Application of gastrodin in preparation of medicine for treating peripheral neuropathy

Abstract

The invention provides an application of gastrodin in preparation of a medicine for treating peripheral neuropathy. According to the application disclosed by the invention, gastrodin can inhibit neuroinflammation mediated by microglia by inhibiting activation of an NF-kB / NLRP3 inflammasome signal channel, so that release of inflammatory factors is reduced, and neuropathy induced by bortezomib is relieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of gastrodin in preparing a medicine for treating peripheral neuropathy. Background Art

[0002] Multiple myeloma (MM) is a common hematologic malignancy characterized by massive plasma cell proliferation accompanied by abnormal immunoglobulin production. MM often leads to multiple organ and tissue damage, including bone destruction, hypercalcemia, anemia, and renal insufficiency, and is associated with high mortality and relapse rates, posing a significant threat to human health. The advent of the proteasome inhibitor bortezomib (BTZ) has ushered in a new era of drug treatment for MM. Its combination with immunomodulators and hormones has significantly improved patient survival, playing an alternative role in the treatment of MM.

[0003] Bortezomib (BTZ) plays an important role in the treatment of multiple myeloma. However, it is known to be neurotoxic, injuring nerve fibers through various pathways and often inducing peripheral neuropathy (BIPN). The incidence of BIPN is high, ranging from 40% to 60%. Patients affected by BIPN may need to reduce their BTZ dose or even discontinue treatment, which severely impacts treatment sustainability and prognosis. However, the exact etiology of BIPN remains incompletely elucidated, and there are no effective preventive or treatment options. Currently, treatment options for BIPN include neuroprotective agents such as vitamin B and methylcobalamin. These drugs have limited clinical efficacy, and dose adjustment and altered route of administration (e.g., intravenous to subcutaneous administration) remain the primary means of alleviating BIPN. Therefore, clarifying the pathophysiological mechanisms of BIPN and identifying therapeutic agents for its treatment are of great clinical significance.

[0004] Neuropathic pain is chronic and closely related to diseases or injuries of the nervous system. Peripheral nerve injury often triggers neuropathic pain, accompanied by significant proliferation of glial cells in the dorsal horn of the spinal cord.

[0005] Studies have shown that although BTZ cannot cross the blood-brain barrier, its accumulation in the dorsal root ganglia can trigger central nervous system glial activation, disrupt glutamate metabolism, and induce inflammation, indirectly leading to central nervous system dysfunction. BTZ has also been reported to increase GFAP expression in the dorsal horn of the spinal cord and alter astrocyte morphology. While inducing neuroinflammation, it can also lead to an imbalance in astrocyte regulation of glutamate transmission, further exacerbating pain responses and nerve damage. Neuroinflammatory responses are key factors in the initiation and maintenance of pain. One study found that after one week of BTZ treatment, the NF-κB pathway was activated in dorsal root ganglion neurons in mice, and transcription levels of the pro-inflammatory cytokines TNF-α and IL-6 reached peaks. Treatment with NF-κB inhibitors or anti-TNF-α antibodies partially reversed BTZ-induced pain symptoms. Notably, NF-κB promotes NLRP3 gene expression and protein synthesis, and further assembles with the ASC adaptor protein and caspase-1 effector protein to form an inflammasome complex, promoting the secretion of multiple inflammatory factors and exacerbating the inflammatory response. Studies have found that BTZ can induce increased NLRP3 expression in dorsal root ganglia through STAT3-related histone acetylation, a pathological change often associated with the development of mechanical pain hypersensitivity. Another study suggests that TNF-α promotes BTZ-induced mechanical allodynia by activating the JNK signaling pathway, an effect that can be alleviated by TNF-α synthesis inhibitors. It is important to note that spinal cord microglia are closely associated with inflammatory responses, and therefore their role in BIPN deserves attention.

[0006] Microglia are a type of immune cell with macrophage functions that reside in the central nervous system. During neuropathic pain, microglia transform from a "resting" form to an "activated" form, with the cell body becoming larger and the processes becoming shorter and thicker. Compared with resting microglia, activated microglia show changes in the expression of surface markers, membrane-bound proteins, or endocytic proteins, and release a variety of inflammatory factors (IL-1β, TNF-α, IL-4, etc.) and a variety of analgesic / pain-inducing substances, leading to changes in the glial microenvironment and resulting in neurotoxicity, which may be related to the occurrence of central sensitization in chronic pain. Among various inflammatory cytokines, the NOD-like receptor (NLR) protein inflammasome in microglia can promote the maturation of IL-1β through the activation of caspase-1 and can recognize "danger or damage-associated molecular patterns" (DAMPs) in response to different types of stimuli. Studies have shown that the IL-1β produced by them is neurotoxic to motor and sensory neurons and is closely related to the occurrence of various diseases. Currently, there are few reports on the role of microglia in BIPN, but there is evidence that BTZ can indeed induce microglial activation. Therefore, it is of great significance to further explore whether the occurrence of BIPN is related to microglial activation and its possible mechanism.

