Application of divalent metal lactate in preparation of product for repairing nerve injury

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of divalent metal lactate in preparation of products for preventing, improving, repairing and treating nerve injury. Preferably, the divalent metal lactate comprises one or a combination of more of calcium lactate, magnesium lactate, zinc lactate and ferrous lactate. The invention provides a novel treatment mode for repairing central and peripheral nerve injury, and the application prospect in the aspects of treatment and repair of central nerve injury caused by trauma and the like or peripheral neuritis or peripheral nerve sensory abnormality caused by viruses, diabetes mellitus, chemotherapeutic drugs and the like is wide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of divalent metal lactate in the preparation of products for preventing, improving, repairing and treating nerve damage. Background Art

[0002] The central and peripheral nervous systems constitute the complete nervous system of the human body. The nervous system plays a leading role in maintaining the homeostasis of the body's internal environment, maintaining the integrity and unity of the body and its coordination and balance with the external environment. With the deepening of social modernization, traffic accidents, sports accidents, etc. are inevitably increasing. At the same time, potential violent incidents and natural disasters may cause damage to the nervous system.

[0003] Spinal cord injury is a serious injury to the central nervous system that can cause sensory and motor impairment in patients, bringing serious burdens to the patient's family and society. Spinal cord injury is a complex and delicate process, which is divided into primary injury and secondary injury according to the pathological process of neuronal injury. Among them, the severity of spinal cord injury is mainly determined in the secondary injury stage. This is mainly because during the secondary injury process, the spinal cord injury microenvironment undergoes significant changes, mainly including increased reactive oxygen levels, damaged blood vessels hindering the delivery of oxygen, leading to hypoxia, increased inflammatory response, etc., coupled with the poor recovery ability of nerves themselves, resulting in the inability of the spinal cord to regenerate naturally after injury. In clinical practice, surgery or rehabilitation training has limited efficacy in patients with spinal cord injury and cannot effectively promote nerve regeneration and functional recovery. Due to the complex pathophysiological process after spinal cord injury, the effects of existing single therapies in the clinic in the treatment of spinal cord injury are not satisfactory. At present, autologous nerve transplantation is considered to be the gold standard for the treatment of nerve defects, but the limited source of donor nerves and the functional defects of the donor nerves have greatly limited its clinical application. Therefore, there is a continuous and urgent clinical demand for drugs for the treatment of spinal cord injury that are safe, effective, low-cost, and easy to administer.

[0004] In addition, the repair and reconstruction of peripheral nerve function, especially long-distance defects, are also global problems in clinical treatment. Autologous or allogeneic nerve transplantation is also a commonly used clinical repair method for the treatment of peripheral nerve injury, but there are a series of problems such as limited donor sources, differences in the structure of donor and recipient nerves, loss of nerve function in the donor area, scars and immune rejection. Peripheral neuritis and peripheral nerve paresthesia are currently the most common peripheral nerve injuries in clinical practice. Peripheral neuritis, also known as peripheral neuritis or polyneuritis, is a distal nerve dysfunction of the limbs caused by various causes. Peripheral neuritis may be caused by a variety of reasons such as nutritional metabolism, drugs and poisoning, vasculitis, tumors, trauma or mechanical compression, and the main manifestations are abnormal sensation, movement and autonomic nerve function within the range of the damaged nerve control. The symptoms of peripheral neuritis include hypoesthesia, pain, numbness or pins and needles, ants, burning, hyperesthesia, etc. Peripheral neuritis may be multiple or single, symmetrical or asymmetrical, and is one of the most common diseases of the nervous system. When the cause of peripheral neuritis is known, treatment targeting the cause can alleviate symptoms and restore damaged nerves, but the effect is poor for peripheral neuritis with unknown causes or refractory primary diseases. Data show that about 40% of patients with peripheral neuritis will have sequelae, with a high disability rate, which seriously affects the patient's physical and mental health and quality of life. At present, there are not many clinical treatments for peripheral neuritis, and they are mainly Western medicine. For example, in clinical practice, peripheral neuritis caused by various reasons can be treated with large doses of B vitamins, such as vitamin B1, B6, B 12 Etc. For patients with obvious pain, analgesics and sedatives can be used. For patients with inflammatory demyelinating lesions, adrenal cortical hormones such as methylprednisone, dexamethasone or hydrocortisone can be used. However, if these drugs are used for a long time, most of them have high toxic side effects, are prone to drug dependence, and are difficult to achieve complete cure.

