Use of egg white lysozyme in the relief of chronic pain

Abstract

The application discloses application of hen egg white lysozyme (HEL) in relieving chronic pain, and experiment results show that HEL significantly and selectively relieves inflammatory pain, neuropathic pain and static mechanical allodynia induced by chemotherapy pain. Meanwhile, experiments show that HEL causes the expression of a mechanical sensitivity ion channel TACAN in the PRKN / TACAN pathway on the cell membrane to be reduced by up-regulating the expression of PRKN protein of primary sensory neurons in the dorsal root ganglion, thereby significantly inhibiting static mechanical allodynia. The application firstly finds that HEL has the effect of relieving static mechanical allodynia and a deep mechanism, and the source of HEL is non-toxic and safe, and has potential application value in preparation of medicines for preventing, treating and relieving chronic pain, and has great clinical significance.

Description

Technical Field

[0001] The present invention relates to a new application of hen egg white lysozyme, in particular to the application of hen egg white lysozyme in alleviating chronic pain. Background Art

[0002] Pain is a complex, subjective, and common clinical symptom and disease. In 1979, the International Association for the Study of Pain defined pain as an unpleasant, subjective sensory and emotional experience associated with tissue damage or potential tissue damage. In 2016, the definition of pain was updated to: Pain is a painful experience caused by actual or potential tissue damage with sensory, emotional, cognitive, and social dimensions. The classification of pain is complex. Based on duration, pain can be divided into acute and chronic pain. Chronic pain is further categorized into the following seven main types: ① chronic primary pain, ② chronic cancer pain, ③ chronic postoperative and post-traumatic pain, ④ neuropathic pain, ⑤ chronic head and maxillofacial pain, ⑥ chronic visceral pain, and ⑦ chronic musculoskeletal pain.

[0003] Clinical studies have shown that mechanical allodynia (Baron, R., Neuropathic pain: a clinical perspective. Handb Exp Pharmacol, 2009(194): p.3-30) is a type of pain secondary to inflammation or nerve damage and caused by harmless mechanical stimulation. At the cellular tissue level, mechanical allodynia is mainly caused by sensitized peripheral sensory neurons in dorsal root ganglion neurons or amplification of pain signals at the spinal cord level. Mechanical allodynia includes static mechanical allodynia caused by harmless Von Frey filaments and dynamic mechanical allodynia caused by brushing. Human psychology studies have shown that static allodynia is subjectively related to the perception of stable pressure stimulation or point stimulation on the skin surface, while dynamic allodynia is related to light touch stimulation (such as wind blowing, dressing). Clinical studies have shown that many patients experience allodynia after inflammation or nerve damage, which has seriously affected their quality of life. Therefore, it is urgent to find potential substances to treat clinical mechanical allodynia.

[0004] Hen Egg Lysozyme (HEL) is a globular protein containing 129 amino acid residues and approximately 14 kDa. It has strong antibacterial properties and the ability to hydrolyze cell wall components of Gram-positive bacteria. Studies have shown that HEL participates in humoral and cellular immunity by promoting the production of related antibodies and enhancing the activity of immune cells, thereby enhancing their antibacterial effects (Murakami, F., T. Sasaki, and T. Sugahara, Lysozymestimulates immunoglobulin production by human-human hybridoma and human peripheral blood lymphocytes. Cytotechnology, 1997. 24(2): p. 177-82). HEL can rapidly increase the body's anti-inflammatory factor levels by inhibiting the release of inflammatory cytokines such as TNF-α and IL-1β. It can also slowly affect the expression of pro-inflammatory genes in macrophages by inhibiting the c-Jun N-terminal kinase (JNK) signaling pathway, thereby regulating the balance between pro-inflammatory and anti-inflammatory states (Tagashira, A., K. Nishi, and T.Sugahara, Lysozyme from hen egg white ameliorates lipopolysaccharide-induced systemic inflammation in mice. Cytotechnology, 2019. 71(2): p. 497-506), ultimately achieving its antibacterial and anti-inflammatory effects. In addition to participating in humoral immunity, HEL also participates in the regulation of cellular immunity, exerting its unique cellular effects in viral infection, tumors, and inflammatory bowel disease. Although HEL plays an important role in the body's antibacterial and anti-inflammatory immune processes, whether it participates in the regulation of pathological pain remains unclear. Summary of the Invention

[0005] The purpose of the present invention is to provide a new use of hen egg white lysozyme in alleviating chronic pain. Experiments have found that HEL significantly and selectively alleviates inflammatory pain, neuropathic pain and static mechanical allodynia induced by chemotherapy drugs, providing a new idea for the treatment of chronic pain and demonstrating the potential application value of hen egg white lysozyme in the preparation of drugs for treating, preventing and alleviating chronic pain.

[0006] PRKN, as an E3 ubiquitin ligase, is involved in the regulation of cellular ion channel function and structure, and is also involved in the regulation of mitochondrial autophagy (Quinn, PMJ, et al., PINK1 / Parkin signalling in neurodegeneration and neuroinflammation. Acta Neuropathol Commun, 2020. 8(1): p. 189). Studies have shown that PRKN is widely distributed in the nervous system and is closely related to the occurrence and development of various neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease (Stichel, CC, et al., Parkinexpression in the adult mouse brain. Eur J Neurosci, 2000. 12(12): p. 4181-94). Furthermore, patients with diabetic static allodynia have decreased PRKN expression in primary sensory neurons (Yamashita, A., et al., Dysregulation of p53 and Parkin Induce Mitochondrial Dysfunction and Leads to the Diabetic Neuropathic Pain. Neuroscience, 2019.416: p. 9-19). TACAN (TMEM120A) is a mechanosensitive ion channel involved in the transmission of mechanical pain signals. Conditional knockout of TACAN in peripheral sensory neurons significantly increases the mechanical pain threshold in mice.