[0007] Gastrodin (Gastrodin) is a traditional Chinese medicinal herb with a long history. Its main component, gastrodin (GAS), has significant pharmacological effects and is widely used to treat chronic pain and various other neuropathies. Clinically, gastrodin is used across multiple disciplines to alleviate neuroinflammatory diseases. Basic experimental studies have demonstrated the neuroprotective and anti-inflammatory properties of gastrodin in animal models of various central nervous system diseases and diabetic peripheral neuropathy. It has been reported that gastrodin exerts neuroprotective effects in surgically treated mice by inhibiting neuroinflammation and microglial activation. In vivo and in vitro studies have shown that gastrodin has a role in the treatment of peripheral nerve injury and demyelinating diseases by acting on Schwann cells. Yang et al. demonstrated the local effects of gastrodin on peripheral nerve injury in a sciatic nerve injury model and found that gastrodin could successfully repair a 10 mm sciatic nerve defect, suggesting its potential for peripheral nerve regeneration. Recently, Song et al., in an animal model of neuroinflammation, found that gastrodin significantly inhibited lipopolysaccharide-induced glial activation and significantly improved neuroinflammatory responses. In addition, studies have reported that gastrodin may exert neuroprotective effects by regulating the TLR4-NF-κB-NLRP3 signaling pathway and reducing glial cell activation. In an in vitro model, researchers found that the expression of NLRP3 inflammasome and IL-1β was significantly increased in mice with nerve injury, and intrathecal administration of gastrodin reversed these changes, suggesting that the neuroprotective effect of gastrodin may be related to the inhibition of neuroinflammation. Separate studies have found that gastrodin can reduce vincristine-induced mechanical hyperalgesia while not affecting the anti-tumor effects of chemotherapy drugs (and may even enhance their efficacy). However, the effect of gastrodin on BIPN has not been reported. Summary of the Invention

[0008] The purpose of the present invention is to provide an application of gastrodin in the preparation of a medicine for treating peripheral neuropathy.

[0009] According to one aspect of the present invention, there is provided a use of gastrodin in the preparation of a medicament for treating peripheral neuropathy.

[0010] In the use provided by the present invention, the peripheral neuropathy includes bortezomib-induced peripheral neuropathy.

[0011] In the application provided by the present invention, the gastrodin inhibits microglia-mediated neuroinflammation by inhibiting the NF-κB / NLRP3 inflammasome signaling pathway.

[0012] In the application provided by the present invention, the gastrodin can inhibit the microglial activation induced by bortezomib and simultaneously inhibit the expression of the inflammatory factor IL-1β.

[0013] In the application provided by the present invention, the gastrodin can inhibit the microglial activation induced by bortezomib and simultaneously inhibit the expression of the inflammatory factor TNF-α.

[0014] In the application provided by the present invention, the gastrodin can reduce the expression levels of IBA-1 and GFAP in the dorsal horn of the spinal cord induced by bortezomib.

[0015] In the application provided by the present invention, the gastrodin can reduce the expression of IL-1β, IL-6 and TNF-α induced by bortezomib, while increasing the expression of IL-10.

[0016] According to another aspect of the present invention, a medicament for treating peripheral neuropathy is provided, comprising gastrodin.

[0017] In the medicine provided by the present invention, the peripheral neuropathy includes bortezomib-induced peripheral neuropathy.

[0018] The medicine provided by the present invention also includes pharmaceutically acceptable excipients.

[0019] The implementation of the present invention has the following beneficial effects: the application of the gastrodin provided by the present invention can inhibit the activation of the NF-κB / NLRP3 inflammasome signaling pathway, thereby inhibiting microglia-mediated neuroinflammation and reducing the release of inflammatory factors, thereby alleviating bortezomib-induced neuropathy, alleviating the symptoms of BIPN patients, ensuring the smooth progress of tumor chemotherapy and improving the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only embodiments of the present invention. Those skilled in the art can also derive other drawings based on the provided drawings without inventive work.

[0021] Figure 1 Shown are the ultrastructures of dorsal root ganglia of mice in each group, including: A: Electron microscopic observation of dorsal root ganglion neurons (scale bar = 2 μm × 6000); B: Electron microscopic observation of satellite glial cells in dorsal root ganglia of mice in each group (scale bar = 1 μm × 12000); white arrows in the figure indicate mitochondrial vacuolization, n = 3;

[0022] Figure 2Shown are changes in myelin sheath thickness (G-ratio) of myelinated fibers in the sciatic nerve. A: Electron microscopic observation of sciatic nerve samples (scale bars = 1 μm, 2 μm); B: Changes in myelin sheath thickness (G-ratio) of myelinated fibers in the sciatic nerve. Compared with the control group; *P < 0.05, n = 3.

[0023] Figure 3 Shown are hematoxylin-eosin (HE) staining of the sciatic nerves of mice in each group. A: Hematoxylin-eosin (HE) staining images of mice in each group. The white arrows in the figure indicate loose and disordered sciatic nerves with axonal deformation (×400); B: Nerve injury scores of mice in each group, *P<0.05, ***P<0.05, n=6;

[0024] Figure 4 Shown are the changes in epidermal nerve fibers in each group of mice, including: A: Immunofluorescence images of PGP9.5-positive nerve fibers in the epidermis of mice in different groups (scale bar = 100 μm); B: Comparison of IENFD in mice in each group; *P < 0.05, **P < 0.01, n = 5;

[0025] Figure 5 Shown are the IBA-1+ fluorescence ratios in the spinal dorsal horn, where A: IBA-1+ immunofluorescence staining in the spinal dorsal horn of mice in each group (scale bar = 100 μm); B: Comparison of the proportion of IBA-1+ fluorescence in the spinal dorsal horn of mice in each group; ***P < 0.001, ns: P > 0.05, n = 6;

[0026] Figure 6 Shown are the GFAP fluorescence ratios in the spinal dorsal horn, where A: GFAP immunofluorescence staining in the spinal dorsal horn of mice in each group (scale bar = 100 μm); B: comparison of the GFAP fluorescence ratios in the spinal dorsal horn of mice in each group; ***P < 0.01, ns: P > 0.05, n = 6;

