More and more in vivo and clinical researches show that hyperglycemia is a common complication after brain or spinal cord injury. Hyperglycemia in brain and spinal cord could aggravate neurologic impairment, which is one of the most important factors affecting patients' prognosis,1-3 meanwhile blood glucose control should not be too strict.4 Therefore, how to control blood glucose after brain or spinal cord injury is a key strategy for therapy. L-lysine monohydrochloride (LMH), an essential amino acid of body, is involved in promoting body development, enhances immunity function, and plays an important role in the metabolic procedure of body. Recent studies showed that LMN also could increase the insulin secretion and regulate the blood glucose level.5-7 However, it is still not clear how LMH affect serum insulin and blood glucose in acute spinal cord injury rats. Here, we explored the effects of LMH on pancreatic islet B cells, serum insulin, and blood glucose level in spinal cord injured rats, in order to provide the experimental basis for the clinical application of LMH in the therapy of spinal cord injury.
Drugs and chemicals
LMH was obtained from Ningjiang Pharmaceuticals, Inc. (China). The radioimmunoreactive insulin kit was purchased from Huaxi diabetes technology institute (China). The insulin anti-serum was obtained from Sigma Chemical Co. (USA). The biotinylated goat-anti-rabbit IgG and avidin-biotin-peroxidase complex were purchased from Vector Co. (USA).
Forty male Wistar rats (120-150 g, 4-6 weeks old), obtained from the Institute of Laboratory Animal Science of Shanghai, were randomly divided into four groups (normal control group, model group, high-dose LMH group, and low-dose LMH group, n=10 each). The rats, which were not performed hemi-transection spinal cord injury, were regarded as normal control group. The other 30 rats were anesthetized by sodium pentobarbital (30 mg/kg, i.p.). Then, the model of spinal cord injured rat was established by hemi-transection at the lower right thoracic spinal cord under aseptic conditions. Next, the rats were immediately administered intraperitoneally with vehicle (normal saline solution) as the model group, or high-dose LMH (621.5 mg/kg equal to LMH 1/8 LD50) as the high-dose LMH group, and low-dose LMH (310.8 mg/kg equal to LMH 1/16 LD50) as the low-dose LMH group.
The rats were sacrificed 48 hours after spinal cord injury. Every blood sample obtained from femoral artery was divided into two tubes. One tube was used to detect insulin content by radioimmunoreactive insulin kit after blood sample centrifuged. The other tube was used to analyze blood glucose level by automatic biochemical analysor.
After the rats were sacrificed, the tail of pancreas was removed, post-fixed overnight at 4°C in Bouin reagents, and then routinely embedded in paraffin. After that, the tissues were cut into 6-μm-thick sections. The islet cells were stained by ABC method. In brief, sections were incubated for 30 minutes in 0.3% H2O2-methanol to inactivate endogenous peroxidase activity and reduce non-specific staining. Then the sections were placed in 0.01 mol/L PBS buffer containing 0.3% Triton X-100 and normal goat serum for 30 minutes and 20 minutes at room temperature, respectively. Following that, the sections were incubated for 24 hours at 4°C with an insulin anti-serum (Sigma Chemical Co.) diluted 1/200 in PBS, washed three times in PBS (5 minutes each) and incubated with biotylinated goat-anti-rabbit IgG (diluted 1/50) for 2 hours at room temperature. Next, they were washed as above and then incubated with avidin-biotinperoxidase complex (ABC kit, Vector Laboratories, USA) for 2 hours at room temperature. After another wash the sections were incubated in chromogen 3,3′-diaminobenzidine tetrahydro-chloride (DAB) for 4-5 minutes. The reaction was stopped in PBS, and the slides were allowed to dry prior to being dehydrated in gradual concentrated ethanol, cleared in xylene and cover-slipped with mounting medium. To control for specificity of immunostaining, some sections were processed as described above but without the primary antibody as negative control. Sections from different treatment groups were processed in parallel to minimize differences.
Quantification of pancreatic islet B insulin positive cells was counted with the aid of a Leica DMIRB/E microscope, equipped with a JVC digital camera (1600 × 1200 dpi in 8 bits) with 400 × magnification, using the Image HPIAS2000 program (Champion Image Co., China). The area of interest was the rectangular image capturing field of the camera. The mean optic density (MOD), and square density (SD) of pancreatic islet B insulin positive cells were made with the help of the Image HPIAS2000 program. The combination of these two indexes can more accurately reflect the change of pancreatic islet B insulin positive cells. The value in each tail of pancreas from individual animal was the means of three adjacent sections, three sides in each section.
The data were presented as mean ± standard deviation (SD). Statistical analyses were conducted using Pearson's χ2 test, one-way analysis of variance (ANOVA), and Duncan's test as a post hoc analysis. P values of less than 0.05 were regarded as statistically significant. All statistical analyses were performed using SPSS 11.5 software (SPSS, USA).
Effects of LMH on serum insulin level in spinal cord injured rats
As shown in Table 1, compared with the normal control group, the serum insulin content was significantly decreased in the model group. After treatment with high-dose LMH (621.5 mg/kg), the serum insulin content was significantly increased as compared with that in model group (P <0.05). Whereas, there was no significant difference in serum insulin content between low-dose LMH (310.8 mg/kg) group and model group. These results showed that LMH could increase the serum insulin in spinal cord injured rats at a dose-dependent manner.