[0005] The inventors found in previous studies that during the degradation of poly-L-lactic acid (PLLA), the molecular structure of PLLA is gradually destroyed and slowly hydrolyzed into lactic acid, which can induce human fibroblasts to increase the production of collagen, resulting in an increase in collagen fibers in the dermis, producing a filling and repairing effect. On this basis, the inventors have found that PLLA and lactic acid and its related lactate compounds have beneficial effects in repairing tissues such as cartilage, connective tissue, tendons, fascia, and nerves (see CN202210028046.9). The present invention is a continuation of previous work, and the present invention has found new uses for divalent metal lactates in the preparation of products for preventing, improving, repairing, and treating nerve damage. Summary of the invention

[0006] In order to overcome the defects and shortcomings in the prior art, the present invention provides a new use of divalent metal lactate. The inventor unexpectedly discovered during the research process that divalent metal lactate can effectively prevent, improve, repair and treat central nervous system and peripheral nerve damage, and based on this, conducted in-depth research and completed the present invention.

[0007] In order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:

[0008] The present invention provides the use of divalent metal lactate in preparing products for preventing, improving, repairing and treating nerve damage, and the tissue with nerve damage is selected from nerve tissue rich in collagen.

[0009] As an optional mode, in the above application, the nerve damage is central nerve damage or peripheral nerve damage, and the prevention, improvement, repair and treatment of nerve damage is nerve regeneration, increase in nerve number and / or nerve repair.

[0010] As an option, in the above application, the central nervous system injury includes spinal cord injury or encephalomyelitis, and the peripheral nerve injury includes peripheral nerve injury, peripheral neuritis or peripheral neuropathy.

[0011] As an optional mode, in the above application, the peripheral neuritis is selected from one or more of the following: facial neuritis, diabetic peripheral neuritis, viral peripheral neuritis, peripheral neuritis caused by chemotherapy drugs or Guillain-Barré syndrome.

[0012] As an optional mode, in the above application, the symptoms of peripheral neuritis include hypoesthesia, pain, numbness or tingling, tingling, burning, hyperesthesia.

[0013] As an optional manner, in the above application, the divalent metal lactate includes one or more combinations of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate.

[0014] As an optional mode, in the above application, the divalent metal lactate is selected from magnesium lactate or a combination of zinc lactate and magnesium lactate.

[0015] Preferably, in the composition, the ratio of the magnesium lactate to the zinc lactate is 1:10-10:1 by weight.

[0016] More preferably, in the composition, the ratio of the magnesium lactate to the zinc lactate is 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 by weight.

[0017] Most preferably, in the composition, the ratio of the magnesium lactate to the zinc lactate is 4:1 by weight.

[0018] As an optional mode, in the above application, the product is selected from one or more of medicines, test kits, health products, and medical devices.

[0019] The concentration of the divalent metal lactate in the product is 10-80 mmol / L, more preferably, the concentration of the divalent metal lactate in the product is 20-60 mmol / L.

[0020] As an optional manner, in the above application, the medical device includes one or more combinations of medical adhesive tape, bandage, gauze, dressing, sponge, and medical suture.

[0021] As an optional mode, in the above application, in the medicine, the divalent metal lactate is an active ingredient, and the medicine further comprises a pharmaceutically acceptable carrier or excipient.

[0022] Preferably, in the medicament, the divalent metal lactate is the only active ingredient.

[0023] As an optional mode, in the above application, the dosage form of the drug includes injection or external preparation.

[0024] Preferably, the external preparation comprises one or more combinations of ointments, creams, patches, sprays, solutions, lotions.

[0025] Preferably, the administration of the product includes one or more of the following: intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration or application.

[0026] In addition, those skilled in the art will appreciate that the use of the preparation, as well as the dosage and volume of administration are related to the age, physical condition and disease of the subject, and can be determined by a clinician based on the circumstances.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a new treatment method for repairing central and peripheral nerve damage that is safe, effective, inexpensive, and simple to administer. In particular, the present invention has found the use of a divalent metal lactate comprising one or more combinations of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate in the preparation of products for preventing, improving, repairing, and treating nerve damage. Therefore, it has broad application prospects in the treatment and repair of central nervous system damage caused by trauma or peripheral neuritis or peripheral nerve paresthesia caused by viruses, diabetes, chemotherapy drugs, etc. DETAILED DESCRIPTION

[0029] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0030] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0032] In the following examples, all experiments were repeated three times, with at least three samples in each group. The experimental data were statistically analyzed using SPSS 21.0 software. Mean ± SD was used for the statistical description of the measurement data, and variance analysis was used for comparison of indicators between groups. P < 0.05 was considered to be statistically significant. When P < 0.05, a pairwise sample test was performed using the LSD-t test.