[0027] Figure 7 The expression of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-10) in the dorsal horn of the spinal cord is shown in Figure 3. A: expression of IL-1β protein in the dorsal horn of the spinal cord of mice in each group; B: expression of TNF-α protein in the dorsal horn of the spinal cord of mice in each group; C: expression of IL-6 protein in the dorsal horn of the spinal cord of mice in each group; D: expression of IL-10 protein in the dorsal horn of the spinal cord of mice in each group; ***P<0.001, **P<0.01, *P<0.05, ns: P>0.05, n=4;

[0028] Figure 8Shown are the colocalization of IBA-1, GFAP, and IL-1β. A: Immunofluorescence co-staining images of IBA-1 (green) and IL-1β (red) (scale bar = 100 μm); B: Immunofluorescence co-staining images of GFAP (green) and IL-1β (red) (scale bar = 100 μm); C: Statistics of the co-staining ratio in each group; D: Colocalization of IBA-1 and IL-1β in the dorsal horn of the spinal cord of mice in each group. ***P < 0.001, *P < 0.05, n = 6;

[0029] Figure 9 The colocalization of IBA-1, GFAP, and TNF-α is shown. A: Immunofluorescence co-staining image of IBA-1 (green) and TNF-α (red) (scale bar = 100 μm); B: Immunofluorescence co-staining image of GFAP (green) and TNF-α (red) (scale bar = 100 μm); C: Statistics of the co-staining ratio in each group; D: Colocalization of IBA-1 and TNF-α in the dorsal horn of the spinal cord of mice in each group; ***P < 0.001, **P < 0.01, n = 6;

[0030] Figure 10 The expression of NF-κB / NLRP3-related pathway proteins was detected by Western blotting. A: p-NF-κBp65, NF-κBp65, NLRP3, ASC, Cleaved-caspase-1, and caspase-1 protein bands in the dorsal angle of the spinal cord of mice in each group; B-E: relative expression levels of p-NF-κBp65 / NF-κBp65, NLRP3, ASC, and Cleaved-caspase-1 / caspase-1 proteins in each group; *P<0.05, **P<0.01, n=3. DETAILED DESCRIPTION

[0031] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The drawings illustrate exemplary embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] Experimental Materials

[0034] experimental animals

[0035] Male C57BL / 6J mice aged 8 weeks (weighing approximately 20-25 g) with normal physiological conditions were selected. C57BL / 6J mice were provided by Hunan Slake Jingda Laboratory Animal Company, with a certificate number of SCXK (Xiang) 2019-0004. The mice were housed in an SPF-grade animal room in the Basic Research Laboratory of Anesthesia and Organ Protection of Zunyi Medical University. The experiment began after the mice had adapted to the environment for one week. During this period, the mice had free access to food, water, and movement. The experiment was carried out in accordance with the protocol approved by the Experimental Animal Ethics Committee of the Affiliated Hospital of Zunyi Medical University. The experimental animal use license number is SYXK (Guizhou) 2021-0004.

[0036] Experimental animal grouping

[0037] Forty C57BL / 6J mice were randomly divided into groups to establish a BIPN mouse model. The experimental groups were divided into a BIPN model group (BTZ group, n=10), a BIPN model group treated with gastrodin (BTZ+GAS group, n=10), and a group receiving gastrodin alone (GAS group, n=10). The mice were compared with a healthy mouse group (Control group, n=10). Mice were housed in individual cages (5 per cage) at a room temperature of (23±2)°C, a humidity of (55%±2%), and a 12-hour alternating light-dark cycle.

[0038] Administration method: Control group: The same dose of normal saline as bortezomib was injected through the tail vein as a control for 4 weeks; BTZ group: According to the previous research results of our research group, bortezomib was administered through the tail vein (0.8 mg / kg, 2q / 7d) to establish a BIPN model, which lasted for 4 weeks; BTZ+GAS group: bortezomib was injected through the tail vein + gastrodin was injected intraperitoneally (100 mg / kg, Qd; Li et al. Brain Res, 2019; Sun et al. International Immuno Pharmacology, 2016), which lasted for 4 weeks; GAS group was injected intraperitoneally with gastrodin (100 mg / kg, Qd) for 4 weeks.

[0039] Statistical Methods: All data are presented as mean ± standard deviation (x-±s) and analyzed using GraphPad Prism 9.0 software. Paired t-tests were used for intra-group comparisons, one-way ANOVA was used for inter-group comparisons, and Tukey's test was used for pairwise comparisons. Repeated-measures ANOVA was used for repeated-measures data. P < 0.05 was considered statistically significant.

[0040] Example 1

[0041] Bortezomib was administered via tail vein injection to mice, and a BIPN mouse model was successfully established after screening. Gastrodin (100 mg / kg, daily) was then administered intraperitoneally to the BIPN mice. Changes in body weight and pain behavior were continuously observed during the dosing period.

[0042] Mechanical pain threshold measurement

[0043] Two days before the experiment, mice were placed on the experimental table at 10:00 am each day for 30 minutes to allow them to acclimate to the environment. On the day of the experiment, mice were placed in a plexiglass cover (with a 2mm×2mm metal mesh at the bottom) and allowed to acclimate for 60 minutes before testing. The room temperature was maintained at 24±1°C, and feces on the metal mesh were cleaned after the mice were quiet. VonFrey fiber (Danmic) was used to stimulate the central area of the sole of the mouse's foot vertically and gradually increase the force starting from 0.008g. The fiber was bent 1cm and maintained for 3-5 seconds. The mouse's pain response (such as licking the foot, sudden contraction, or shaking the foot, etc.) was observed. If the mouse did not show the above reaction, the stimulation was increased by one level of g until a pain response appeared. Each measurement was repeated 5 times with a 3-minute interval in between. When the mouse showed the above reaction at least 3 times, the g number at that time was recorded as 1 mechanical pain threshold. Each mouse was measured 6 times (3 times for the left and right hind paws respectively), with an interval of 3 minutes between each time, and the average value of the 6 times was calculated as the mechanical withdrawal reflex threshold (Austin et al. Jove-journal of Visualized Experiments, 2012).