[0007] The present invention conducted animal modeling experiments, which showed that HEL upregulated the expression of PRKN protein in primary sensory neurons in the dorsal root ganglion (DRG), causing a decrease in the expression of the mechanosensitive ion channel TACAN in the PRKN / TACAN pathway on the cell membrane, thereby significantly and selectively alleviating inflammatory pain, neuropathic pain, and static mechanical allodynia induced by chemotherapy drugs.

[0008] Based on the experimental results of the present invention, the present invention proposes the following technical solutions:

[0009] Disclosed is a use of hen egg lysozyme (HEL) in the preparation of a medicine for preventing, treating or alleviating chronic pain.

[0010] In the above application, the content of hen egg white lysozyme in the unit dose of the drug for preventing, treating or alleviating chronic pain is a therapeutically effective amount.

[0011] In the above application, the drug is administered to humans or animals, including mice. When administered to mice, the effective dosage of hen egg white lysozyme is 400 mg / kg mouse.

[0012] In the above application, the chronic pain is static mechanical tactile allodynia.

[0013] In the above application, the chronic pain includes inflammatory pain, neuropathic pain or pain caused by chemotherapy drugs.

[0014] Furthermore, the present invention provides a drug for preventing, treating or alleviating chronic pain, wherein the active ingredient of the drug includes hen egg white lysozyme. The drug can use hen egg white lysozyme as a single active ingredient or it can be compounded with other active ingredients.

[0015] In the above medicine, the content of hen egg white lysozyme in a unit dose of the medicine is a therapeutically effective amount.

[0016] The above-mentioned medicine can be a medicine for humans or a medicine for animals, and the animals can be various animals.

[0017] Among the above drugs, when the drug is used for mice, the dosage of each egg white lysozyme is 400 mg / Kg mouse.

[0018] In the above-mentioned medicine, the chronic pain includes inflammatory pain, neuropathic pain or pain caused by chemotherapy drugs.

[0019] The dosage forms of the above drugs are tablets, capsules, granules, oral liquids, pills, aerosols or injections.

[0020] The egg white lysozyme used in the present invention is derived from food, is non-toxic and safe, may not be tolerated and has little side effects. Animal chronic pain analgesia experiments have shown that the egg white lysozyme has a strong analgesic effect on chronic pain such as inflammatory pain, neuropathic pain and chemotherapy pain, indicating that the analgesic effect of the egg white lysozyme is strong and low in toxicity. Its mechanism of action is different from that of opioid or aspirin analgesics commonly used in clinical practice, and it has low addictiveness.

[0021] The present invention discovered for the first time that HEL has the effect of alleviating static touch-induced allodynia and its deep mechanism provides a theoretical basis for the treatment of chronic pain. It proves that HEL has potential application value in preparing drugs for preventing, treating and alleviating chronic pain, and has great clinical significance.

[0022] The beneficial effects of the present invention are:

[0023] 1. Egg white lysozyme is an extract derived from food with potential clinical therapeutic value. It significantly expands the safe therapeutic window and may be non-tolerable, addictive, and have minimal side effects. Its mechanism of action may be different from that of commonly used opioid or aspirin analgesics in clinical practice.

[0024] 2. The hen egg white lysozyme of the present invention can be derived from natural food hen egg white, which is a common raw material and can thus promote the development of related industries.

[0025] 3. The present invention confirms for the first time that hen egg white lysozyme has the effect of intervening in static mechanical allodynia caused by chronic pain, including inflammatory pain, neuropathic pain, and pain caused by chemotherapy drugs, and can be used to prepare therapeutic drugs for chronic pain.

[0026] 4. Egg white lysozyme upregulates the expression of PRKN protein in primary sensory neurons in dorsal root ganglia, causing a decrease in the expression of the mechanosensitive ion channel TACAN in the PRKN / TACAN pathway on the cell membrane, thereby significantly inhibiting static mechanical touch-induced pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Diagram of the mouse modeling method and design process.

[0028] Drug intervention was administered 7 days prior to model establishment, via oral gavage at a dose of 400 mg / kg once daily, continuing until behavioral testing was completed after model establishment. The inflammatory pain model (CFA model) was administered for 13 days, with behavioral testing conducted on the first, third, and fifth days of model establishment. The neuropathic pain model (SNL model) was administered for 18 days, with behavioral testing conducted on the fourth, seventh, and tenth days of model establishment. The chemotherapeutic pain model (VCR model) was administered for 18 days, with behavioral testing conducted on the fourth, seventh, and tenth days of model establishment. The behavioral test consisted of a mechanical threshold test, using an up-and-down method to measure the mechanical withdrawal threshold of mice. Briefly, mice were placed in a plexiglass cover on a 10 × 10 mm wire mesh plate. Von Frey filaments of increasing strength were applied to the mid-point of the hind paw for 6–8 seconds, with a 5-minute interval between stimulations. A positive response was considered if the mouse withdrew its paw or licked it immediately after the test period or upon removal of the filament. Paw lifts induced by physical activity were not considered positive responses. The upper limit of the number of tests for each mouse is 5 times. If there are ≥3 positive reactions among 5 times, the mouse is considered sensitive to this stimulation intensity. If there are ≥3 negative reactions, the mouse is considered insensitive. The lowest sensitive stimulation intensity of the mouse is recorded as the mechanical pain threshold of the mouse.

[0029] Figure 2 This figure shows the effect of HEL on relieving inflammatory pain.

[0030] The specific method of establishing the CFA model is as follows: mice are anesthetized by inhalation of 2% isoflurane, disinfected with 0.5% iodine, and then 20 μl of complete Freund's Adjuvant (CFA) is injected into the center of the palmar side of the hind paw using a 1 ml syringe. If the plantar swelling occurs, the model is successfully established and can be used for subsequent experiments.