Effects of LMH on blood glucose level in spinal cord injured rats
Next we detected the effects of LMH on blood glucose in spinal cord injury rats. As shown in Table 1, compared with the control group, the blood glucose level was significant increased in the model group. After treatment with high-dose LMH, the blood glucose level was significant decreased (P <0.05). However, there was no significant difference in blood glucose level between low-dose LMH group and model group. These results indicate that LMH also could decrease blood glucose level in spinal cord injured rats at a dose-dependent manner.
Effects of LMH on islet B cells in spinal cord injured rats
As shown in Figure, the insulin positive staining islet B cells were distributed in the center of islet. The positive staining was observed in cytoplasm of islet B cells, but not in nucleus of islet B cells, other islet cells, or exocrine cells in pancreas. The control section showed negative staining. The immunostaining intensity and the insulin positive staining number of islet B cell in model group were significant lower than those in the control group. After treatment with high-dose LMH, the insulin immunostaining intensity and the positive staining number of islet B cell were significant higher than those in model group. There were no significant differences in the immunostaining intensity and the positive staining number of islet B cell between low-dose LMH group and model group. Using the Image HPIAS2000 program, we analyzed the MOD and SD of islet B cells. The quantitative analysis data were consistent with the above results (Table 2).
It is well known that hyperglycemia is a common complication after brain or spinal cord injury. The finding that hyperglycemia after brain or spinal cord injury is subject to the following interpretations. The hyperglycemia after brain or spinal cord injury could be considered as a stress state. In stress state, the adrenergic nerve is excited, and then results in an obviously increase in catecholamines. Simultaneously, the increasing of glucagon, growth hormone, and cortical hormone in blood will result in the decreasing of glucose utilization.8,9 In addition, the decomposition of lipid and protein is increased, the synthesis of hepatic glycogen is decreased, and the decomposition of hepatic glycogen is also increased in stress state. These alterations result in the increasing of blood glucose. In the present study, we found that in spinal cord injured rats, the serum insulin contents were decreased, the blood glucose levels were increased, and the immunostaining intensities of islet B cells were attenuated, when compared with normal control rats. These results further demonstrate that spinal cord injury can result in hyperglycemia which is associated with the attenuation of synthesis and secretion of insulin in islet B cells. The underlying mechanism is involved in the obvious increasing of catecholamines induced by the excitation of hypothalamic-pituitaryadrenocortical axis and adrenergic nerve in stress state. The catecholamines could inhibit the synthesis and secretion of insulin through affecting receptors in islet B cells, then cause the decreasing of insulin content, and then result in the increasing of blood glucose.10
Several reports have demonstrated that amino acids, especially for lysine, arginine, and leucine, could stimulate the synthesis and secretion of insulin in islet B cells.5,6 However, it is still not clear whether LMN could affect the insulin contents and blood glucose levels in spinal cord injury rats. In the present study, we first investigated the effects of LMH on the insulin contents and blood glucose levels in spinal cord injury rats. Our results indicated that LMH could increase the insulin contents, decrease the levels of blood glucose, and enhance the insulin immunostaining of islet B cells in spinal cord injured rats, suggesting that LMH might regulate the synthesis and secretion of insulin through islet B cells. In addition, statistical data showed that high-dose LMH had a much stronger effects than low-dose LMH, suggesting LMH could enhance the secretion function of islet B cells at a dose-dependent manner. Although how exactly LMH promotes the synthesis and secretion of insulin in islet B cells has yet to be elucidated, some previous reports could give us inspiration. Sener et al11 demonstrated that islet B cells could intake some amino acids through amino acid transport system. In physiological status, one islet cell can intake LMH about (2.8±0.4) pmol which is obviously higher than arginine and ornithine. In addition, their data also showed that intracellular concentration of LMH in islet B cells was significant higher than extracellular concentration, suggesting the metabolism of islet B cells might be LMH-dependent.
The hyperglycemia after injury to the spinal cord is one of the most important factors affecting prognosis, and persistent hyperglycemia has been considered as an independent prognosis parameter.2,12 Thus, more and more attention is being paid to the hyperglycemia after injury to the brain and spinal cord. Previous studies indicated that insulin could promote the proliferation of many kinds of cells, including spinal cord cells, contribute to nerve repair, and play an important role in nervous system as a kind of neurotransmitter.13,14 Additionally, recent reports showed that insulin also could inhibit cellular apoptosis.15 Here, we found that LMH not only increased the synthesis and secretion of insulin, but also decreased the blood glucose level, suggested that LMH had a beneficial effect on spinal cord injury through increasing the insulin content and decreasing the blood glucose level. Other reports demonstrated that LMH could decrease the morbidity of stroke in hereditary hypertension rat,16 facilitate the benzodiazepine by binding its receptor, and had anti-convulsion and neuroprotective effects.17,18 Moreover, some novel findings indicated that LMH could reduce intracellular oxidative stress and had a potential preventive and therapeutic effect on diabetes and its complication.19-21 These results suggest that LMH might be a promising neuroprotective drug and should be further studied.
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