[0044] Thermal pain threshold measurement

[0045] Two days before the experiment, the mice were placed on the experimental table for 30 minutes at 2 pm every day to allow them to adapt to the environment. On the day of the experiment, the mice were placed on a 2mm thick glass plate with a light-transmitting glass plate with a hole on the top of the outer cover before testing and the glass plate was kept dry. The indoor temperature was 24±1°C, and the mice were allowed to adapt to the environment for 60 minutes. After the mice were quiet, the thermal radiation light source was turned on (the maximum intensity was set to 40 and the maximum time was 20s) to illuminate the central area of the soles of the mice. When the mice showed reactions such as licking their feet or retracting their feet, the light source was immediately cut off, and the reaction time was recorded. Each mouse was measured six times (three times for the left hind paw and three times for the right hind paw respectively). The two measurements before and after were separated by 5 minutes, and a total of six measurements were made. The average of the six times was finally taken as the thermal retraction reflex threshold.

[0046] The results of mouse body weight and behavior showed that: (1) Body weight: Compared with the control group, the body weight of the BTZ group decreased significantly (P < 0.05); the body weight of the GAS group did not change significantly (P > 0.05). After treatment with gastrodin, the body weight of the mice in the BTZ + GAS group increased (P < 0.05). (2) Thermal pain threshold: Compared with the control group, the thermal pain threshold of the BTZ group decreased significantly (P < 0.05); the GAS group did not change significantly (P > 0.05). After treatment with gastrodin, the thermal pain threshold of the mice in the BTZ + GAS group increased significantly (P < 0.05). (3) Mechanical pain threshold: Compared with the control group, the mechanical pain threshold of the BTZ group decreased significantly (P < 0.05); the GAS group did not change significantly (P > 0.05). After treatment with gastrodin, the mechanical pain threshold of the mice in the BTZ + GAS group increased significantly (P < 0.05).

[0047] The results showed that Gastrodin could partially alleviate the systemic toxicity caused by the chemotherapy drug bortezomib, partially improve the thermal hyperalgesia in mice, and BTZ-induced mechanical hyperalgesia in mice could be partially improved by GAS.

[0048] Example 2

[0049] Transmission electron microscopy was used to examine the damage of neurons and satellite cells in the dorsal root ganglion (DRG) of the mice in the above groups, as well as the myelin sheath thickness and pathological damage of the myelinated fibers of the sciatic nerve.

[0050] Sampling: After the completion of the preliminary experiments, the mice were anesthetized with isoflurane. Continue to observe the righting reflex reaction of the mice. When it completely disappears and the mice are in a deep anesthesia state, fix the mice, cut the xiphoid process and surrounding skin muscles of the mice with curved scissors, puncture the diaphragm and open the chest cavity to fully expose the mouse heart. Use an injection needle to pierce the left ventricle of the mouse, and place the other end of the needle in a container filled with PBS through a peristaltic pump. At the same time, use microscissors to cut the right atrial appendage of the mouse, turn on the peristaltic pump and pump in 50ml of PBS. When the color of the mouse liver turns white, turn off the peristaltic pump. Then change the perfusion liquid to 4% paraformaldehyde, and stop perfusion when the mouse body is stiff. Separate the dorsal root ganglia of the mouse on the operating table, and immediately place the sciatic nerve in glutaraldehyde for fixation. The subsequent operation steps are as follows:

[0051] 1) Fixation: Remove the tissue from glutaraldehyde and fix it in 1% osmium tetroxide.

[0052] 2) Dehydration: Pyruvic acid was used as a dehydrating agent to perform gradient dehydration on the tissue samples. The dehydration process started at a 30% concentration and then increased step by step, with each level acting for 15 minutes, including 3 times at a 100% concentration.

[0053] 3) Embedding and Sectioning: After the dehydration step, the tissue sample is infiltrated at room temperature for approximately 2 hours to allow the embedding agent to fully penetrate the tissue. The sample is then embedded in epoxy resin and cured at an appropriate temperature to form a resin block. After embedding, the resin block is sliced ultrathinly using a microtome to a thickness of approximately 60 to 90 nm.

[0054] 4) Staining: Stain the ultrathin sections in a solution containing uranyl acetate for 20 minutes. After staining, rinse and air-dry the sections. Then, continue staining in a lead citrate solution for 5 minutes. Rinse again and air-dry the sections to ensure no residual liquid remains before proceeding to the next step.

[0055] 5) Observation: Observe the sections and collect images using a transmission electron microscope.

[0056] Experimental results

[0057] Transmission electron microscopy was used to examine the damage of dorsal root ganglia in various mice, and the results showed that Gastrodin could reduce the damage of dorsal root ganglia in BIPN mice. Compared with the Control group, the neurons in the dorsal root ganglia of mice in the BTZ group showed morphological abnormalities, and a large number of mitochondrial swellings accompanied by vacuolation appeared in the neurons (Neuron, Neu) and their surrounding satellite glial cells (Satellitecells), and the cristae structure was broken and dissolved. Compared with the BTZ group, after the intervention of Gastrodin, the structure of neurons and satellite cells in the BTZ+GAS group partially returned to normal. The mitochondrial cristae structure was regularly arranged, no abnormal deposits were found in the matrix, the outer membrane contour was not broken, and mitochondrial vacuolation was occasionally observed. Compared with the Control group, no abnormal changes were observed in the dorsal root ganglion neurons and satellite cells in the GAS group (such as Figure 1 (A), (B)).