[0033] Example 1: Effects of divalent metal lactate on sciatic nerve injury model animals 1. Establishment of sciatic nerve injury model and experimental grouping

[0034] SD rats (male and female, 1:1), 8 weeks old, weighing 200-220g, a total of 40, were placed in a temperature-controlled room (22±2℃). The animals were anesthetized by intraperitoneal injection of 3.0% (w / v) sodium pentobarbital (0.2ml / 100g). After complete anesthesia, the left buttocks of the rats were prepared, and the prepared skin was disinfected with alcohol cotton balls and iodine cotton balls. Then an oblique incision was made, and the left sciatic nerve was exposed by blunt separation in the gluteal muscle gap. The sciatic nerve was clamped with a hemostatic forceps 0.5cm away from the lower edge of the piriformis muscle 3 times (10s each time, 10s interval), and the muscle sutures next to the clamp were marked with surgical sutures, and the muscles and skin were sutured immediately and disinfected again. After surgery, the animals were randomly divided into 4 groups, 10 in each group. The rats were housed in cages and then given intraperitoneal injections of drugs or saline according to the grouping, once a day for 4 consecutive weeks. The experimental groups and treatment methods are shown in Table 1. The general condition of the rats, skin ulcers around the incision, and functional recovery of the limbs on the operated side were closely observed after surgery.

[0035] Table 1: Experimental groups and treatments

[0036]

[0037]

[0038] 2. Footprint Collection and Sciatic Nerve Index Scoring

[0039] A homemade rat gait recording box was built, with a passage of 90 cm in length, 15 cm in width, and 20 cm in height. A 15 cm wide continuous recording paper was placed at the bottom. At 1, 2, 3, and 4 weeks after surgery, the soles of the rats’ hind feet were painted with colored non-toxic dyes, and the rats walked on the continuous recording paper to leave bilateral footprints. The footprints of the experimental foot (E) and the normal foot (N) were selected, and the following variables were measured: ① print length (PL), which refers to the distance from the heel to the toe; ② toe spread (TS), which refers to the distance from the first toe to the fifth toe; ③ inter toe distance (IT), which refers to the distance from the first toe to the fourth toe. The above three variables were substituted into the following formula to calculate the sciatic nerve function index (SFI). When SFI = 0, it is normal, and SFI = -100, it is complete injury. The calculation formula is as follows: SFI = -38.3×(EPL-NPL) / NPL+109.5×(ETS-NTS) / NTS+13.3×(EIT-NIT) / NIT-8.8

[0040] At each time point after surgery (1 week, 2 weeks, 3 weeks and 4 weeks), rat footprints were collected and SFI was calculated to evaluate the motor function and recovery of rats in each group. The results are shown in Table 2. The results showed that at 2 weeks, 3 weeks and 4 weeks after surgery, the SFI of the low, medium and high doses of magnesium lactate groups were significantly better than that of the control group treated with normal saline (Table 2, medium dose magnesium lactate group P < 0.01, low dose and high dose magnesium lactate groups P < 0.05). This shows that low, medium and high doses of magnesium lactate have certain therapeutic effects and can promote the recovery of sciatic nerve function. At the same time, at 2 weeks, 3 weeks and 4 weeks after surgery, the SFI of the medium dose magnesium lactate group was significantly better than that of the low and medium dose magnesium lactate groups (P < 0.05), indicating that medium dose magnesium lactate has a better therapeutic effect on nerve injury and is more conducive to the recovery of nerve function.

[0041] Table 2: Sciatic nerve index of each group at different time points after surgery (n=10)

[0042]

[0043]

[0044] Note: a Compared with group A, P < 0.05; aa Compared with group A, P < 0.01; b Compared with group B and group D, P<0.05.

[0045] 3. Real-time Quantitative PCR

[0046] 3.1 Extraction of total RNA

[0047] Remove the sciatic nerve, wash it in DEPC-treated water, add 0.5mL Trizol, grind it thoroughly, and centrifuge to obtain the supernatant. Add 0.1mL chloroform, centrifuge to obtain the supernatant. Add 0.8mL isopropanol, centrifuge to discard the supernatant. Add 1mL 75% ethanol, centrifuge to discard the supernatant. Add 20μL DEPC-treated water to dissolve the RNA. Use a nucleic acid protein detector to measure the concentration and purity of the total RNA solution.

[0048] 3.2 Real-time quantitative PCR reaction

[0049] The expressions of Caspase-3 and GAP-43 in the sciatic nerve tissues of each group were detected by RT-qPCR.