[0031] The horizontal axis in the figure indicates the time (days) of behavioral testing after modeling, and the vertical axis indicates the threshold value (g) for mechanical sensitivity. The triangle represents the HEL treatment group, which was given 400 mg / kg egg white lysozyme orally for 13 consecutive days and underwent CFA modeling on one hind paw. The square represents the CFA model group, which was given an equal volume of saline orally and underwent CFA modeling on one hind paw. The circle represents the control group, which was given an equal volume of saline orally. Behavioral test data were analyzed using two-way repeated measures ANOVA followed by Bonferroni post hoc test. P A value < 0.05 was considered statistically significant.

[0032] Figure 3 This figure shows the effect of HEL on alleviating neuropathic pain.

[0033] The specific method of establishing the SNL model is as follows: mice were anesthetized by inhalation of 2% isoflurane, shaved with a shaver, and prepared for skin disinfection with 0.5% iodine. The skin was cut on the left side of the L4-S1 spinal cord stage, a 1-2 cm surgical incision was made, the L5 and L6 nerves were found, the nerves were ligated with 4-0 silk sutures, and the muscles and skin were sutured in layers to complete the model for subsequent experiments.

[0034] The horizontal axis in the figure indicates the time (days) of behavioral testing after modeling, and the vertical axis indicates the threshold for mechanical sensitivity (g). The triangle represents the HEL treatment group, which was given 400 mg / kg egg white lysozyme orally for 18 consecutive days and underwent SNL modeling on one side. The square represents the SNL model group, which was given an equal volume of saline orally and underwent SNL modeling on one side. The circle represents the control group, which was given an equal volume of saline orally. Behavioral test data were analyzed using a two-way repeated measures ANOVA followed by a Bonferroni post hoc test. P A value < 0.05 was considered statistically significant.

[0035] Figure 4 This is a diagram showing the effect of HEL on alleviating pain caused by chemotherapy drugs.

[0036] The specific method of establishing the VCR model is as follows: an appropriate amount of vincristine is dissolved in normal saline to a concentration of 0.1 mg / kg and injected intraperitoneally for 7 consecutive days to establish a chemotherapy pain model. The model is completed and used for subsequent experiments.

[0037] The horizontal axis in the figure indicates the time (days) of behavioral testing after modeling, and the vertical axis indicates the threshold for mechanical sensitivity (g). The triangles represent the HEL treatment group, which was given 400 mg / kg egg white lysozyme by gavage for 18 consecutive days and underwent VCR modeling. The squares represent the VCR model group, which was given an equal volume of saline by gavage and underwent VCR modeling alone. The circles represent the control group, which was given an equal volume of saline by gavage. Behavioral test data were analyzed using a two-way repeated measures ANOVA followed by a Bonferroni post hoc test. P A value < 0.05 was considered statistically significant.

[0038] Figure 5 RNA-seq screening of differentially expressed genes in HEL to alleviate inflammatory pain.

[0039] The figure shows the transcriptome sequencing results after modeling. The CFA model was built according to Figure 1 As shown, samples were collected on ice on day 5 after modeling. DRG samples from each group were placed in enzyme-free EP tubes and sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for RNA-seq. The figure shows a gene heatmap, with the control group, CFA model group, and HEL-treated group shown from left to right, and the group-specific differences in seven genes from top to bottom. Specifically, after CFA modeling, seven genes were downregulated in the CFA group, while after HEL intervention, gene levels increased in the CFA+HEL group. Colors indicate changes in levels, with lighter colors indicating greater differences.

[0040] Figure 6 This is a graph showing the changes in the differentially expressed genes detected by qPCR.

[0041] The implementation method is the same Figure 1 As shown, samples were collected on ice on the 5th day after CFA modeling. DRGs of each group were placed in enzyme-free EP tubes for conventional qPCR. The qPCR results are shown in the figure. The horizontal axis represents the differentially expressed genes in each group. The circle represents the control group (control), the square represents the CFA model group (CFA+Veh), and the triangle represents the HEL treatment group (CFA+HEL). The vertical axis represents the mRNA expression level. One-way ANOVA followed by Tukey's multiple comparisons test method was used for data analysis. * P < 0.05, ** P< 0.01,*** P < 0.001.

[0042] Figure 7 This is a graph showing the changes in the protein levels of various TACAN components after Western Blotting detection of PRKN knockdown and HEL intervention.

[0043] The figure shows the changes in TACAN protein levels in various components after intrathecal injection of PRKN-siRNA and oral gavage intervention with HEL. Figure 7 AC represents the relative expression of TACAN protein in DRG tissue membrane protein, cytoplasmic protein and total protein, respectively. The protein WB strips are shown above the statistical graph. The upper strip represents the expression of TACAN, and the lower strip represents the expression of the internal reference protein. Among them, the internal reference of membrane protein is TfR, and the internal reference of cytoplasmic protein and total protein is β-actin. The statistical graph shows the statistics of the relative expression of TACAN. The horizontal axis represents different groups. The circle represents the control group (control), the square represents the knockdown group (PRKN-siRNA), and the triangle represents the treatment group (PRKN-siRNA+HEL). The vertical axis represents the relative expression of TACAN. One-way ANOVA followed by Tukey's multiple comparisons test method was used for data analysis. P A value < 0.05 was considered statistically significant.

[0044] Figure 8 This is a diagram showing how HEL regulates static mechanical touch-induced pain through the PRKN / TACAN pathway.

[0045] The horizontal axis represents the group, with circles representing the control group (control), squares representing the overexpression group (TACAN-over), triangles representing the treatment group (TACAN-over+HEL), and diamonds representing the re-intervention group (TACAN-over+HEL+PRKN-siRNA). The vertical axis represents the mechanical threshold g of each group. Behavioral test data were analyzed using two-way repeated measures ANOVA followed by Bonferroni post hoc test. P A value < 0.05 was considered statistically significant.

