Application of dietary fiber inulin in preparation of medicine for preventing prediabetic peripheral neuropathy

Abstract

The invention provides application of dietary fiber inulin in preparation of a medicine for preventing prediabetic peripheral neuropathy, and belongs to the technical field of medicines. The invention provides medicine application of dietary fiber inulin, experiments prove that the dietary fiber inulin can effectively improve hyperalgesia, hyperalgesia hypersensitivity reaction and sensory nerve action potential change of peripheral neuropathy mice in the early stage of diabetes mellitus, and meanwhile, can remarkably repair sciatic nerve myelin sheath and axonal lesions, so that the dietary fiber inulin can be used for treating diabetes mellitus. The traditional Chinese medicine composition has the effect of remarkably improving prediabetic peripheral neuropathy.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to application of dietary fiber inulin in preparing a medicine for preventing prediabetic peripheral neuropathy. Background Art

[0002] Diabetes is a group of metabolic diseases characterized by high blood sugar. High blood sugar is caused by defects in insulin secretion or its biological function, or both. Long-term high blood sugar in diabetes can lead to chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves.

[0003] Peripheral neuropathy (DPN) is a syndrome characterized by sensory loss, muscle weakness and atrophy, decreased tendon reflexes, and vasomotor symptoms. This lesion affects the conduction and perception of the nervous system, leading to dysfunction in different parts of the body. More and more evidence shows that peripheral neuropathy is common in the prediabetes stage, especially in the state of marginal hyperglycemia. As the course of the disease increases, the clinical manifestations of peripheral neuropathy are: starting from the front end of the toes, symmetrical pain and numbness in both feet, and developing proximally. This chronic pain often presents with hyperalgesia, paresthesia, and spontaneous pain such as tingling, burning, cold, and electric shock pain. Patients may even develop mental illnesses such as irritability and depression.

[0004] Inulin is a polymer of oligofructose and also a dietary fiber. So far, there is no report on the use of dietary fiber inulin for preventing prediabetic peripheral neuropathy. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of dietary fiber inulin for preparing a medicine for preventing prediabetic peripheral neuropathy.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows: Application of dietary fiber inulin in the preparation of drugs for preventing prediabetic peripheral neuropathy.

[0007] Preferably, the drug uses dietary fiber inulin as the main active ingredient and has the following uses: Improve hyperalgesia and allodynia as well as changes in sensory nerve action potentials; Repair of sciatic nerve myelin and axonopathy; Promotes the expression of proteins related to sciatic nerve myelination.

[0008] Preferably, the drug is administered orally.

[0009] Beneficial effects of the present invention: The present invention provides the use of dietary fiber inulin in the preparation of a drug for preventing prediabetic peripheral neuropathy, and provides a new drug use of dietary fiber inulin. According to the embodiment of the present invention, the dietary fiber inulin can effectively improve the hyperalgesia and hyperalgesia reaction and the changes in sensory nerve action potential in mice with prediabetic peripheral neuropathy, and can significantly repair the myelin and axonal lesions of the sciatic nerve. The above results show that dietary fiber inulin has the effect of significantly improving prediabetic peripheral neuropathy, can be used to prepare a drug for preventing prediabetic peripheral neuropathy, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 To establish the DPN model.

[0011] Figure 2 To investigate the effects of dietary fiber inulin on allodynia, thermal hyperalgesia and sciatic nerve electrophysiology in DPN mice.

[0012] Figure 3 The ultrastructure of sciatic nerve tissue of mice in each experimental group under transmission electron microscope.

[0013] Figure 4 The results of immunohistochemical staining of MBP and P0 in situ of sciatic nerve of mice in each experimental group. DETAILED DESCRIPTION