[0058] To investigate possible structural changes in the myelin sheaths of peripheral nerve fibers in each group of mice, transmission electron microscopy was used to observe the ultrastructure of sciatic nerves, and the myelin sheath thickness of myelinated nerve fibers was quantified using the G-ratio (the ratio of the inner diameter of the myelin sheath to the outer diameter of the myelin sheath). The results showed that gastrodin could ameliorate BTZ-induced peripheral nerve injury in mice. Compared with the control group, the myelin sheaths of myelinated nerve fibers in the BTZ group showed loosening of the myelin lamellae in some regions, thinning of the overall myelin sheath thickness, and a larger G-ratio (0.71±0.06 vs 0.55±0.05, P<0.01), which was statistically significant. Compared with the BTZ group, the G-ratio in the BTZ+GAS group decreased significantly after treatment with gastrodin (0.56±0.02 vs 0.71±0.06, P<0.05), indicating an overall thickening of the myelin sheath, with only a few regions showing mild loosening of the myelin lamellae. Compared with the Control group, the myelin sheath of myelinated nerve fibers in the GAS group showed no obvious morphological abnormalities, and the G-ratio did not change significantly (0.59±0.04 vs 0.54±0.02 P>0.05), with no statistical difference. (e.g. Figure 2 AB).

[0059] Example 3

[0060] Observation of pathological changes of myelinated nerve fibers by HE staining

[0061] Fresh sciatic nerves were harvested, and residual blood and hair removed from the surface. The tissues were then quickly transferred to a 4% paraformaldehyde fixative to maintain structural integrity. After 24 hours, the tissues were subjected to a gradient dehydration process, paraffin-embedded, and sectioned. The subsequent staining procedures were as described below.

[0062] 1) Dewaxing and hydration: Dewaxing in dewaxing solution for 1 hour, soaking in anhydrous ethanol for 10 minutes, then soaking in gradient alcohol for 5 minutes each, and finally rinsing with running water for 5 minutes

[0063] 2) Hematoxylin staining: Differentiation was performed after 10 minutes of hematoxylin staining

[0064] 3) Eosin staining: Soak in eosin staining solution for 2 minutes

[0065] 4) Dehydration: Gradient alcohol dehydration

[0066] 5) Sealing: After draining, add a drop of neutral resin and seal the slide with a coverslip.

[0067] 6) After staining, observe the pathological changes of the sciatic nerve under a microscope and take photos.

[0068] The pathological changes of the sciatic nerves of the four groups of mice were detected by hematoxylin-eosin (HE) staining. The results showed that compared with the Control group, the sciatic nerves in the BTZ group were disordered and loose with inflammatory cell infiltration, the nerve fibers were edematous and irregular in shape, the Schwann cells were irregularly arranged, the myelin sheaths were unevenly colored and most of them were swollen. Compared with the BTZ group, after intervention with gastrodin, the sciatic nerves of the BTZ+GAS group mice were relatively neatly arranged, only some sciatic nerve fibers were swollen, the inflammatory cell infiltration was less, and the Schwann cells were relatively regular. Compared with the Control group, no obvious pathological changes were observed in the sciatic nerves of the GAS group mice. (e.g. Figure 3 )

[0069] Example 4

[0070] Intraepidermal nerve fiber staining

[0071] The footpad skin of mice was collected, and the epidermal peripheral nerve fibers were labeled with PGP9.5. The density of epidermal nerve fibers (IENF) was detected by immunofluorescence staining, and the changes in IENF density were compared among the mice in each group.

[0072] PGP9.5 is a protein gene product, and its corresponding antibody can specifically recognize and localize to nerve fibers, thereby achieving visual labeling of neural structures in tissues. After the anesthetized mouse is perfused, the skin of the center area of the right hind paw of the mouse is taken (be sure to remove the fascia), with an area of approximately 3×3 mm 2 , and quickly place in fixative to fix. Prepare a primary antibody working solution by mixing PGP9.5 with antibody diluent at a ratio of 1:600 for standby use. Stain according to the fluorescent staining steps in Example 6 below.

[0073] Intraepidermal nerve fibers can innervate the nerve structures of the dermis and epidermis, and protein gene product 9.5 (PGP9.5) can label nerve axons. The results of labeling the nerve fibers in the footpad tissue of mice using PGP9.5 antibody showed that compared with the Control group, the intraepidermal nerve fiber density (IENFD) in the BTZ group was significantly reduced (16.17±1.84 vs 25.17±4.53, P<0.01), and the difference was statistically significant; the IENFD of mice in the GAS group was slightly reduced (18.17±3.55 vs 25.17±4.53, P<0.05), and the difference was statistically significant. Compared with the BTZ group, the IENFD of mice in the BTZ+GAS group increased significantly after intervention with gastrodin (22.50±4.04 vs 16.17±1.84, P<0.05), with statistical differences (such as Figure 4 AB).

[0074] Example 6

[0075] Immunofluorescence was used to detect the changes in the expression of microglial markers (IBA-1) and astrocyte markers (GFAP) in the dorsal horn of the spinal cord of mice in each group.