[0046] Figure 9 This figure shows the changes in TACAN in membrane proteins after TACAN overexpression virus, HEL, and PRKN-siRNA intervention.

[0047] The protein WB bands are above the statistical graph. The upper one is the expression level of TACAN, and the lower one is the expression level of TfR internal reference protein. The statistical graph shows the statistics of TACAN relative to TfR expression. The horizontal axis represents each group. The circle represents the control group (control), the square represents the overexpression group (TACAN-over), the triangle represents the treatment group (TACAN-over+HEL), and the diamond represents the re-intervention group (TACAN-over+HEL+PRKN-siRNA). The vertical axis represents the relative expression level of TACAN. One-way ANOVA followed by Tukey's multiple comparisons test method was used for data analysis. P A value < 0.05 was considered statistically significant. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0049] In the following examples, unless otherwise specified, all contents are expressed in percentage by mass.

[0050] Example 1 Study on the Alleviating Effect of HEL on Inflammatory Pain

[0051] 1.1 Materials and Reagents:

[0052] Egg white lysozyme, Sigma, Catalog No. 62971-10g-F, diluted with 0.9% saline and administered at a dose of 400 mg / kg. Complete Freund's adjuvant (CFA), Sigma, Catalog No. F5881-10ML.

[0053] 1.2 Animals

[0054] Male C57BL / 6 mice, 6-8 weeks old, were provided by the Laboratory Animal Center of Sun Yat-sen University. Animals were randomly assigned and housed in individual cages with free access to food and water. The room temperature was controlled at 24 ± 1°C, the relative humidity was 50%-60%, and a 12-h day / night cycle was maintained to match the animals' circadian rhythms. Experimental procedures were conducted in accordance with national animal experimentation regulations and with minimal animal distress. Mice were acclimated to the behavioral testing laboratory for 3 days before use in formal experiments.

[0055] 1.3 Methods

[0056] Fifteen mice were randomly divided into three groups according to their body weight: control group, CFA model group (CFA+veh), and HEL treatment group (CFA+HEL). The experiment was divided into three groups, with 5 mice in each group. Figure 1 As shown, each group of mice received the following interventions:

[0057] 1) The HEL treatment group was given 400 mg / kg HEL solution by gavage daily starting 7 days in advance. 7 days later, 20 μl of complete Freund's adjuvant was injected into the left hind paw, followed by continuous gavage of 400 mg / kg HEL solution.

[0058] 2) The CFA model group received an injection of 20 μl of complete Freund's adjuvant into the left hind paw and an equal volume of normal saline by gavage;

[0059] 3) The control group was gavaged with the same volume of normal saline.

[0060] Static mechanical thresholds were measured on days 1, 3, and 5 after CFA model establishment. The mechanical withdrawal thresholds of mice were measured using an up-and-down method. Mice were placed in a plexiglass cover on a 10 × 10 mm wire mesh plate. Von Frey filaments of increasing strength were applied to the mid-foot of the hind paw for 6–8 seconds, with a 5-minute interval between stimulations. A positive response was considered if the mouse withdrew its paw or licked it immediately after the fiber was removed or within the test period. Paw lifts induced by physical activity were not considered positive responses. This threshold reflects chronic pain caused by inflammation. Each mouse was tested a maximum of 5 times. A positive response of ≥3 out of 5 was considered sensitive to the stimulus intensity, while a negative response of ≥3 was considered insensitive. The lowest stimulus intensity to which the mouse was sensitive was recorded as the mechanical pain threshold.

[0061] 1.4 Results

[0062] The mechanical thresholds of mice in each group at different times are as follows Figure 2 The results showed that in the CFA model, after administration of HEL, the mechanical threshold of mice was improved on the 1st, 3rd and 5th day, with statistical significance. P <0.001,** P <0.01,*** P <0.05, indicating that HEL has a significant analgesic effect on inflammatory pain.

[0063] Example 2 Study on the Alleviating Effect of HEL on Neuropathic Pain

[0064] 2.1 Materials and Reagents

[0065] Egg white lysozyme, Sigma, catalog number 62971-10g-F, was diluted with 0.9% saline and administered at a dose of 400 mg / kg.

[0066] 2.2 Animals

[0067] Male C57BL / 6 mice, 6-8 weeks old, were provided by the Laboratory Animal Center of Sun Yat-sen University. Animals were randomly assigned and housed in individual cages with free access to food and water. The room temperature was controlled at 24 ± 1°C, the relative humidity was 50%-60%, and a 12-h day / night cycle was maintained to match the animals' circadian rhythms. Experimental procedures were conducted in accordance with national animal experimentation regulations and with minimal animal distress. Mice were acclimated to the behavioral testing laboratory for 3 days before use in formal experiments.

[0068] 2.3 Methods

[0069] Fifteen mice were randomly divided into three groups according to their body weight: control group, SNL model group (SNL+veh), and HEL treatment group (SNL+HEL). The experiment was divided into three groups, with 5 mice in each group. Figure 1 As shown, each group of mice received the following interventions:

[0070] 1) The HEL treatment group was given 400 mg / kg HEL solution by gavage daily starting 7 days in advance. 7 days later, the left L5 / L6 nerve was ligated, and then 400 mg / kg HEL solution was continued by gavage daily.

[0071] 2) The SNL model group underwent left L5 / L6 nerve ligation and received an equal volume of normal saline by gavage;

[0072] 3) The control group was gavaged with the same volume of normal saline.

