CN111803621A

CN111803621A - Application of insulin gene enhancer binding protein-1 in preparing medicine for treating diabetic peripheral neuropathy

Info

Publication number: CN111803621A
Application number: CN202010490676.9A
Authority: CN
Inventors: 孙勤; 徐晨鑫; 张敏; 杨帆; 曾俊
Original assignee: Sichuan Provincial Peoples Hospital
Current assignee: Sichuan Provincial Peoples Hospital
Priority date: 2020-06-02
Filing date: 2020-06-02
Publication date: 2020-10-23

Abstract

The invention discloses an application of insulin gene enhancer binding protein-1 in preparing a medicine for treating diabetic peripheral neuropathy, which well improves the damage and pathological changes of peripheral nerve functions by adopting a mode of overexpression of insulin gene enhancer binding protein-1 (ISL 1) in vivo, shows that ISL1 has the potential of treating diabetic peripheral neuropathy, and the symptom improvement can reach the expectation, thereby providing a theoretical basis for ISL1 to treat diabetic peripheral neuropathy and meet the actual treatment requirement.

Description

Application of insulin gene enhancer binding protein-1 in preparing medicine for treating diabetic peripheral neuropathy

Technical Field

The invention relates to the technical field of medical treatment, in particular to application of insulin gene enhancer binding protein-1 in preparing a medicament for treating diabetic peripheral neuropathy.

Background

Diabetes is one of the most serious chronic diseases in the world, affecting the lives of millions of people worldwide. From mild asymptomatic distal sensory neuropathy to severe radiculopathy, is associated with diabetic neuropathy syndromes.

Diabetic Peripheral Neuropathy (DPN) is one of the most common chronic complications of diabetes. Diabetic peripheral neuropathy is a heterogeneous disease with complex pathophysiological characteristics, the pathogenesis of which is influenced by oxidative stress. In addition, many years of research have shown that oxidative stress, neuroinflammation and apoptosis play a crucial role in the pathogenesis of diabetic peripheral neuropathy. Clinical manifestations of diabetic peripheral neuropathy include elevated vibration and thermal sensation thresholds, which progress to sensory loss with peripheral nerve fibrosis. Painful diabetic neuropathy is one of diabetic peripheral neuropathy, seriously affects the quality of life of diabetic patients, and aggravates economic burden. Also, diabetic peripheral neuropathy causes foot ulcers, which is one of the main causes of the increase in the number of amputations in diabetic patients in recent years.

The current treatment for DPN mainly employs comprehensive approaches, such as: (1) influence of control risk factors such as smoking cessation, alcohol withdrawal and the like; (2) the blood sugar is strictly controlled to reach the standard, and the neurotoxicity of high sugar is reduced; (3) dilating blood capillary and improving microcirculation around nerve; (4) supplementing neurotrophic factors such as mecobalamin; (5) administration of analgesic drugs to ameliorate symptoms; however, the above-mentioned therapeutic measures have not yet satisfied the therapeutic requirements for diabetic peripheral neuropathy. In the case of mecobalamin supplementation, homocysteine can be converted into methionine, and the methionine is involved in the process of synthesizing thymine by deoxynucleoside, so that nucleic acid and protein synthesis is promoted, thus possibly promoting axonal transport, axonal regeneration and myelination, preventing axonal degeneration and repairing damaged nerve tissues. Clinically, however, mecobalamin has extremely slow effect of improving symptoms of patients with diabetic peripheral neuropathy, and the symptoms of patients can be improved only after the patients take the medicine for 6 months. Moreover, the improvement effect of partial patients can not reach the expectation no matter how fast the effect is. Therefore, new methods for treating diabetic peripheral neuropathy are continuously searched, and the method is of great importance for improving the life quality of diabetic patients and reducing medical expenses.

The discovery of a new cytokine provides a new potential target for treating peripheral neuropathy, Insulin gene enhancer binding protein-1 (ISL 1) is an important transcription factor, is mainly expressed in pancreatic secretory cells, heart, neurons and the like, can influence the secretion of Insulin, glucagon and the like of mammals, and has an important regulation effect on the development of heart and neurons, and in recent years, ISL1 is also found to be related to the development of some malignant tumors. Although the biological function of ISL1 has been gradually revealed, it has not been involved in the improvement of diabetic peripheral neuropathy.

Disclosure of Invention

Aiming at the problem that the prior art is difficult to meet the treatment requirement of diabetic peripheral neuropathy, the invention provides the application of insulin gene enhancer binding protein-1 in preparing a medicament for treating diabetic peripheral neuropathy.

The invention establishes a diabetic rat model accompanied with diabetic neuropathy, then utilizes a lentiviral vector to over-express ISL1 gene in the rat model body, and after a DM rat over-expresses ISL1 protein, the expression: (1) an increase in animal pain threshold and nerve conduction velocity; (2) the sciatic neurons of animals are arranged more closely, Schwann cells have more complete structures, large and round nuclei and increased chromosomes; (3) increased expression of Nerve Growth Factor (NGF) and brain-derived neurotrophic factor (BDNF) in the sciatic nerve.