[0050] Primer design

[0051] Caspase-3 upstream primer 5`-GAGCTTGGAACGGTACGATA-3`

[0052] Downstream primer 5`-CCGTACCAGAGCGAGATGAC-3`

[0053] GAP-43 upstream primer 5`-GTGTGTGAGCCTGTCCTCTC-3`

[0054] Downstream primer 5`-AAAACCGGGGTACAGTGCAA-3`

[0055] Caspase-3 plays a key role in the process of cell apoptosis and is also a potential drug target. Studies have shown that Caspase-3 can severely inhibit DNA replication, transcription and damage repair functions, ultimately leading to irreversible apoptotic effects in cells. Therefore, reducing the activation of Caspase-3 can effectively inhibit the process of cell apoptosis. The results of real-time quantitative PCR of Caspase-3 are shown in Table 3. The results showed that there was no significant change in the expression of Caspase-3 mRNA in the normal saline control group at each time period. Compared with the same time period of this group, the expression of Caspase-3 mRNA in the low, medium and high doses of magnesium lactate groups was significantly downregulated on the first, third and seventh days after modeling (P<0.05). At the same time, within 7 days after modeling, the Caspase-3 mRNA levels in the low, medium and high doses of magnesium lactate groups gradually decreased over time (P<0.05). Compared with the low-dose group, the expression of Caspase-3 mRNA in the medium and high doses of magnesium lactate groups was significantly downregulated on the first, third and seventh days (P<0.05). Compared with the high-dose group, the expression of Caspase-3 mRNA in the medium-dose magnesium lactate group was significantly reduced at each time point (P<0.05). After sciatic nerve injury, the expression of Caspase-3 in rat neurons was significantly enhanced, and the number of apoptotic neurons also increased. After the injection of magnesium lactate, Caspase-3 protein can be reduced, reducing the occurrence of apoptosis caused by nerve injury.

[0056] Table 3: Comparison of Caspase-3 mRNA expression at different time points after modeling and at different time points after surgery

[0057]

[0058] Note: Compared with group A, a P<0.05; compared with group B, b P<0.05; compared with group D, d P<0.05; compared with the first day of the same group on the third day, e P<0.05; compared with the 3rd day in the same group on the 7th day, f P<0.05.

[0059] GAP-43 is a membrane phosphoprotein that is widely present in neurons and is one of the hallmarks of neuronal regeneration. It can play a key role in the regenerative response of neurons by regulating the actin cytoskeleton. The results of real-time quantitative PCR of GAP-43 are shown in Table 4. The results showed that there was no significant change in the expression of GAP-43 mRNA in the saline control group. Compared with the same time in this group, the expression of GAP-43 mRNA in the low, medium and high doses of magnesium lactate groups increased significantly on the first, third and seventh days after modeling (P<0.05). At the same time, within 7 days after modeling, the level of GAP-43 mRNA in the low, medium and high doses of magnesium lactate groups gradually increased over time, reaching the highest level on the seventh day (P<0.05). Compared with the low-dose group, the expression of GAP-43 mRNA in the medium and high doses of magnesium lactate groups was significantly upregulated on the first, third and seventh days (P<0.05). Compared with the high-dose group, the expression of GAP-43 mRNA in the medium-dose magnesium lactate group was significantly increased at each time point (P<0.05). Magnesium lactate can increase the expression of GAP-43, alleviate sciatic nerve injury in rats, and promote nerve regeneration.

[0060] Table 4: Comparison of GAP-43 mRNA expression at different time points after modeling and at different time points after surgery

[0061]

[0062] Note: Compared with group A, a P<0.05; compared with group B, b P<0.05; compared with group D, d P<0.05; compared with the first day of the same group on the third day, e P<0.05; compared with the 3rd day in the same group on the 7th day, f P<0.05.

[0063] Example 2: Effects of divalent metal lactates on spinal cord injury model animals

[0064] 1. Establishment of rat spinal cord injury model

[0065] SD rats (male and female, 1:1), 8 weeks old, weighing 200-220g, a total of 40, were placed in a temperature-controlled room (22±2℃). The animals were anesthetized by intraperitoneal injection of 3.0% (w / v) sodium pentobarbital (0.2mL / 100g). After complete anesthesia, the anesthetized animals were fixed in a prone position, and routine disinfection was performed after skin preparation. A longitudinal incision (2-3cm) was made on the dorsal midline with the twelfth thoracic vertebra (T12) as the base point, and the incision was made from the outside to the inside until the subcutaneous fascia, and the dorsal lamina of the rat T9-11 was accurately removed to expose the dura mater of the spinal cord. The middle part of the exposed spinal cord was clamped with an aneurysm clip for 40s to prepare a spinal cord injury (SCI) model. During the experiment, the bladder of the rats in the spinal cord injury group was squeezed to help them urinate once in the morning and evening. After urination, the perineum was cleaned and kept dry. Limbs soaked in urine were cleaned and dried in time, and the body position was changed frequently. In addition, to prevent infection, gentamicin (2000U / kg·d) was routinely injected intramuscularly 3 days after surgery. After surgery, the animals were randomly divided into 4 groups, 10 rats in each group, and the rats were housed in separate cages. Subsequently, they were given intraperitoneal injections of drugs or saline according to the grouping, once a day for 4 consecutive weeks. The experimental groups and treatment methods are shown in Table 5.

