Diabetic peripheral neuropathy (DPN) is a common microvascular complication of diabetes associated with high disability rate and low quality of life. Tang-Luo-Ning (TLN) is an effective traditional Chinese medicine for the treatment of DPN. To illustrate the underlying neural protection mechanisms of TLN, the effect of TLN on electrophysiology and sciatic nerve morphology was investigated in a model of streptozotocin-induced DPN, as well as the underlying mechanism. Sciatic motor nerve conduction velocity and digital sensory nerve conduction velocity were reduced in DPN and were significantly improved by TLN or
Diabetic peripheral neuropathy (DPN) is a common microvascular complication of diabetes and affects more than 50% of diabetic patients [
As an important neuron in peripheral nerve system, SCs have become a center for many research studies in recent years as they play a key role in myelination, neurotrophic support, homeostasis, and repair after peripheral nerve injury [
As is well known, the incidence of DPN could be attributed to a multiple mechanisms. Traditional Chinese medicine (TCM), which has the advantage of hitting multiple targets at the same time, may be a good choice for such therapeutic agents [
In our study, due to multiple-target characteristic of Chinese herbal compound, microarray expression analysis was used to screen cell-survival related genes and pathways regulated by TLN, followed further validation by quantitative real time polymerase chain reaction (qPCR) and western blot.
TLN recipe mainly contains the ingredients from three different traditional Chinese herbs: the dried root of
Male Sprague Dawley rats weighting 180 g–220 g were purchased from Vital River Laboratories (Beijing, China, certificate No. SCXK 2006–0009). The animals were housed in separate cages at constant temperature (20–22°C) and humidity (50%–67%) under a 12-hour light/dark cycle in specific pathogen free animal laboratory of BUCM. The animals had free access to standard chow diet (Vital River, Beijing, China) and sterilized drinking water during the period of this experiment. The experimental protocol was approved by the ethics committee of BUCM. All animal experiments were conducted in accordance with the NIH guide for the care and use of laboratory animals (NIH Publication No. 80-23; revised 1978).
After one week of acclimatization, rats were given STZ (Sigma, 60 mg/kg body weight), and 72 hours later rats with fasting blood glucose more than 16.7 mmol/L were considered successfully induced for diabetes. The diabetic rats were randomly divided into three groups including STZ induced diabetic group (STZ group), Tang-Luo-Ning group (TLN group), and alpha-lipoic acid group (
Sciatic motor nerve conduction velocity (MNCV) and distal digital sensory nerve conduction velocity (SNCV) were measured after induction of anesthesia with 10% chloral hydrate (300–350 mg/kg) at two points (10 weeks and 20 weeks after intervention). Body temperature was maintained at 37°C with a warming pad. For sciatic measurement, the left sciatic motor conduction system was stimulated at the sciatic notch where sciatic nerve exits and at ipsilateral ankle via bipolar electrodes with a width of 0.1 ms. The threshold was determined when the compound muscle action appeared or disappeared, and we set at 1.5-fold above the threshold as stimulus intensity. The recorded bipolar electrodes were placed at the first interosseous muscle of the hind-paw, as well as reference electrode was placed 1 cm away from the recording electrode but between the stimulating and the recording electrode. Hind-limb SNCV was recorded in the digital nerve to the second toe by stimulating with a square-wave pulse duration of 0.05 ms using the smallest intensity current that resulted in a maximal amplitude response. The sensory nerve action potential was recorded behind the medial malleolus. The maximal SNCV was calculated from the latency to the onset of the initial negative deflection and the distance between stimulating and recording electrodes. The latencies of action potentials were measured as described below by physiological data recording system (MacLab/400, ADI, Australia) and dual-beam memory oscilloscope (VC-10, Nihon Kohden Corporation, Japan). Average conduction time was calculated after 7–10 measurements. Sciatic MNCV and distal digital SNCV were measured and calculated based on the method [
Approximately 0.1 cm of sciatic nerve was removed from the lower edge of the left piriformis and cut into pieces about 1 mm3 by volume. After prefixing with cold, 4% glutaraldehyde nerve tissues were fixed with 1% osmic acid and dehydrated and embedded in 1 : 100 mixture of Epon 812 and 100% aceton. Semithin sections were cut and stained with toluidine blue before they were further cut into 50 nm ultrathin sections using an ultramicrotome (8800, LKB, Bromma, Sweden). After double staining with uranyl acetate and lead nitrate, morphological changes of the sciatic nerves were observed by transmission electron microscopy (H-600, Hitachi, Japan). Images were taken by imaging systems (Moticam 2306, Motic Instruments Inc., Canada).