[0073] Behavioral assessments were performed on days 4, 7, and 10 after SNL model establishment. The mechanical withdrawal threshold (MPT) of the mice was measured using an up-and-down method. Mice were placed in a plexiglass enclosure on a 10 × 10 mm wire mesh plate. Von Frey filaments of increasing strength were applied to the mid-foot of the mouse for 6–8 seconds, with a 5-minute interval between stimulations. Paw withdrawal or licking during the test period or immediately after the filament removal was considered a positive response; paw lifts induced by physical activity were not considered positive responses. The threshold reflects chronic pain caused by nerve injury. Each mouse was tested a maximum of five times. A positive response of ≥3 out of 5 was considered sensitive to the stimulus intensity, while a negative response of ≥3 was considered insensitive. The lowest stimulus intensity to which the mouse was sensitive was recorded as the mechanical pain threshold.

[0074] 2.4 Results

[0075] The mechanical thresholds of mice in each group at different times are as follows Figure 3 The results showed that in the SNL model, after HEL was administered, the mechanical threshold of mice was improved on the 4th, 7th and 10th days, with statistical significance. P <0.001,**P <0.01,*** P <0.05, indicating that HEL has a significant analgesic effect on neuropathic pain.

[0076] Example 3 Study on the Effect of HEL on Relieving Pain Induced by Chemotherapy Drugs

[0077] 3.1 Materials and Reagents

[0078] Egg white lysozyme (Sigma, catalog number 62971-10g-F) was diluted with 0.9% saline and administered at a dose of 400 mg / kg. Vincristine (VCR) was obtained from Shenzhen Wanle Pharmaceutical Co., Ltd., batch number 2011V2.

[0079] 3.2 Animals

[0080] Male C57BL / 6 mice, 6-8 weeks old, were provided by the Laboratory Animal Center of Sun Yat-sen University. Animals were randomly assigned and housed in individual cages with free access to food and water. The room temperature was controlled at 24 ± 1°C, the relative humidity was 50%-60%, and a 12-h day / night cycle was maintained to match the animals' circadian rhythms. Experimental procedures were conducted in accordance with national animal experimentation regulations and with minimal animal distress. Mice were acclimated to the behavioral testing laboratory for 3 days before use in formal experiments.

[0081] 3.3 Methods

[0082] Fifteen mice were randomly divided into three groups according to their body weight: control group, VCR model group (VCR+veh), and HEL treatment group (VCR+HEL). The experiment was divided into three groups, with 5 mice in each group. Figure 1 As shown, each group of mice received the following interventions:

[0083] 1) The HEL treatment group received intraperitoneal injection of 0.1 mg / kg VCR solution for 7 consecutive days and oral gavage of 400 mg / kg hen egg white lysozyme solution every day;

[0084] 2) The VCR model group received an intraperitoneal injection of 0.1 mg / kg VCR solution and an equal volume of normal saline by gavage;

[0085] 3) The control group was gavaged with the same volume of normal saline.

[0086] Behavioral assessments were performed on days 4, 7, and 10 after VCR model establishment. The mechanical withdrawal threshold (MPT) of the mice was measured using an up-and-down method. Mice were placed in a plexiglass cover on a 10 × 10 mm wire mesh plate. Von Frey filaments of increasing strength were applied to the mid-foot of the mouse for 6–8 seconds, with a 5-minute interval between stimulations. Paw withdrawal or licking during the test period or immediately after the filaments were removed was considered a positive response; paw lifts induced by physical activity were not considered positive responses. The threshold reflects the severity of chronic pain induced by chemotherapy. Each mouse was tested a maximum of 5 times. A positive response of ≥3 out of 5 was considered sensitive to the stimulus intensity, while a negative response of ≥3 was considered insensitive. The lowest stimulus intensity to which the mouse was sensitive was recorded as the mechanical pain threshold.

[0087] 3.4 Results

[0088] The mechanical thresholds of mice in each group at different times are as follows Figure 4 The results showed that in the VCR model, after administration of HEL, the mechanical threshold of mice was improved on the 4th, 7th and 10th days, with statistical significance. P <0.001,** P <0.01,*** P <0.05, indicating that HEL has a significant analgesic effect on chronic pain caused by chemotherapy drugs.

[0089] Example 4 Screening of HEL Targets for Alleviating Chronic Pain

[0090] 4.1 Materials and Reagents

[0091] Egg white lysozyme, Sigma, Catalog No. 62971-10g-F, diluted with 0.9% saline and administered at a dose of 400 mg / kg. Complete Freund's adjuvant, Sigma, Catalog No. F5881-10ML.

[0092] 4.2 Animals

[0093] Male C57BL / 6 mice, 6-8 weeks old, were provided by the Laboratory Animal Center of Sun Yat-sen University. Animals were randomly assigned and housed in individual cages with free access to food and water. The room temperature was controlled at 24 ± 1°C, the relative humidity was 50%-60%, and a 12-h day / night cycle was maintained to match the animals' circadian rhythms. Experimental procedures were conducted in accordance with national animal experimentation regulations and with minimal animal distress. Mice were acclimated to the behavioral testing laboratory for 3 days before use in formal experiments.

[0094] 4.3 Methods

[0095] Thirty-six mice were randomly divided into three groups based on body weight, namely, a control group (control), a CFA model group (CFA+veh), and a HEL-treated group (CFA+HEL). The experiment was divided into three groups, with 12 mice in each group. Each group of mice received the following interventions:

[0096] 1) The HEL treatment group was given 400 mg / kg hen egg white lysozyme solution by gavage daily starting 7 days in advance. 7 days later, 20 μl of complete Freund's adjuvant was injected into the left hind paw, followed by continuous gavage of 400 mg / kg hen egg white lysozyme solution.

[0097] 2) CFA model group: 20 μl complete Freund's adjuvant was injected into the left hind paw and the same volume of normal saline was administered orally;

[0098] 3) The control group was gavaged with the same volume of normal saline.

[0099] On the 5th day after the CFA model was established, the mouse DRG tissues were dissected and placed in enzyme-free EP tubes for RNA-seq and q-PCR quantitative analysis, respectively.