[0066] Table 5: Experimental groups and treatments

[0067]

[0068] 2. Motor system score (BBB score)

[0069] The neurological function assessment method proposed by American researchers Basso et al., namely the BBB scoring method, was used to evaluate the limb function of rats after spinal cord injury. The method is: put the animal into an open basin, tap the basin wall to make it crawl, and observe the animal's hip, knee, and ankle joints walking, driving movement and coordination. The results are listed in Table 6.

[0070] As shown in Table 6, rats in each group had different degrees of functional recovery after magnesium lactate treatment, and the medium-dose magnesium lactate group recovered the fastest. One day after surgery, there was no statistically significant difference in the BBB scores of rats in each group (P>0.05); the BBB scores of each group gradually increased over time, and the difference gradually increased. At 4 weeks after surgery, the medium-dose magnesium lactate group had the highest score, followed by the high-dose magnesium lactate group, the low-dose magnesium lactate group, and the saline-treated control group had the lowest score. There was no statistically significant difference between the low-dose and high-dose magnesium lactate groups (P=0.213), and the differences in the other groups were statistically significant (P<0.05).

[0071] Table 6: BBB scores before and after surgery in each group

[0072]

[0073] Note: a Compared with group A, P < 0.05; b Compared with group B, P < 0.05; c Compared with group C, P < 0.05; d Compared with group D, P<0.05.

[0074] 3. Real-time Quantitative PCR

[0075] The injured spinal cord of 0.5 cm was cut with the injured area as the center, and RT-PCR was performed to measure the gene expressions of brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE).

[0076] 3.1 Extraction of total RNA

[0077] Add 1mL to the homogenizer tube. Place 100mg of tissue in the homogenizer tube, add RNA extraction solution and grind thoroughly, centrifuge and take the supernatant for later use. Add 250μL of chloroform, centrifuge and take the supernatant. Add 0.8mL of isopropanol, centrifuge and discard the supernatant. Add 1mL of 75% ethanol, centrifuge and discard the supernatant. Add 15μL of RNase-free water to dissolve RNA. Use a nucleic acid protein detector to measure the concentration and purity of the total RNA solution.

[0078] 3.2 Reverse transcription reaction

[0079] 3.3 Real-time quantitative PCR reaction

[0080] Primer design

[0081] BDNF upstream primer 5`-GTCAAGTGCCTTTGGAGCCT-3`

[0082] Downstream primer 5`-CATGGGATTGCACTTGGTCTC-3`

[0083] GFAP upstream primer 5`-AGTCGGCGAGTTACCAGGAG-3`

[0084] Downstream primer 5`-TTAATGACCTCGCCATCCCG-3`

[0085] NSE upstream primer 5`-TATCCTGGAGAACAGCGAAGC-3`

[0086] Downstream primer 5`-GACAAAGTCCTGGTAGAGTGCCC-3`

[0087] BDNF is the most widely distributed and abundantly expressed neurotrophic factor in the mammalian brain. It plays an important role in the normal growth, development and plasticity of synapses and can inhibit cell apoptosis by activating downstream pathways. According to Table 7, it can be found that 1 week after surgery, the expression of BDNF in the saline-treated control group was statistically significant in the low, medium and high doses of magnesium lactate groups (P<0.05). There was no statistically significant difference in BDNF expression between the low-dose magnesium lactate group and the high-dose magnesium lactate group (P=0.628). 4 weeks after surgery, except for the difference between the saline-treated control group and the low-dose magnesium lactate group (P=0.820), the differences between the other groups were statistically significant (P<0.05). At 4 weeks after surgery, the differences in the expression of GFAP and NSE among the four groups were statistically significant (P<0.05). As an important neurotransmitter, BDNF plays an important role in the growth and differentiation of neurons and the regulation of synaptic plasticity by binding to specific receptors and activating downstream pathways. As can be seen from Table 7, divalent metal lactate can increase the expression of BDNF, which can then reduce neuronal apoptosis after spinal cord injury by activating downstream anti-apoptotic pathways and participate in the neural remodeling process.