RNA extraction and microarray were performed according to manufacturer’s protocols by Shanghai Biotechnology Co., Ltd. TRIZOL Reagent (Catalogue number 15596-018, Life technologies, Carlsbad, CA, USA) was added into sciatic nerve, and total RNA was isolated using RNeasy Kit (Qiagen). RNA integrity number (RIN) and 28 s/18 s ratio were determined by Agilent BioAnalyzer 2100. RNAs with RIN ≥ 7.0 and 28S/18S > 0.7 were deemed to be of sufficient quality. RNA concentration and A260/A280 ratio were determined by Nanodrop ND-1000. RNAs were purified by QIAGEN RNeasy Kit, followed by cDNA synthesis and cRNA fluorescent labeling, purifying, and shearing. Probes were hybridized with Rat Gene Expression Microarray slides (4 × 44 K microarray, Agilent, Catalogue number G2519F-014879) in a hybridization oven (Agilent G2545A). After thorough washing, array slides were scanned by Agilent scanner with both 100% and 10% PMT setting. Results were analyzed by Agilent software.
With threshold of fold change between experimental samples (TLN group) and control samples (STZ group, CTL group) set at 2, genes that were differentially regulated by TLN were identified. Genes that were first downregulated >2-fold (compared with CTL) by STZ and then upregulated >2-fold (compared with STZ) by TLN were considered as upregulated genes, and the downregulated genes were opposite. Differentially regulated genes by TLN were analyzed using an online SAS system for hierarchical clustering, Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis (enrichment
To confirm microarray data and further screen cell survival related pathway, Nrg1, GRB2-associated binding protein 1 (Gab1), mammalian target of rapamycin (Mtor), and phosphoinositide-3-kinase, catalytic, beta polypeptide (Pik3cb) were selected for validation by qPCR. Primers were designed by Sheng Gong Biotech (Shanghai, China) using Primer Premier 5.0 software (PREMIER Biosoft International, CA, USA). The sequences of Nrg1, Pik3cb, Mtor, Gab1, and GAPDH were the following: Nrg1: forward primer 5′-TGGCACATCCATCCAAATAC-3′, reverse primer 5′-GTAGCATGCTGCTGGGTCTA-3′; Pik3cb: forward primer 5′-AGATGTTGCTCAGCTTCAGG-3′, reverse primer 5′-TT CATCACTCATCTGTCGCA-3′; Mtor: forward primer 5′-AGA ACCACATGCCACACAGT-3′, reverse primer 5′-CTTTGGCATTTGTGTCCATC-3′; Gab1: forward primer 5′-CTCCTGAGACCACAAAGCAA-3′, Reverse primer 5′-AACGCTAGCTGCTTCTCACA-3′; GAPDH: Forward primer 5′-CAACTCCCTCAAGATTGTCAGCAA-3′, Reverse primer: 5′-GGCATGGACTGTGGTCATGA-3′. RNA was reverse transcribed by M-MLV Reverse Transcriptase (CK2801A, Takara) according to manufacturer’s protocols, and qPCR was performed on qPCR machine (ABI 7500, Life Technologies, USA) with the following program settings: 94°C for 15 min, 94°C for 15 s, 60°C for 34 s, and 72°C for 15 s for 40 cycles followed by 72°C for 10 min. All qPCRs were performed in duplicate, and relative expression was calculated using the
To determine the expression level of (P-) ErbB2, extracellular signal-regulated kinase (Erk), P-Erk, protein kinase B (PKB/Akt), P-Akt, bad, and P-bad Ser112/Ser136, the sciatic nerve tissues were quickly pulverized in prechilled mortar and then fractionated by SDS-PAGE. 40
Differential gene expression and bioinformatics analysis were performed using online SAS analysis system provided by Shanghai Biotechnology Corporation. All other data were statistically analyzed by SPSS13.0 software. Average value ± standard deviation (Mean ± SD) were calculated, one-way ANOVA (analysis of variance) and LSD-
Compared with the STZ group, an obvious improvement of sciatic MNCV and distal digital SNCV were observed after 10 weeks of TLN treatment (Figure
Effects of TLN and
In control rats, there were fair-arranged nerve fibers, mainly with myelinated nerve of thick myelin and large axon diameter simultaneously. In diabetic rats, serious demyelination, axial degeneration and reduced number of myelinated nerve fibers were observed on transmission electron microscope, and the pathological injury in this study corresponded to typical morphological changes of DPN. Likewise, the pathologic and morphological changes in the sciatic nerves of diabetic rats treated with TLN and
Transmission electron micrographs showing the ultrastructure of sciatic nerve fiber (×10000) (a) CTL rats: intact myelinated axon. (b) STZ rats: serious demyelination, axial degeneration, and Schwann cell proliferation. (c) TLN rats: moderate segmental demyelination and axial degeneration. (d)
Using an online SAS system, cluster analysis was performed and the resultant heat map allowed us to visualize the differential gene expression patterns between the experimental groups (Figure
Cell survival-related pathways regulated by TLN.
Pathway name | Positive and negative genes | Enrichment test |
---|---|---|
ErbB signaling pathway | Nrg1 |
0.0121 |
Neurotrophin signaling pathway | Pik3cb |
|
Phosphatidylinositol signaling system | Pik3cb | 0.0057 |
Enrichment test
Major genes in cell survival-related pathways upregulated by TLN.