[0076] Remove mouse spinal cord tissue and trim the ends with scissors, leaving the lumbar enlargement intact. Immediately place in fixative and cool overnight. The next day, dehydrate the sample with 30% sucrose solution and wait until the sample sinks to the bottom before proceeding.

[0077] After dehydration, air-dry the spinal cord tissue and place it in a square mold. Add pre-chilled OCT (Optimal Cutting Temperature) composite embedding medium to thoroughly infiltrate the tissue, and use a notebook to record the corresponding position of each specimen group. Store the embedded tissue in a -20°C refrigerator. Once frozen, use a cryostat to slice and collect spinal cord tissue sections from the L4-L5 segment. Slice thickness should be adjusted based on experimental results.

[0078] Fluorescent staining steps

[0079] 1) Arrange the collected spinal cord sections neatly on a glass slide and allow to dry naturally at room temperature for 10 minutes. Wash with 1× PBS three times, 5 minutes each wash.

[0080] 2) Serum blocking: After natural drying, the sections on the slides were circled with a hydrophobic histochemical pen and blocked with 1% BSA at room temperature for 2 h.

[0081] 3) Add primary antibody: Discard the blocking solution, blot any remaining blocking solution with absorbent paper, and circle the section again with a hydrophobic paintbrush. Add the primary antibody working solution and incubate at 4°C overnight.

[0082] 4) Add secondary antibody: The next day, discard the primary antibody and wash away any remaining primary antibody with 1x PBS. After air drying, add secondary antibody and incubate at room temperature in the dark for 2 hours.

[0083] 5) Sealing: After 2 hours, discard the secondary antibody, wash the residual secondary antibody with 1× PBS, and seal the slides with mounting medium after drying.

[0084] 6) Observation: An Olympus BX61WI fluorescence microscope was used to observe and take pictures.

[0085] Immunofluorescence was used to stain the microglial marker IBA-1 in the dorsal horn of the spinal cord. The results showed that compared with the control group, the expression of IBA-1 in the dorsal horn of the spinal cord of the BTZ group mice was significantly increased (2.75±0.21 vs 0.73±0.23, P<0.001), with statistical significance; the expression of IBA-1 in the GAS group did not change significantly (0.85±0.14 vs 0.73±0.23, P>0.05), with statistical significance; compared with the BTZ group, the expression of IBA-1 in the BTZ+GAS group was significantly reduced after intervention with Gastrodin (2.06±0.38 vs 2.75±0.21, P<0.001), with statistical significance. (As Figure 5 ).

[0086] Immunofluorescence was used to quantitatively analyze the staining of GFAP, a marker of astrocytes in the dorsal horn of the spinal cord. The results showed that compared with the control group, the expression of GFAP in the dorsal horn of the spinal cord of mice in the BTZ group was significantly increased (5.90±0.44 vs 2.13±0.26, P<0.01), and the difference was statistically significant; the expression of GFAP in the GAS group did not change significantly (2.63±0.71 vs 2.13±0.26, P>0.05), and the difference was not statistically significant; compared with the BTZ group, the expression of GFAP in the BTZ+GAS group was significantly reduced after intervention with Gastrodin (3.60±0.50 vs 5.90±0.44, P<0.01), and the difference was statistically significant. (As shown in Figure 2). Figure 6 AB).

[0087] Example 7

[0088] Enzyme-linked immunosorbent assay (ELISA) was used to detect the changes in inflammatory factors (TNF-α, IL-1β, IL-6, and IL-10) in the dorsal horn of the spinal cord of mice in each group.

[0089] ELISA was used to detect the expression of inflammatory factors. Sample collection procedures were as described in 1.25. Only PBS was perfused, not 4% paraformaldehyde. After perfusion, fresh L4-L6 spinal cord tissue was obtained from the mouse and placed on ice until ready for use. The following procedures were followed:

[0090] 1) Tissue homogenization: Rinse fresh spinal cord samples with pre-chilled PBS. Mix the spinal cord sample with PBS at a ratio of 1:9. Disrupt the tissue using an ultrasonic disruptor on ice. Centrifuge at 5000 g for 20 min at 4°C. Collect the supernatant for later use.

[0091] 2) Take out the reagent box 30 minutes before use and allow it to return to room temperature.

[0092] 3) Dilute the standard sample in series.

[0093] 4) Sample Addition: Arrange the standard and sample wells appropriately and label them accordingly. Add 100 μL of the prepared standard solution and sample solution to the corresponding wells of the ELISA plate, avoiding bubbles. Set up a blank control well and add an equal volume of diluent to the corresponding wells as a zero reference. Perform all sample additions at room temperature and maintain a consistent addition speed to ensure comparability and accuracy of the experimental results. After sealing the plate, place the ELISA plate in a constant temperature incubator at 37°C for 1 hour.

[0094] 5) Remove the ELISA plate, discard the liquid (no need to wash the plate), and add the biotin-labeled antibody solution to each well, ensuring that the sample is added evenly and without bubbles, and incubate again for 1 hour.

[0095] 6) Tap the plate gently on absorbent paper to remove excess liquid and then add washing solution to wash.

[0096] 7) Add enzyme conjugate working solution.

[0097] 8) After shaking off the liquid in the wells, rinse with detergent. After rinsing, gently tap on absorbent paper to remove any residual liquid. Add substrate and continue incubation.

[0098] 9) Add stop solution and wait for the plate to return to room temperature before measuring the OD value.