[0088] Table 7: Relative expression of BDNF in the spinal cord of rats 1 week and 4 weeks after surgery

[0089]

[0090] Note: a Compared with group A, P < 0.05; b Compared with group B, P < 0.05; d Compared with group D, P<0.05.

[0091] GFAP expression is an important reference indicator for the growth, proliferation and differentiation of spinal cord nerve cells and is used as a therapeutic target for acute spinal cord injury. According to Table 8, it can be found that 1 week after surgery, the expression of GFAP in the control group treated with normal saline was statistically significant in the low, medium and high doses of magnesium lactate (P<0.05). The difference in GFAP expression among the four groups was statistically significant 1 week after surgery (P<0.05). The difference in GFAP expression among the four groups was statistically significant 4 weeks after surgery (P<0.05). GFAP is a specific marker protein for astrocytes. Astrocytes are the most numerous cells in the central nervous system, so they have a higher expression level after surgery. Compared with other groups, the expression of GFAP in the medium dose of divalent lactate group was significantly reduced, indicating that it inhibits the proliferation of astrocytes and then inhibits the formation of glial scars, which can promote axon regeneration and neural reconstruction. .

[0092] Table 8: Relative expression of GFAP in the spinal cord of rats 1 week and 4 weeks after surgery

[0093]

[0094] Note: a Compared with group A, P < 0.05; b Compared with group B, P < 0.05; d Compared with group D, P<0.05.

[0095] NSE is an enzyme that plays a key role in the respiration of nerve cells and is widely present in brain tissue and other peripheral nerve cells. NSE can participate in the body's glycolysis metabolism. When the body is damaged by hypoxia, ischemia, poisoning, etc., NSE will be released and can enter the blood circulation with the destruction of the blood-brain barrier. According to Table 9, it can be found that one week after surgery, the expression of NSE in the control group treated with normal saline was statistically significant in the three groups of low, medium and high doses of magnesium lactate (P<0.05). There was no statistically significant expression of NSE in the three groups of low, medium and high doses of magnesium lactate one week after surgery (P>0.05). The difference in the expression of NSE among the four groups was statistically significant at 4 weeks after surgery (P<0.05). The medium and high doses of divalent lactate groups can increase the expression level of NSE, among which the medium dose group has a more significant effect on increasing NSE. Since NSE participates in the catalytic reaction in the process of cellular glucose metabolism, the increased expression of NSE can improve the energy metabolism of cells and promote the recovery of damaged cells.

[0096] Table 9: Relative expression of NSE in the spinal cord of rats 1 week and 4 weeks after surgery

[0097]

[0098] Note: a Compared with group A, P < 0.05;b Compared with group B, P < 0.05; d Compared with group D, P<0.05. Example 3: Effects of divalent metal lactate on autoimmune neuritis model animals 1. Establishment of autoimmune neuritis model and experimental grouping

[0099] 40 SD rats (male and female, 1:1 ratio), 8 weeks old, weighing 200-220 g, were placed in a temperature-controlled room (22 ± 2 °C). 180－199 , 1mg H37Ra, 100μL saline, 100μL IFA mixture was fully emulsified and injected into the subcutaneous part of the soles of both hind limbs (100μL per sole) as the injection dose for one rat. After the model was successfully established, rats in each group were gavaged with the corresponding drugs or saline once a day for 4 consecutive weeks.

[0100] Table 10: Experimental groups and treatments

[0101]

[0102] 2. Behavioral scoring

[0103] During the administration period, the rats were observed and their behavior was scored daily. 0 points, no clinical symptoms; 5 points, quadriplegia or death.

[0104] On the first day after the administration of saline, some rats in the autoimmune neuritis group began to show varying degrees of neurological damage symptoms, including lack of energy, unclean fur, and redness and swelling of both hind feet. On the fifth day, all rats showed varying degrees of autoimmune neuritis symptoms, with muscle tension loss, tail dragging, and paralysis. On the eighth day, the autoimmune neuritis symptoms of rats reached a peak, and the highest neurological deficit score was 4.0±0.5. On the sixth day of administration, some rats in the low-dose magnesium lactate group showed varying degrees of neurological damage symptoms. On the eleventh day, all rats showed varying degrees of autoimmune neuritis symptoms. On the fifteenth day, the autoimmune neuritis symptoms of rats reached a peak, and the highest neurological deficit score was 3.0±0.5. After that, the autoimmune neuritis symptoms of rats gradually eased. On the 28th day, the neurological deficit score was 0-1. On the 10th day of administration, some rats in the medium-dose magnesium lactate group showed varying degrees of neurological damage symptoms; on the 16th day, all rats showed varying degrees of autoimmune neuritis symptoms; on the 21st day, the symptoms of autoimmune neuritis in rats reached a peak, and the neurological deficit score was the highest, which was 2.0±0.5. Afterwards, the symptoms of autoimmune neuritis in rats gradually eased; on the 25th day, the neurological deficit score was 0. On the 8th day of administration, some rats in the high-dose magnesium lactate group showed varying degrees of neurological damage symptoms; on the 12th day, all rats showed varying degrees of autoimmune neuritis symptoms; on the 16th day, the symptoms of autoimmune neuritis in rats reached a peak, and the neurological deficit score was the highest, which was 2.5±0.5; afterward, the symptoms of autoimmune neuritis in rats gradually eased; on the 33rd day, the neurological deficit score was 0-1. The above results show that magnesium lactate has a certain therapeutic effect on neurological deficits caused by autoimmune neuritis, among which the effect of medium-dose magnesium lactate is the most significant.

