digital images [standard BMP (bitmap) file format]. Calibration can be carried out under conditions of 250 times. Measuring results: To compare the positive area and positive ratio, as well as the optical density (OD) in the immunohistochemical image among different groups. Each slice was analyzed for at least three visual fields.

Evaluation of staining: Two observers were blinded to the identity of the slides (CJ, GH) and performed all the assessments. The positive staining of CGT expression in the cytoplasm and/or the nucleus was present with brown particles. The intensity was greater than the non-specific background which can be considered as the standard. The result was judged semi-quantitatively by using a grading system. We randomly observed 10 high-power microscope (250 x) fields. One hundred cells per field, a total of 1000 cells, and the positive cells ＜5% were regarded as 0, 5%-24% as 1, 25%-49% as 2, ＞50% as 3, and cells without stained were 0, with faint yellow were 1, brown yellow were 2, brown were 3. The positive level can be judged according to both the results of multiplying: negative (0, ﹣), weak positive (1-2, +), moderate positive (2-4, ++), strongly positive (＞4, +++). For the analyses presented in this article, the positive expression of CGT was defined as moderate and intense immunohistochemical staining.

1.7 Statistical analysis

All results were expressed asStatistical analyses were performed with the SPSS 15.0 software (SPSS, Inc1, Chicago, IL, USA). Statistical differences were assessed by One-way ANOVA of variance with Tukey or Dunnett's post-hoc test for multiple-group comparisons. Student'st-test was used for two-group comparisons. APvalue of ＜0.05 was considered to be statistically significant.

2 Results

2.1 Blood glucose and body weight

During the experiment, the model group, the EA non-acupoint group, and the EA acupoint group eachhad one rat died, and 5 rats were selected from each group and involved in the result analysis. Before modeling, there were no significant differences in comparing body weight and blood glucose among the four groups (P＞0.05). After modeling and 12 treatments, the blood glucose levels and body weight of the rats from the other three groups were significantly higher than that of the normal group (P＜0.01). There were no significant differences between the model group and the EA groups in comparing the blood glucose level and body weight at any time points (P＞0.05). The blood glucose levels and body weight had no significant differences between the two EA groups at any time points(P＞0.05), (Table 1, 2).

Table 1. Comparison of the body weight

Table 1. Comparison of the body weight

Note: Compared with the normal group at the corresponding time point, 1)P＜0.05, 2)P＜0.01

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Table 2. Comparison of the blood glucose (, mmol/L)

Table 2. Comparison of the blood glucose (, mmol/L)

Note: Compared with the normal group at the corresponding time point, 1)P＜0.05, 2)P＜0.01

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2.2 Analysis of the CGT protein expression

The results of the semi-quantitative analysis of CGT mRNA expressions in the rats’ sciatic nerve of each group after EA intervention for 12 times were compared. The results are shown in Table 3, Figure 1.

The numbers of positive cells in the CGT mRNA expressions in the model group, the EA acupoint group, and the EA non-acupoint group after modeling were significantly reduced as compared with that in the normal group (P＜0.05). After intervention, the number of positive cells in the CGT mRNA expressions in EA acupoint group was significantly higher than that in the model group and the EA non-acupoint group (P＜0.05). The number of positive cells in the CGT mRNA expression in the EA acupoint group tended to increase to approach the normal level after intervention (Table 3).

2.3 CGT mRNA expression

The Ct values of CGT mRNA expression of sciatic nerves of DPN rats in the model group, the EA non-acupoint group, and the EA acupoint group were significantly increased compared with that in the normal group (P＜0.01). After EA intervention, the value in the EA acupoint group significantly decreased approaching the normal level, and was significantly lower than those in the model group and the EA non-acupoint group (P＜0.05), (Figure 1, 2).

Table 3. Comparison of the consolidated immunohistochemical scores

3 Discussion

DPN is generally considered to be one of the most common complications of diabetes mellitus, equally affecting both types of diabetes[13-15]. Studies suggest that about 30% of patients with diabetes mellitus are affected by DPN and 16%-26% of diabetic patients experience chronic pain. Sensory polyneuropathy is also a major complication of type 1 diabetes. Both conditions are still lacking adequate treatments to our current knowledge[16-17].