[0099] The activation of spinal cord glial cells is closely related to the occurrence and development of neuroinflammatory response. In order to further explore the expression of related inflammatory factors after the activation of spinal cord dorsal horn glial cells and whether gastrodin has the effect of inhibiting the expression of these inflammatory factors, we used ELISA to quantitatively measure the expression of inflammatory factors IL-1β, IL-6, TNF-α and IL-10. The results showed that compared with the control group, the expression levels of IL-1β, IL-6, and TNF-α in the BTZ group were significantly increased (IL-1β: 89.87±3.89 vs 34.52±3.95, P < 0.001; IL-6: 41.80±1.72 vs 18.47±2.51, P < 0.001; TNF-α: 738.5.4±19.30 vs 498.8±38.12, P < 0.001), with statistical differences; the expression level of the anti-inflammatory factor IL-10 showed a downward trend (29.55±0.53 vs 39.90±4.40, P > 0.05), but no statistical difference was found. Compared with the BTZ group, the expression levels of IL-1β, IL-6 and TNF-α in the BTZ+GAS group were significantly decreased after intervention with gastrodin (IL-1β: 40.61±7.25 vs 89.87±3.89, P<0.001; IL-6: 29.59±0.67 vs 41.80±1.72, P<0.01; TNF-α: 350.6±35.71 vs 738.5.4±19.30, P<0.001); the expression level of IL-10 was significantly increased (52.96±7.38 vs 29.55±0.53, P<0.01), and the differences were statistically significant. (e.g. Figure 7 AD)

[0100] Next, in order to determine the source of inflammatory factors, immunofluorescence staining was used to co-localize IL-1β (red fluorescence) with IBA-1 (green fluorescence) and GFAP (green fluorescence). The results showed that compared with GFAP, IL-1β was mainly co-localized with IBA-1, suggesting that the increase in IL-1β may be related to the activation of microglia. Compared with the control group, the number of IBA-1 and IL-1β co-stained positive cells in the BTZ group was significantly increased (71.96±3.45 vs 33.24±4.27, P < 0.001), and the difference was statistically significant. Compared with the BTZ group, the number of IBA-1 and IL-1β co-stained positive cells in the BTZ+GAS group was significantly reduced after intervention with gastrodin (52.00±2.52 vs 71.96±8.44, P < 0.05), suggesting that BTZ-induced microglial activation can be inhibited by gastrodin, and the release of IL-1β was also significantly reduced. (such as Figure 8 AD)

[0101] Colocalization of TNF-α (red fluorescence) with IBA-1 (green fluorescence) and GFAP (green fluorescence) showed that, compared with GFAP, TNF-α primarily colocalized with IBA-1, suggesting that increased TNF-α expression may be associated with microglial activation. Compared with the control group, the number of cells positive for IBA-1 and TNF-α co-staining in the BTZ group was significantly increased (68.06±1.69 vs 45.27±1.96, P<0.01), with statistically significant differences. Compared with the BTZ group, the number of cells positive for IBA-1 and TNF-α co-staining in the BTZ+GAS group after treatment with gastrodin was significantly decreased (57.10±1.81 vs 68.06±1.69, P<0.05), suggesting that gastrodin can inhibit BTZ-induced microglial activation and suppress the expression of the inflammatory cytokine TNF-α.

[0102] (like Figure 9 AD).

[0103] Example 8

[0104] Western blot (WB) was used to detect the expression of proteins related to the NF-κB / NLRP3 inflammasome signaling pathway: the expression of p-NF-κBp65, NF-κBp65, NLRP3, ASC, Cleaved-caspase-1, and pro-caspase-1 proteins in the dorsal horn of the spinal cord of the above four groups of mice were detected.

[0105] 1) Tissue Protein Extraction: Spinal cord tissue was removed from the freezer, weighed, and minced into a 1.5 ml EP tube (operate on ice). RIPA and PMSF (100 μl of RIPA and 1 μl of PMSF per 10 mg of spinal cord tissue) were added to the tube. After lysing on ice for 20 min, the tissue was sonicated for 2-3 seconds using an ultrasonic disruptor (power setting 20). A total of three cycles of sonication were performed. Once the tissue was completely disrupted, it was centrifuged and the supernatant was temporarily stored in a 4°C refrigerator.

[0106] 2) BCA protein quantification: First, remove the supernatant of the sample and dilute it in a proportional gradient using PBS buffer, diluting it 5-fold, 10-fold, and 20-fold respectively. Place the diluted samples on ice for later use. Add the standard and the sample diluted with PBS buffer to a 96-well plate and mark it. Then add the colorimetric solution, close the lid tightly, place it in the incubator, and set the time. After incubation, wait for the 96-well plate to return to room temperature and measure the OD value on the machine. Set the loading system to 40ug / 20ul and unify the concentration of each histone. After adding the loading buffer, boil the protein, denature the protein, and store it in aliquots in an ultra-low temperature refrigerator.

[0107] 3) Electrophoresis: Soak the glass plate in pure water. Rinse the plate with detergent, then rinse with pure water to remove any detergent foam. Dry the plate in a dryer until dry and free of water marks. Install and secure the plate in the corresponding rack. Prepare 10% separating gel and add it to the plate. Use anhydrous ethanol to press the gel line to maintain a level surface. After approximately 20 minutes, when the separating gel solidifies, tilt the gel rack to remove the anhydrous ethanol and blot any remaining liquid with filter paper. Add the prepared stacking gel and insert the comb vertically. Allow to stand at room temperature for approximately 20 minutes. Install the prepared gel into the electrophoresis tank, add running buffer, and slowly remove the comb. Use a 20µl pipette to remove the gel filaments from the lanes before loading the samples. Protein loading: From left to right, Maker, Control, BTZ, BTZ+GAS, and GAS groups. Set the voltage to 80V and begin electrophoresis. After 30 minutes, adjust the voltage to 120V for Maker separation. Stop electrophoresis after Maker separation is complete.