[0100] 4.4 Results

[0101] To further analyze how HEL alleviates static mechanical allodynia, we performed RNA-seq analysis to screen for differential gene expression in DRG tissues. Figure 5 As shown in Figure 3, there are 7 differentially expressed genes. The gene expression in the CFA model group is down-regulated, while the gene expression in the CFA+HEL group is up-regulated. To confirm the changes in these 7 genes, we further performed q-PCR analysis. Figure 6 The data showed that Prkn mRNA decreased most significantly in the CFA group and recovered most significantly in the CFA+HEL group, suggesting that PRKN may be a molecule potentially involved in HEL regulation of static mechanical touch-evoked allodynia.

[0102] Example 5 Study on the potential molecular mechanism of PRKN in HEL inhibition of static mechanical allodynia

[0103] 5.1 Materials and Reagents:

[0104] Egg white lysozyme, Sigma, Catalog No. 62971-10g-F, diluted in 0.9% saline and administered at a dose of 400 mg / kg. TARGETplus Mouse Park2 siRNA, Dharmacon, Catalog No. L-065413-01-0010. BCA Protein Quantification Kit, Thermo Fisher Scientific, Catalog No. 23227. Minute™ Plasma Membrane Protein and Cellular Fraction Isolation Kit, Invent, Catalog No. SM-005-50T. 10% TGX Rapid Gel Kit, Bio-Rad, Catalog No. 1610183. Anti-TMEM120A antibody, Bioss, Catalog No. bs-19952R. β-actin, Abcam, Catalog No. ab8226. BSA, Sigma, Catalog No. B2064-100G. Ultrasensitive Luminescent Detection Kit, Yazyme, Cat. No. 01592050. Urethane, MacLean, Cat. No. U820333-100g. RIPA Lysis Buffer, Beyotime, Cat. No. P0013B.

[0105] 5.2 Instruments and Equipment

[0106] Gel electrophoresis and electrotransfer system, brand model Bio-RAD 1658033; ultrasonic cell disruptor, brand model SONICS VCX130; microplate reader, brand model Bio-Rad iMark; fully automatic chemiluminescence image analysis system, brand model Tanon-5200; ultraspeed refrigerated centrifuge, brand model Thermo fresc021.

[0107] 5.3 Animals

[0108] Male C57BL / 6 mice, 6-8 weeks old, were provided by the Laboratory Animal Center of Sun Yat-sen University. Animals were randomly assigned and housed in individual cages with free access to food and water. The room temperature was controlled at 24 ± 1°C, the relative humidity was 50%-60%, and a 12-h day / night cycle was maintained to match the animals' circadian rhythms. Experimental procedures were conducted in accordance with national animal experimentation regulations and with minimal animal distress. Mice were acclimated to the behavioral testing laboratory for 3 days before use in formal experiments.

[0109] 5.4 Methods

[0110] Eighteen mice were randomly divided into three groups based on body weight: a control group (PRKN-siRNA + veh), and a treatment group (PRKN-siRNA + HEL). The experiment was divided into three groups, with six mice in each group. Each group of mice received the following interventions:

[0111] 1) The treatment group received daily oral administration of 400 mg / kg HEL solution starting 7 days in advance. Seven days later, 10 μl of PRKN-siRNA was injected intrathecally, followed by continuous oral administration of 400 mg / kg HEL solution.

[0112] 2) The knockdown group received an intrathecal injection of 10 μl of PRKN-siRNA and an equal volume of saline by gavage;

[0113] 3) The control group was gavaged with the same volume of normal saline.

[0114] Forty-eight hours after intrathecal injection of 10 μl of PRKN-siRNA, mouse DRG tissue was dissected and dissolved in cold RIPA buffer. Protein components were separated by gel electrophoresis and incubated on a PVDF membrane with primary antibodies against TACAN (Rabbit, 1:1000, Novus), TfR (Mouse, 1:1000, Abcam), and β-actin (Mouse, 1:2000, Affinity) at 4°C overnight. The following day, immune complexes were detected by incubation with secondary antibodies against Rabbit (1:10,000) and Mouse (1:10,000) at room temperature for 1 hour.

[0115] Intrathecal injection of PRKN-siRNA: Centrifuge the synthesized PRKN-siRNA to allow the powder to reach the bottom of the tube. Add 20 μl of 5% glucose solution for every 5 nmol of siRNA. Add 1 μg / μl of Rvg-9R solution at a ratio of 2.5 μg PRKN-siRNA: 7.5 μg Rvg-9R, mix well, and incubate at room temperature for 10-15 min. The experimental mice were anesthetized by inhalation of 2% isoflurane. After skin preparation and disinfection, the L6 spinous process was exposed and the needle was inserted into the subarachnoid space at the L5 / L6 interval. The tail swing indicated that the needle had entered correctly. The needle was pulled out and a 15 cm section of PE-10 tube was clamped with curved forceps and slowly inserted along the small hole where the needle had been inserted. The PR-10 tube was slowly pushed forward about 1 cm. If cerebrospinal fluid was seen flowing out of the PE-10 tube, it indicated that the tube was correctly placed. 10 μl of the prepared PRKN-siRNA solution was aspirated with a 20 μl microsyringe, pushed into the PE-10 tube and injected into the sheath. The solution was kept for 10 minutes, the PE-10 tube was pulled out, bleeding was stopped, and the muscles and skin were sutured in layers. The mice were fed under normal conditions after surgery. After 48 hours, they could be used for subsequent experiments.

[0116] Western blot method:

[0117] 1) Reagent preparation

[0118] Glue dispensing system (Bio-Rad)

[0119] Separation gel: Using the Bio-Rad gel preparation system, 3.5 ml of solution A, 3.5 ml of solution B, 35 μl of 10% APS, and 3.5 μl of TEMED.