[0105] 3. Detection of inflammatory factor levels

[0106] Existing theories believe that inflammatory factors are one of the factors that aggravate the condition of autoimmune neuritis. IL-17, a characteristic cytokine secreted by Th17 cells, participates in the occurrence of autoimmune diseases by inducing target organ inflammatory cascade reactions. Blood was collected from the retro-orbital venous plexus of rats and the supernatant was obtained by centrifugation. The concentrations of IL-17 and IFN-γ in serum were determined by an enzyme marker, and the results are listed in Table 11. Compared with the autoimmune neuritis group gavaged with normal saline, the levels of IL-17 and IFN-γ in the serum of rats gavaged with magnesium lactate were significantly reduced (P < 0.05). Compared with the low and high doses of magnesium lactate groups, the levels of IL-17 and IFN-γ in the medium dose of magnesium lactate group were significantly reduced (P < 0.05). This shows that magnesium lactate can reduce the secretion of inflammatory cytokines caused by autoimmune neuritis, among which the effect of medium dose of magnesium lactate is the most significant.

[0107] Table 11: Comparison of IL-17 and IFN-γ levels in serum of rats in each group

[0108]

[0109] Note: a Compared with group A, P < 0.05; b Compared with group B, P < 0.05; d Compared with group D, P<0.05.

[0110] In the above application, the results of other divalent metal lactates including calcium lactate, zinc lactate, ferrous lactate and various combinations are similar to the above magnesium lactate results, which will not be repeated here.

[0111] Example 4: Effects of divalent metal lactate on autoimmune encephalomyelitis model animals 1. Establishment of autoimmune encephalomyelitis model and experimental grouping

[0112] 40 SD rats (male and female, 1:1 ratio), 8 weeks old, weighing 200-220 g, were placed in a temperature-controlled room (22±2°C). 35-55 The peptide was diluted to 6 mg / mL in PBS and mixed with an equal volume of CFA. A 5 mL syringe was connected using a dual-channel connector and repeatedly pushed and pulled to mix to make MOG 35-55 Oil-in-water antigen emulsion. Experimental rats were immunized with antigens and the antigen emulsion was injected subcutaneously at multiple points on the back, 4 points per rat, with a total injection of 0.2 mL of antigen emulsion (MOG 35-55 Peptide content was 600 μg per rat); 0 hours and 48 hours after the antigen emulsion immunization, PTX was intraperitoneally injected as a sensitizer to increase the incidence of the model. After the model was successfully established, each group of rats was gavaged with the corresponding drug or saline once a day for 4 consecutive weeks.

[0113] Table 12: Experimental groups and treatments

[0114]

[0115] 2. Neurological deficit score

[0116] During the administration period, the rats were observed and neurological deficits were scored daily: 0 points, no clinical symptoms; 5 points, moribund or dead.