Therefore, compared with the prior art, the invention has the following beneficial effects:

the overexpression of the protein refers to that the expression level of the corresponding protein is higher than the conventional level, generally speaking, the overexpression of the protein can cause diseases, but aiming at diabetic peripheral neuropathy, the damage of peripheral nerve functions and pathological injury are well improved by adopting a mode that insulin gene enhancer is combined with protein-1 to be overexpressed in vivo, so that the technical prejudice is broken through, ISL1 has the potential of treating the diabetic peripheral neuropathy, and the symptom improvement effect can be expected, so that a theoretical basis is provided for ISL1 to treat the diabetic peripheral neuropathy and meet the actual treatment requirement.

Detailed Description

The present invention is further illustrated by the following examples, which include, but are not limited to, the following examples.

Examples

1. Construction of DM rat, Lentiviral vector overexpressing ISL1

Male SD rats of SPF grade 5 weeks old were selected and randomly divided into two groups, one group was given a normal diet named normal group (n = 10), the other group was given a high fat diet, and the rats fed with the high fat diet were intraperitoneally injected with 0.45% STZ solution (35 mg/kg) 6 weeks later, followed by monitoring blood glucose, and if the blood glucose was >16.7mmol/L for 3 consecutive days, it was considered that the diabetic model rats were successfully constructed.

A lentiviral vector (LV-ISL 1) and a control empty viral vector (LV-NC) were constructed which overexpressed ISL1 protein. The lentiviral vector was transfected into DM rats by tail vein injection. The DM rats are divided into 3 groups according to different intervention modes, and each group comprises 8 rats; LV-ISL1 group rats were transfected with a lentiviral vector expressing ISL1 protein, LV-NC group rats were transfected with an empty viral vector (i.e., a viral vector not expressing ISL 1), and DM group was injected tail vein with an equal amount of saline.

2. ISL1 can increase pain threshold of DM rat and peripheral nerve conduction velocity

Measuring the motor nerve conduction velocity of the rat by using an electrode method; fixing the rat in a prone position after anesthesia, placing a stimulation electrode on the incisal track of the right ischium of the rat, placing a recording electrode on the second toe of the right foot, and placing a reference electrode on the midpoint of the two electrodes; single pulse square wave stimulation is carried out, the pulse width is 0.1m, the stimulation intensity is 1.5 times of the threshold value, and the interval between two times of stimulation is more than 5 s; latency (time taken for an action potential to be conducted from a motor nerve to a distal muscle) was measured and recorded, and the measurement was repeated 5 times, and the mean value thereof was calculated; the distance between the stimulating electrode and the recording electrode is measured and the motor conduction velocity (m/s) = distance between two points/latency is calculated.

The pain sensation of rats was evaluated as paw with paw latency (PWL) and paw with paw threshold (PWT) after thermal stimulation. PWL and PWT were measured and compared for each group of animals at 0, 7, 14, 21, 28 days.

As a result, from day 14, the DM rats exhibited a decrease in PWL, a decrease in PWT, and a decrease in motor nerve conduction velocity, as compared to the normal rats, indicating that the DM rats exhibited hypoalgesia, a decrease in pain threshold, and peripheral nerve function impairment. Compared with LV-NC rats, LV-ISL1 rats have prolonged PWL, increased PWT and increased motor nerve conduction velocity, which indicates that ISL1 improves peripheral nerve function damage of DM rats.

3. ISL1 for improving peripheral neuropathological injury of DM rat

Sciatic nerves of rats in each group are separated, and observation by an electron microscope shows that sciatic nerves of rats in DM group have fibrosis, loose structure, larger gaps, decreased density of myelinated nerve fibers and uneven distribution compared with sciatic nerves of rats in normal group. The LV-ISL1 group rats had uniform distribution and arrangement of sciatic nerve fibers, Schwann cell structure was intact, and nerve myelin was observed compared with LV-NC rats. This indicates that the DM rats exhibited pathological damage to the peripheral nerves, while ISL1 ameliorated the pathological damage to the peripheral nerves in DM rats.

4. ISL1 improving the expression of nerve growth factor in DM rats

Nerve Growth Factor (NGF) and brain-derived neurotrophic factor (BDNF) belong to mammalian neurotrophic factors and have important protective effects on the development, growth and differentiation of nerves of animals.

And detecting the expression of the neurotrophic factors in sciatic nerve fibers of each group of rats by using an RT-qPCR (reverse transcription-quantitative polymerase chain reaction) and Western blot method. Reduced NGF and BDNF expression in sciatic nerve of DM rats compared to normal group; the expression of the above-mentioned factors was increased in the LV-ISL1 group as compared with that in the LV-NC group. This indicates that ISL1 can increase the expression of NGF and BDNF and thus have neuroprotective effects.

The above examples demonstrate that the in vivo over-expression of the ISL1 protein can improve diabetic peripheral neuropathy by establishing a diabetic rat model and overexpressing ISL1 through lentivirus transfection, and show that the ISL1 protein has clinical application potential in treating diabetic peripheral neuropathy.

The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.

Claims

1. The application of insulin gene enhancer binding protein-1 in preparing medicine for treating diabetic peripheral neuropathy.