[0014] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0015] Example 1: Study on the preventive effect of dietary fiber inulin on prediabetic peripheral neuropathy Experimental content The present invention does not specifically limit the source of the dietary fiber inulin, and the conventional commercially available dietary fiber inulin in the art can be used. 4-week-old SPF female leptin receptor mutant db / db mice were selected as diabetic peripheral neuropathy (DPN) models. According to fasting blood glucose (FBG) and oral glucose tolerance test (OGTT), the mice were randomly divided into: prediabetes group (PDM group), inulin intervention prediabetes group (INU / PDM group), diabetic group (DM group), inulin intervention diabetic group (INU / DM group). At the same time, 4-week-old female C57BL / 6J mice were set as normal control group. Inulin (INU) intervention group, each mouse was gavaged at 5% (wt / wt) for 6 consecutive weeks; the model group and blank control group were gavaged with an equal amount of normal saline every day for 6 weeks. During the dietary fiber intervention period, the body weight and blood glucose level of each group of mice were monitored regularly; the changes in the latency of pain threshold of each group of mice were monitored using the von Frey fiber analgesia instrument and the thermal radiation test. After the intervention, the behavioral performance and sciatic nerve action potential of DPN mice were monitored, and the ultrastructural changes of the sciatic nerve of DPN mice and the expression of myelin protein in the sciatic nerve of DPN mice were observed.

[0016] Experimental methods Monitoring behavioral performance and sciatic nerve action potential in DPN mice 1.1 Monitoring the mechanical withdrawal threshold (PWT) Each mouse was placed individually on an experimental mesh pad composed of an independent plexiglass box and a wire mesh. After the mouse was observed to adapt to the environment and stop exploring behavior, the PWT of each mouse was detected by the up and down method. The tactile sensitivity of mice was evaluated by the gram of von Frey filaments. Different gram of Von Frey Filaments (vFFs) represents different strengths. The filaments were vertically stimulated between the third and fourth metatarsal bones or the lateral sole of the right hind paw of the mouse until the filaments were slightly bent. The mice were observed to see whether there were obvious reactions such as paw withdrawal, shaking and licking, which were considered to be similar to pain perception. Each filament was tested 10 times, with at least 30 seconds between each time. At least 6 or more similar pain perception reactions in 10 tests were recorded as positive. The minimum gram that elicits a positive reaction is usually used as PWT, and it is used to measure the degree of mechanical allodynia in mice.

[0017] 1.2 Monitoring thermal withdrawal latency (PWT) Thermal hyperalgesia was assessed by measuring the paw stretch latency (PWL) of mice to harmful thermal stimulation using a PL-200 plantar analgesia tester. The experimental environment was kept quiet, and the mice were placed in a glass cover to familiarize themselves with the environment and the instrument for 20 minutes before the measurement began. A 5mm diameter heat light source was placed vertically on the sole of the right hind paw of the mouse to observe whether the mouse showed obvious pain-like reactions such as paw retraction, shaking and licking. Each thermal radiation automatically terminated for 12 seconds to avoid possible tissue damage. At the same time, a thermal radiation experiment was performed every 1 minute to prevent behavioral disorders and thermal sensitivity. The PWT of each mouse was the average of 3 measurements, and thermal hyperalgesia was measured by measuring the PWT of the mouse.

[0018] 1.3 Monitoring the electrophysiological activity of the sciatic nerve in DPN mice Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The body temperature of mice was maintained at 37°C to reduce animal stress caused by anesthesia. After anesthesia, the hair of the right lower limb of the mouse was removed and dissected, and small incisions were made at the bilateral sciatic notch and ankle. The muscle tissue was slowly separated layer by layer with a glass needle until the sciatic nerve of the mouse was fully exposed. The experiment was monitored by a 0.2ms rectangular pulse of the BL-420S biological function measurement system. The sciatic notch of the sciatic nerve was stimulated by ultra-micro stimulation (1V) with a pole spacing of about 10mm through a bipolar needle electrode. After stimulation, a bipolar recording electrode was used to record the compound action potential (CAP). During the experiment, physiological saline was intermittently dripped on the exposed sciatic nerve dissection to ensure smooth conduction. The appearance of 6 continuous stable waveforms was used as the standard for stopping the experiment. At this time, the following measurements and calculations were performed: ① Measure the distance between the stimulating electrode and the recording electrode, represented by S. ② Determine the action potential latency recorded by the two electrodes and calculate the difference (the time difference between the two peaks), expressed as Δt. ③ Measure the sensory nerve action potential amplitude (SNAPamplitudes), that is, the distance between the action potential trough and the peak toe. The sensory nerve conduction velocity (SNCV, in meters per second) is calculated as the ratio of the distance between the two bipolar recording electrodes to the time difference between the two waiting times, that is, V=S / Δt.