Figure 1. Expression of CGT mRNA by HE staining

Figure 2. CGT mRNA expression

We realized that nerve conduction was greatly facilitated by myelin, a lipid-rich membrane that wraps around the axon in higher vertebrates, while myelin is formed by oligodendrocytes in the CNS and by Schwann cells in the PNS, suggesting the essential role of CGT modification in maintaining myelin function and structures. As a consequence of metabolic dysfunctions, DPN develops in an early responsive ‘metabolic phase’ and a late nonresponsive ‘structural phase’. The early DPN is characterized by peripheral nerve dysfunctions, reversible by common euglycemic therapies. The late DPN presents irreversible neuropathic symptoms, such as thermal hypoalgesia and mechanical allodynia, associated with a reduction in neurotrophic support to PNS neurons and with neuronal sufferance and atrophy[17]. Furthermore, our earlier studies found that CGT mRNA level in sciatic nerve of diabetic neuropathy rats was increased after functional and morphological abnormality[11]. Therefore, we speculated that CGT was essential for the development and functional maintenance of PNS, which was primarily targeted by DPN, and was indicated as a possible therapeutic target for peripheral neuropathies.

Traditional Chinese acupuncture and its Western derivate EA have proven effective in the treatment of neuralgia, and their neurophysiologic correlates have become the subjects of extensive investigations[5,18]. Many studies have proven that the increased nerve growth factor in sensory neurons of early diabetic rats is corrected via EA and the actions of EA on the inhibitory neurotransmitter γ-aminobutyric acid (GABA has a role in the generation and maintenance of diabetes-induced sensory dysfunctions and the activity-dependent regulation of spinal NGF synthesis)had also been reported[19-20]. Moreover, extensive clinical studies have shown that EA is effective in the treatment of neuralgia in diabetes[19]. However, the specific mechanism of EA in treating DPN is not very clear until now. Our research hypothesis, therefore, is that the action of EA on diabetic neuropathy should be mediated through the CGT signaling pathway.

In this study, we found that the conduction velocity of sciatic nerve in DPN rats was recovered significantly, and the number of CGT positive cells increased after EA acupoints treatment, while there was no significant change after non-acupoint EA treatment. The CGT mRNA expression level of the sciatic nerve was inversely proportional to the Ct value of mRNA. After EA acupoint treatment, compared with the model group, the CGT mRNA expression of sciatic nerve in the EA acupoint group was higher, while there was no significant change in the EA non-acupoint group. The results of this study also confirmed that the conduction velocity of sciatic nerve in the model group was slowed down, reflecting that the function and structure of the myelin sheath in diabetes were obviously abnormal, and the numbers of positive CGT cells and mRNA expression of the sciatic nerve decreased in the model group compared to that in the normal group. It indicates that there should be a certain correlation between the abnormal number of CGT protein positive cells and mRNA expression of the sciatic nerve and sciatic nerve neuropathy in rats. Furthermore, some studies demonstrated that CGT mRNA expression was down-regulated for damaged myelin, but appeared up-regulated for the re-formation of myelin[21]. Our study suggests that the decreased number of positive cells and anomalous mRNA expression of CGT protein in sciatic nerve of DPN rats were corresponding to the slowed conduction velocity (SNCV, MNCV) of sciatic nerve, and shows that the number of positive cells and mRNA expression of CGT protein in DPN were reduced. It can be considered as one of the reasons for the slower conduction velocity of sciatic nerve after the myelin is injured.

Zusanli (ST 36) from the Stomach Meridian of Foot Yangming and Shenshu (BL 23) from the Bladder Meridian of Foot Taiyang are commonly used in human acupuncture to treat neuralgia[22-23]. According to the theory of TCM, by tonifying yin-yang of kidney and dredging qi and blood of meridians, EA can improve metabolic disorders of diabetes, increase the conduction velocity of sciatic nerve caused by nerve injuries, and promote the recovery of neurological function in DPN. Furthermore, EA acupoint treatment does no harm to the sciatic nerve in rats.

However, our study has some limitations. The relatively smaller sample size and shorter treatment time cannot provide sufficient evidence for the treatment of DPN. Obviously, it is necessary to carry out further study to explore the specific mechanism of EA acupoint treatment of DPN by expanding the sample size, prolonging intervention time, and adopting multicenter, randomized, double-blinded methods. In addition, there are lots of drugs for the treatment of nerve injuries at present, such as nerve growth factor (gangliosides) and gene therapies. How can we combine acupuncture and medicine treatments in a proper way in treating DPN? It is worthy of further study.

Conflict of Interest

There is no potential conflict of interest in this article.

Acknowledgments

This work was supported by Postgraduates Innovation Ability Training Special Scientific Research Project of Shanghai University of Traditional Chinese Medicine.

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Date: September 28, 2013