[0120] Stacking gel: Using the Bio-Rad gel preparation system, prepare 1.5 ml of solution A, 1.5 ml of solution B, 15 μl of 10% APS, and 3 μl of TEMED.

[0121] 1× electrophoresis buffer: Weigh 3.03 g Tris Base, 14.4 g glycine, and 1 g SDS, and dilute to 1 L with deionized water.

[0122] 1× transfer buffer: Weigh 3.03 g Tris Base and 14.4 g glycine, add 200 ml methanol, and dilute to 1 L with deionized water.

[0123] 1× TBST buffer: Weigh 8.8 g of NaCl, add 2.35 ml of 20% Tween 20 solution and 10 ml of 1 M Tris•HCl solution (pH = 7.5), and add deionized water to make up to 1 L.

[0124] Western blotting blocking solution: 0.5 g of skim milk powder or BSA was dissolved in 10 ml of TBST solution.

[0125] Western blotting primary antibody diluent: Dissolve 0.5 g of BSA in 10 ml of TBST solution and add primary antibodies according to the antibody instructions (TACAN, Bioss, Rabbit, 1:1000; β-actin, Abcam, Mouse, 1:2000; TfR, Abcam, Mouse, 1:1000).

[0126] Western blotting secondary antibody diluent: Dissolve 0.5 g of skim milk powder or BSA in 10 ml of TBST solution. Add the secondary antibody corresponding to the primary antibody (GM, KPL, 074-1806, 1:10,000; G-Rb, KRL, 074-1506, 1:10,000) and mix well.

[0127] 2) Tissue protein extraction

[0128] Total protein extraction: After anesthetizing mice with 10% urethane (0.3-0.4 ml), perfuse with 0.01 M PBS. Rapidly remove DRG tissue from the mice on an ice pack. Remove any remaining blood clots in cold PBS. Add an appropriate amount of RIPA lysis buffer containing protease inhibitors. Ultrasonicate on ice and centrifuge at 14,800 rpm for 20 min at 4°C. Aliquot the supernatant and store at -80°C.

[0129] Membrane protein and cytoplasmic protein extraction: Membrane proteins were extracted according to the standard procedures of the membrane protein extraction kit. After anesthetizing the mice with 10% urethane (0.3-0.4 ml), perfuse the mice with 0.01 M PBS, quickly remove the mouse DRG tissue on an ice pack, add 200 μl of solution A, grind with a plastic rod for 1 min, then add 300 μl of solution A, incubate on ice for 10 min, centrifuge at 14000 rpm in a 4℃ centrifuge for 30 min, vortex mix, centrifuge at 3000 rpm for 1 min, take the supernatant into another EP tube, centrifuge at 14000 rpm in a 4℃ centrifuge for 30 min, aspirate the supernatant and save it, which is the cytoplasmic protein, then add 200 μl of solution B, vortex mix, centrifuge at 10000 rpm in a 4℃ centrifuge for 5-10 min, transfer the supernatant to another new EP tube, add 1.6 ml of ice-bath PBS, centrifuge at 14000 rpm for 30 min, discard the supernatant, and retain the precipitate, which is the membrane protein. Add an appropriate amount of RIPA lysis buffer, aliquot and save it for later use.

[0130] 3) Determination of protein concentration using BCA kit

[0131] Prepare an appropriate amount of BCA working solution based on the number of samples, using a ratio of Solution A to Solution B of 50:1. Prepare a series of standard protein concentration gradients according to the kit instructions. Add the standard and sample proteins sequentially to a 96-well plate, adding 200 μl of BCA working solution to each well. Incubate at 37°C with slow shaking for 30 minutes, and measure protein concentration on a microplate reader.

[0132] 4) Gel electrophoresis and membrane transfer

[0133] Add the protein sample to the loading buffer at a ratio of 4:1 and cook at 100°C for 6 minutes. Add the protein to the gel wells and run at 80 V for 30-40 minutes. Then, run at 100 V for an appropriate amount of time, depending on the protein's molecular weight. After electrophoresis, place the gel and PVDF membrane in a transfer chuck and transfer at 300 mA for an appropriate amount of time, depending on the protein's molecular weight.

[0134] 5) Immune response

[0135] Block at room temperature with slow shaking for 1 hour. Remove the blocking solution, add the primary antibody, and incubate overnight at 4°C. Recover the primary antibody and wash the membrane three times with TBST (10 minutes each). Then, add the secondary antibody and incubate at room temperature with slow shaking for 1 hour. Wash the membrane three times with TBST (10 minutes each).

[0136] 6) Exposure

[0137] In a dark room, mix equal amounts of Luminescent Solution A and Solution B. Apply the luminescent solution evenly to the membrane. Use a CCD camera to capture the bands. Once clear bands are visible, photograph the marker under white light and save the image. Analyze the image using Tanon GIS.

[0138] 5.5 Results

[0139] TACAN, as a mechanosensitive ion channel, is involved in the transmission of mechanical pain signals. Conditional knockout of TACAN in peripheral sensory neurons significantly increases the mechanical threshold of mice. We hypothesized that PRKN is involved in the molecular mechanism by which HEL reduces static touch-evoked allodynia. Therefore, we injected PRKN-siRNA intrathecally and, after HEL intervention, detected the expression changes of each component protein TACAN. Western blotting results showed that compared with the control group, Figure 7 As shown in AB, in DRG tissue, PRKN-siRNA increased the membrane expression of TACAN but reduced its cytoplasmic accumulation. After HEL intervention, the expression of TACAN on the cell membrane decreased, while its cytoplasmic accumulation increased, returning to a level similar to that of the normal group. Figure 7 As shown in Figure C, PRKN-siRNA did not alter the expression of total TACAN in DRG neurons, and HEL intervention did not affect the changes in total TACAN expression. These data suggest that HEL increases TACAN membrane trafficking by downregulating PRKN, thereby alleviating static mechanical allodynia.