[0019] Observation of ultrastructural changes of sciatic nerve in DPN mice Fixation: Take 1 cubic millimeter of sciatic nerve tissue and place it in 2.5% glutaraldehyde fixative for 2 hours, add 0.1M sodium dimethylarsenate buffer to soak and rinse 4 times, each rinse for 15 minutes, add 1% osmium acid solution to soak the sciatic nerve tissue for 2 hours, then add 0.1M sodium dimethylarsenate buffer to rinse repeatedly, each rinse for 15 minutes, and rinse twice.

[0020] Infiltration: After fixing the sciatic nerve tissue, place it in an infiltration solution prepared by an incomplete embedding solution and propylene oxide in a ratio of 1:1, and place it at room temperature for 1 hour; then soak the sciatic nerve in an infiltration solution prepared by an incomplete embedding solution and propylene oxide in a ratio of 2:1 for 1 hour; finally, soak it in a complete embedding solution overnight.

[0021] Embedding and polymerization: trim the sciatic nerve tissue, embed it in an embedding plate filled with complete embedding solution, and place it in an incubator at 60°C for 48 h.

[0022] Sectioning and staining: The located sciatic nerve embedded block was sectioned at a thickness of 50 nm and stained with uranyl acetate for 30-45 min; then stained again with lead citrate for 30 min.

[0023] Detection of myelin protein expression in sciatic nerve of DPN mice After paraffin removal, the paraffin sections were boiled in 10 mM citrate buffer (pH 6.0), rinsed with ddH2O, and immersed in ddH2O containing 0.3% H2O2 for 10 min. After rinsing with PBS, they were blocked with 5% normal goat serum for 1 h and incubated with primary antibodies (myelin basic protein, MBP 1:200; myelin protein zero, P0 1:200, Abcam) at 4°C overnight. The next day, the tissue sections were rinsed with PBS three times and incubated with secondary antibodies goat anti-mouse (1:100, ThermoFisher) and goat anti-rabbit (1:100, ThermoFisher) at 37°C for 1 h. DAB was used as the color developer and the results were observed under an inverted microscope.

[0024] Experimental Results Figure 1 For the establishment of the DPN model, Figure 1 A: Curve of the change of mechanical withdrawal reflex threshold over time in mice. Figure 1 B: Curve of the change of the latency of thermal withdrawal reflex over time in mice. Figure 1 C: Action potential latency of mice, Figure 1 D: sensory nerve conduction velocity, Figure 1 E: Amplitude of sensory nerve action potential. Control: normal control group, PDM: prediabetes group, DM: diabetic group. ** P <0.05,*** P <0.001 for PDM group compared with Control group. ## P <0.01, ### P <0.001 for the DM group compared with the Control group. It can be seen that compared with the normal control group, the mechanical withdrawal reflex threshold of mice ( Figure 1 A) and thermal withdrawal latency ( Figure 1 B) In the prediabetes group (PDM) and the diabetic group (DM group), the levels gradually decreased over time and were significantly different from those in the control group at week 6 ( P <0.001). Sciatic nerve action potentials also showed similar results ( Figure 1 C), indicating that the DPN model is reliable.