[0117] On the 5th day after the administration of normal saline, some rats in the autoimmune encephalomyelitis group began to show different degrees of neurological damage symptoms; on the 9th day, all rats showed different degrees of autoimmune encephalomyelitis symptoms, with muscle tension loss, tail dragging and paralysis; on the 12th day, the symptoms of autoimmune encephalomyelitis in rats reached a peak, and the highest neurological deficit score was 3.5±0.5. On the 7th day of administration, some rats in the low-dose magnesium lactate group showed different degrees of neurological damage symptoms; on the 12th day, all rats showed different degrees of autoimmune encephalomyelitis symptoms; on the 16th day, the symptoms of autoimmune encephalomyelitis in rats reached a peak, and the highest neurological deficit score was 2.5±0.5; after that, the symptoms of autoimmune encephalomyelitis in rats gradually eased; on the 28th day, the neurological deficit score was 0-1. On the 12th day of administration, some rats in the medium-dose magnesium lactate group showed varying degrees of neurological impairment symptoms; on the 18th day, all rats showed varying degrees of autoimmune encephalomyelitis symptoms; on the 23rd day, the symptoms of autoimmune encephalomyelitis in rats reached a peak, and the neurological deficit score was the highest, which was 1.5±0.5. Afterwards, the symptoms of autoimmune encephalomyelitis in rats gradually eased; on the 26th day, the neurological deficit score was 0. On the 10th day of administration, some rats in the high-dose magnesium lactate group showed varying degrees of neurological impairment symptoms; on the 15th day, all rats showed varying degrees of autoimmune encephalomyelitis symptoms; on the 19th day, the symptoms of autoimmune encephalomyelitis in rats reached a peak, and the neurological deficit score was the highest, which was 2.5±0.5; afterward, the symptoms of autoimmune encephalomyelitis in rats gradually eased; on the 31st day, the neurological deficit score was 0-1. Magnesium lactate plays a certain therapeutic role in the neurological deficits caused by autoimmune encephalomyelitis, among which the effect of medium-dose divalent lactate is the most significant.

[0118] 3. Detection of inflammatory factor levels

[0119] After the rats were killed, spinal cord tissue was taken, homogenized with tissue lysis solution, and the supernatant was centrifuged to determine the concentrations of TNF-α, IL-6, and IL-β. The results are listed in Table 13. Compared with the autoimmune encephalomyelitis group gavaged with normal saline, the levels of TNF-α, IL-6, and IL-β in the spinal cord of rats gavaged with magnesium lactate were significantly reduced (P < 0.05). Compared with the low and high doses of magnesium lactate groups, the levels of TNF-α, IL-6, and IL-β in the medium dose of magnesium lactate group were significantly reduced (P < 0.05). This shows that magnesium lactate can reduce the secretion of inflammatory cytokines caused by autoimmune encephalomyelitis, thereby reducing the damage to myelin tissue, and the effect of medium dose of magnesium lactate is more significant.

[0120] Table 13: Comparison of TNF-α, IL-6, and IL-β levels in the spinal cord of rats in each group

[0121]

[0122] Note: a Compared with group A, P < 0.05; b Compared with group B, P < 0.05; d Compared with group D, P<0.05.

[0123] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of divalent metal lactate in the preparation of products for preventing, improving, repairing and treating nerve damage, characterized in that: The tissue in which nerve damage occurs is selected from nerve tissue rich in collagen.

2. The use according to claim 1, characterized in that: The nerve damage is central nerve damage or peripheral nerve damage, and the prevention, improvement, repair and treatment of nerve damage is nerve regeneration, increase in nerve number and / or nerve repair.

3. The use according to claim 2, characterized in that: The central nervous system injury includes spinal cord injury or encephalomyelitis, and the peripheral nervous system injury includes peripheral nerve injury, peripheral neuritis or peripheral neuropathy.

4. The use according to claim 3, characterized in that: The peripheral neuritis is selected from one or more of the following: facial neuritis, diabetic peripheral neuritis, viral peripheral neuritis, peripheral neuritis caused by chemotherapy drugs or Guillain-Barré syndrome, and the symptoms of the peripheral neuritis include hypoesthesia, pain, numbness or tingling, tingling, burning, and hyperesthesia.

5. The use according to any one of claims 1 to 4, characterized in that: The divalent metal lactate includes one or more combinations of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate.

6. The use according to claim 5, characterized in that: The divalent metal lactate is selected from magnesium lactate or a combination of zinc lactate and magnesium lactate. Preferably, in the composition, the ratio of zinc lactate to magnesium lactate is 1:10-10:1 by weight.

7. The use according to claim 1, characterized in that: The product is selected from one or more of drugs, kits, health products, and medical devices. Preferably, the concentration of the divalent metal lactate in the product is 5-250 mmol / L, and more preferably, the concentration of the divalent metal lactate in the product is 30-150 mmol / L.

8. The use according to claim 7, characterized in that: The medical device includes one or more combinations of medical adhesive tape, bandage, gauze, dressing, sponge, and medical suture.

9. The use according to claim 7, characterized in that: In the medicine, divalent metal lactate is an active ingredient, and the medicine further comprises a pharmaceutically acceptable carrier or excipient.

10. The use according to claim 7, characterized in that: The dosage form of the drug includes injection or external preparation. Preferably, the external preparation includes one or more combinations of ointments, creams, patches, sprays, solutions, lotions. Preferably, the administration methods of the product include one or more of the following: intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration or application.

Images