[0140] Example 6 Study on the Effect of HEL on Alleviating Static Mechanical Allodynia via the PRKN / TACAN Pathway

[0141] 6.1 Materials and Reagents:

[0142] Egg white lysozyme (Sigma, Catalog No. 62971-10g-F) was diluted with 0.9% saline and administered at a dose of 400 mg / kg. TARGETplus Mouse Park2 siRNA (Dharmacon, Catalog No. L-065413-01-0010) was used. TACAN overexpression virus (Raav-hSyn-TMEM120A-2A EGFP-WPRE-PA) was used by Privy Council, Catalog No. PT4858.

[0143] 6.1 Animals

[0144] Male C57BL / 6 mice, 6-8 weeks old, were provided by the Laboratory Animal Center of Sun Yat-sen University. Animals were randomly assigned and housed in individual cages with free access to food and water. The room temperature was controlled at 24 ± 1°C, the relative humidity was 50%-60%, and a 12-h day / night cycle was maintained to match the animals' circadian rhythms. Experimental procedures were conducted in accordance with national animal experimentation regulations and with minimal animal distress. Mice were acclimated to the behavioral testing laboratory for 3 days before use in formal experiments.

[0145] 6.2 Methods

[0146] Twenty-four mice were randomly divided according to body weight into four groups: a control group (control), an overexpression group (TACAN-over + veh), a treatment group (TACAN-over + HEL), and a re-intervention group (TACAN-over + HEL + PRKN-siRNA). The experiment was divided into four groups, with six mice in each group. Each group received the following interventions:

[0147] 1) The re-intervention group was injected intrathecally with TACAN overexpression virus. 21 days after infection, HEL was gavaged daily. 7 days later, 10 μl of PRKN-siRNA was injected intrathecally. 48 hours later, behavioral tests were performed and samples were collected. HEL gavage was continued until the day of collection.

[0148] 2) The treatment group received intrathecal injection of TACAN overexpressing virus and, 21 days after infection, continued daily gavage with HEL;

[0149] 3) The overexpression group received intrathecal injection of TACAN overexpression virus, and 21 days after infection, the patients were gavaged with an equal volume of normal saline;

[0150] 4) The control group was gavaged with the same volume of normal saline.

[0151] Forty-eight hours after intrathecal injection of 10 μl of PRKN-siRNA, behavioral testing was performed. The mechanical withdrawal threshold (MPT) of the mice was measured using an up-and-down method. Mice were placed in a Plexiglas cover on a 10 × 10 mm wire mesh plate. Von Frey filaments of increasing strength were applied to the mid-point of the hind paw for 6–8 seconds, with a 5-minute interval between stimulations. Paw withdrawal or licking during the test period or immediately after the filaments were removed was considered a positive response; paw lifts induced by physical activity were not considered positive. This threshold reflects the severity of chronic pain induced by chemotherapy. After behavioral testing, DRG tissue was dissected and dissolved in cold RIPA buffer. After separation by gel electrophoresis, the samples were incubated on PVDF membranes with primary antibodies against TACAN (Rabbit, 1:1000, Novus), TfR (Mouse, 1:1000, Abcam), and β-actin (Mouse, 1:2000, Affinity) at 4°C overnight. The next day, immune complexes were detected by incubation with corresponding secondary antibodies. Western blotting procedures and intrathecal siRNA injection were performed as described in Section 5.4.

[0152] Intrathecal AAV injection method: Dilute the purchased AAV with normal saline to the appropriate titer and mix well for later use. Anesthetize the experimental mouse with 2% isoflurane inhalation. After skin preparation and disinfection, expose the L6 spinous process and puncture the subarachnoid space at the L5 / L6 interspace with a needle. A flick of the tail indicates correct needle entry. Remove the needle and, while using curved forceps, grasp a 15 cm section of PE-10 tubing and slowly insert it along the small hole where the needle was inserted. Slowly advance the PE-10 tubing forward about 1 cm. If cerebrospinal fluid is seen flowing out of the PE-10 tubing, it indicates that the tubing is correctly placed. Use a 20 μl syringe to draw 10 μl of the prepared TACCAN-AAV solution, push it into the PE-10 tubing, and inject it into the sheath. Leave it for 10 minutes, remove the PE-10 tubing, stop bleeding, and suture the muscles and skin in layers. Feed the mouse under normal conditions after surgery. After 21 days, the mouse can be used for subsequent experiments.

[0153] 6.3 Results

[0154] Behavioral data show that Figure 8 As shown in Figure 2, HEL pretreatment of TACAN-overexpressing mice alleviated static mechanical allodynia. After feeding HEL to TACAN-overexpressing mice, PRKN-siRNA induced static mechanical allodynia again. Figure 9As shown in Figure 3, HEL was able to reduce the increased membrane expression of TACAN in DRG tissue, and PRKN-siRNA reversed the expression of TACAN. Together, these data indicate that the PRKN / TACAN pathway is selectively involved in the process of HEL-mediated static mechanical allodynia.

Claims

1. Use of hen egg white lysozyme in the preparation of a medicament for preventing, treating or alleviating neuropathic pain.

2. The use according to claim 1, characterized in that: The neuropathic pain is pain caused by chemotherapy drugs.

3. The use according to claim 1, characterized in that: In the unit dose of the drug for preventing, treating or alleviating neuropathic pain, the content of hen egg white lysozyme is a therapeutically effective amount.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine is used for human or animal body.

5. The use according to claim 4, characterized in that: When the drug is a drug for preventing, treating or alleviating neuropathic pain in mice, the effective dosage of hen egg white lysozyme per administration is 400 mg / kg mouse.

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