[0025] Figure 2 The effects of dietary fiber inulin on allodynia, thermal hyperalgesia and sciatic nerve electrophysiology in DPN mice. Figure 2 A: Effects of dietary intervention on mechanical allodynia, Figure 2 B: Evaluation of the dietary fiber inulin treatment on thermal hyperalgesia in diabetic neuropathic pain, Figure 2 C: Representative traces of compound action potential (CAP) in sciatic nerve of db / db mice after 6 weeks of dietary fiber inulin intervention. Figure 2 D: Dietary fiber inulin increases sciatic nerve sensory nerve conduction velocity (SNCV), Figure 2 E: Dietary fiber inulin has a promoting effect on sensory nerve action potential (SNAP).* P <0.05 and ** P <0.01 means compared with the PDM group, ## P <0.01 and #### P <0.0001 compared with the DM group. Figure 2 As shown in A and B, as the intervention time prolonged, the supplementation of dietary fiber inulin significantly delayed the decreasing trend of PWT in the INU / PDM group and the INU / DM group compared with the untreated model group. Similarly, during the treatment, dietary fiber inulin significantly increased the latency to thermal stimulation in the INU / PDM group and the INU / DM group compared with the untreated group. To further evaluate the effect of dietary fiber inulin on DPN, nerve conduction measurements showed that the sensory nerve conduction velocity (SNCV, Figure 2 D) and sensory nerve action potential (SNAP, Figure 2 Compared with the model, dietary fiber inulin intervention significantly improved these indicators, especially in the INU / PDM group ( Figure 2 C). The above results indicate that dietary fiber inulin effectively improves hyperalgesia and allodynia in DNP mice, and the changes in sensory nerve action potential reverse myelinated nerve fiber damage.

[0026] Figure 3 The ultrastructure of the sciatic nerve tissue of mice in each experimental group under transmission electron microscopy. Figure 3 A: PDM group, Figure 3 B: INU / PDM group, Figure 3 C: DM group, Figure 3 D: INU / DM group. The red arrows are Schwann cells and the black arrows are myelin layers. Bar×2000μm. It can be seen that in the DM group ( Figure 3 C) The myelin sheath of the sciatic nerve of mice was swollen and deformed, with local vacuolar degeneration, loss of light and dark concentric circle structure, layered deformation of myelinated fiber lamellae, and partial axonal separation and edema. In contrast, the DF / DM dietary fiber intervention group ( Figure 3 The sciatic nerve edema and vacuolar deformation of the PDM group were less than those of the DM group, and only some of the myelinated fibers were slightly deformed. At the same time, axonal changes were not obvious. Figure 3 A) Compared with the DF / PDM group, the sciatic nerve myelin sheath ( Figure 3 B) It presents a concentric circular layered structure with alternating light and dark, and is arranged regularly. The myelinated nerve fibers are dense and uniform, with only mild vacuolar degeneration. The above results show that dietary fiber inulin can significantly repair sciatic nerve myelin and axonal lesions.

[0027] Figure 4 The results of immunostaining analysis of MBP and P0 in situ of sciatic nerve. Figure 4 A Left: MBP expression in PDM group; Figure 4 A right: MBP expression in INU / PDM group, Figure 4 B Left: MBP expression in DM group; Figure 4 B right: MBP expression in INU / DM group, Figure 4 C Left: P0 expression in the PDM group; Figure 4 C right: P0 expression in INU / PDM group, Figure 4 D Left: P0 expression in DM group; Figure 4 D right: P0 expression in INU / DM group, Figure 4 E is the expression of MBP in PDM group, INU / PDM group, DM group, and INU / DM group. Figure 4 F is the P0 expression results of the PDM group, INU / PDM group, MBP expression, DM group, and INU / DM group. * P <0.05 and ** P <0.01 means compared with the PDM group, # P <0.05 indicates compared with the DM group. Compared with the corresponding model group, the dietary fiber inulin intervention groups (including INU / PDM and INU / DM) ( Figure 4 MBP expression was upregulated in the sciatic nerve of the INU / DPM group (right side of A and B). Figure 4 The P0 protein level in the PDM group was significantly decreased compared with that in the right side of C ( Figure 4 Myelin protein P0 showed similar results throughout the diabetic period ( Figure 4 D).

[0028] What is disclosed above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. Application of dietary fiber inulin in the preparation of drugs for preventing prediabetic peripheral neuropathy.

2. The use of the dietary fiber inulin according to claim 1 in the preparation of a drug for preventing prediabetic peripheral neuropathy, characterized in that: The drug uses dietary fiber inulin as the main active ingredient (purity of 91%) and has the following uses: (a) Improve hyperalgesia and allodynia as well as changes in sensory nerve action potential; (b) Repair of sciatic nerve myelin and axonal lesions; (c) Promotes the expression of proteins related to sciatic nerve myelination.

3. The use of the dietary fiber inulin according to claim 2 in the preparation of a drug for preventing prediabetic peripheral neuropathy, characterized in that: The administration route of the drug is oral administration.

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