[0108] 4) Electrotransfer: Cut the PVDF membrane according to the length of the bands after electrophoresis and activate it in methanol. Place the filter paper, sponge, and transfer clip into an aluminum plate filled with transfer buffer. Place the transfer clip with the black side facing down on top of the sponge. Place the filter paper on top of the sponge and the glue on top of the filter paper. Cover with the PVDF membrane (taking care to remove any air bubbles), then cover with the filter paper and sponge. Secure the clip and install it in the transfer tank. Add the electrophoresis buffer and transfer to a foam box filled with ice. Transfer the membrane at a constant current of 300mA for 2 hours.

[0109] 5) Blocking: Block with blocking solution at room temperature for 2 hours.

[0110] 6) Antibody Incubation: Wash the blocked PVDF membrane six times, 5 minutes each time. After washing, place the membrane in the prepared primary antibody solution and place it on a shaker at 4°C. The next day, recover the primary antibody and wash the membrane six times, 5 minutes each time. After washing, add the secondary antibody and incubate on a shaker at room temperature for 1 hour. After 1 hour, recover the secondary antibody and wash the membrane again six times, 5 minutes each time.

[0111] 7) Development: Prepare a developing solution in the dark, place the PVDF in the prepared developing solution, and expose it to light.

[0112] 8) Band analysis: ImageJ software was used to analyze the grayscale value of the bands.

[0113] Western Blot analysis of proteins involved in the NF-κB / NLRP3 pathway was performed semi-quantitatively. The results showed that compared with the control group, the BTZ group had a significantly increased p-NF-κBp65 / NF-κBp65 ratio (1.12±0.04 vs 0.59±0.14, P<0.05); a significantly increased NLRP3 expression (1.02±0.02 vs 0.74±0.04, P<0.05); a significantly increased ASC expression (1.03±0.01 vs 0.68±0.09, P<0.05); and a significantly increased cleaved-caspase-1 / caspase-1 ratio (1.15±0.06 vs 0.72±0.03, P<0.01). The differences were statistically significant. After intervention with gastrodin, the expression of p-NF-κBp65 / p-NF-Kbp65 ratio was significantly decreased (0.68±0.05 vs 1.12±0.04, P<0.05); NLRP3 expression was significantly decreased (0.73±0.05 vs 1.02±0.02, P<0.01); ASC expression was significantly decreased (0.64±0.02 vs 1.03±0.01, P<0.05); Cleaved-caspase-1 / caspase-1 ratio was significantly decreased (0.72±0.02 vs 1.15±0.06, P<0.01), and the differences were statistically significant. Compared with the control group, there was no significant difference in the expression of related pathway proteins in the GAS group, including p-NF-κBp65 / NF-κBp65 (0.61±0.14 vs 0.59±0.14, P>0.05); NLRP3 (0.73±0.05 vs 0.74±0.04, P>0.05); ASC (0.49±0.11 vs 0.68±0.09, P>0.05); Cleaved-caspase-1 / caspase-1 (0.78±0.07 vs 0.72±0.03, P>0.05). The above results indicate that gastrodin can inhibit the expression of proteins related to the NF-κB / NLRP3 signaling pathway, which suggests that gastrodin alleviates BTZ-induced microglia-related neuroinflammation, which may be related to the NF-κB / NLRP3 inflammasome signaling pathway. (e.g. Figure 10 AE).

[0114] Furthermore, those skilled in the art will appreciate that although some embodiments herein include certain features included in other embodiments but not other features, combinations of features from different embodiments are intended to be within the scope of the present invention and to form different embodiments. For example, in the claims below, any of the claimed embodiments may be used in any combination.

[0115] It should be noted that the above embodiments illustrate rather than limit the invention, and that those skilled in the art may devise alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between brackets should not be construed as limiting the claims. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The present invention may be implemented by means of hardware comprising several different elements and by means of appropriately programmed computers. In a unit claim enumerating several means, several of these means may be embodied by the same item of hardware. The use of the words first, second, and third etc. does not indicate any order. These words may be interpreted as names.

Claims

1. Application of gastrodin in the preparation of medicines for treating peripheral neuropathy.

2. The use according to claim 1, characterized in that The peripheral neuropathy includes bortezomib-induced peripheral neuropathy.

3. The use according to claim 2, characterized in that The gastrodin inhibits microglia-mediated neuroinflammation by inhibiting the NF-κB / NLRP3 inflammasome signaling pathway.

4. The use according to claim 2, characterized in that The gastrodin can inhibit the microglial activation induced by bortezomib and the expression of the inflammatory factor IL-1β.

5. The use according to claim 2, characterized in that The gastrodin can inhibit the microglial activation induced by bortezomib and simultaneously inhibit the expression of the inflammatory factor TNF-α.

6. The use according to claim 2, characterized in that The gastrodin can reduce the expression levels of IBA-1 and GFAP in the spinal cord dorsal horn induced by bortezomib.

7. The use according to claim 2, characterized in that The gastrodin can reduce the expression of IL-1β, IL-6 and TNF-α induced by bortezomib, and increase the expression of IL-10.

8. A drug for treating peripheral neuropathy, characterized in that: Including gastrodin.

9. The drug according to claim 8, characterized in that The peripheral neuropathy includes bortezomib-induced peripheral neuropathy.

10. The drug according to claim 8, characterized in that Pharmaceutically acceptable excipients are also included.

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