Probe Id | Gene Id | Gene symbol | Gene name | FC: STZ versus CTL | FC: TLN versus STZ | FC: |
---|---|---|---|---|---|---|
A_44_P438143 | 112400 | Nrg1 | Neuregulin 1 | 0.3638 | 2.2264 | 2.1083 |
A_43_P12820 | 85243 | Pik3cb | Phosphoinositide-3-kinase, catalytic, and beta polypeptide | 0.3298 | 3.3562 | 2.8317 |
A_44_P191924 | 56718 | Mtor | Mechanistic target of rapamycin (serine/threonine kinase) | 0.3786 | 2.2988 | 1.9853 |
A_44_P176342 | 361388 | Gab1 | GRB2-associated binding protein 1 | 0.2205 | 4.8388 | 3.8136 |
FC: fold change.
Heat map showed the differentially expressed genes regulated by TLN between four groups. Red color indicates over expressed genes, whereas green color indicates the opposite. The color scale bar is shown. Four groups: CTL group: nonstreptozotocin-induced group, STZ group: streptozotocin-induced diabetic group, TLN group: Tang-Luo-Ning group, and
GO analysis and significantly altered categories regulated by TLN. (a) Gene ontology category of biological process for differentially expressed genes regulated by TLN. (b) The significant GO categories for differently expressed genes. The vertical axis represents the GO category, and the horizontal axis represents the number of genes changed in each category. There are 12 categories that were significantly altered (enrichment
Cell-survival genes (Nrg1, Mtor, Gab1, and Pi3kcb) identified from the microarray analysis were selected and performed qPCR for validation. Results showed that the TLN intervention upregulated the Nrg1, Mtor, and Gab1 expression levels, while the expression pattern of Pi3kcb were not consistent with the microarray data (Table
Effect of TLN and
In brief, we focused on Nrg1/ErbB2 pathway and do further validation by western blot. It is demonstrated that there was little variation of total ErbB2, Erk, Bad, Akt, and P-Akt after STZ injection (
Effect of TLN and
Taken together, these results from western blot and qPCR demonstrated that TLN could enhance NRG/ErbB2→Erk/Bad dependent SCs-survival signal pathway (Figures
The present study provides direct evidence for a crucial role of TLN in function and morphology of sciatic nerve after diabetes. The protective effects of the compound recipe TLN may be attributed to promote SCs-survival through the activation of Nrg1/ErbB2→Erk/Bad (Ser112) signal pathway by increasing the phosphorylation of ErbB2, Erk, and Bad (Ser112) at a protein level and mRNA expression of Nrg1, Mtor, and Gab1. This finding supports the hypothesis that TLN was a critical regulator in SCs survival and recovery of peripheral nerve regeneration ability. Recently there has been a great deal of interest in SCs due to the key role of it in myelination regeneration and repair after peripheral nerve injury.
DPN is a long-term common complication of diabetes. In this study, STZ was used to induce diabetic neuropathy model, which was widely performed in Sprague—Dawley rats [
In fact, ErbB2 signal pathway was most closely linked to SCs survival among the three screened signal pathways by microarray (Table
The binding and activation of Nrg1 and ErbB2/ErbB3 coreceptor to activate the downstream signal pathways is very important to transmit SCs survival signal. Being a family member of polypeptide growth/differentiation factors containing an epidermal growth factor-like motif, Nrg1 could activate membrane-associated ErbB tyrosine kinase receptors and mediate SCs differentiation, proliferation, and migration through binding and activation of a heterodimeric ErbB2/ErbB3 coreceptor [
In addition, after binding to the high-affinity ErbB2/ErbB3 coreceptor complex, Nrg could potentially activate several distinct signaling pathways, mainly including p21Ras/Raf-1/MEK/Erk cascade [
Despite a marked improvement in the function and morphology of sciatic nerve with TLN intervention after STZ injection, administration of TLN did not completely halt the progression of sciatic nerve injury. This suggests that there may be other signaling pathways involved in pathogenesis of nerve damage. Furthermore, although the Nrg1/ErbB2 receptor dependent SCs-survival effect of TLN is explicit in this
In conclusion, the findings of the current study provide support to the hypothesis that compound recipe TLN could inhibit SC apoptosis, which lead to an improvement in sciatic MNCV, distal digital SNCV and sciatic nerve pathological injury, suggesting TLN offering a promising alternative medicine for the DPN patients to delay the progression. The mechanism of the protective effect of TLN on promoting SCs survival may be partly due to activate Nrg1/ErbB2→Erk/bad signal pathway.
The authors declare that they have no conflict of interests, financial or otherwise.
This work was supported by National Natural Science Foundation of China (NSFC) (no. 30873253). Thus, the authors thank NSFC for financial support. The authors also thank Shanghai Biotechnology Co., Ltd for providing technical assistance related to the study.