CN114903984A - Application of hen egg white lysozyme in relieving chronic pain - Google Patents

Abstract

The invention discloses application of hen egg white lysozyme in relieving chronic pain, and the experiment result shows that HEL remarkably and selectively relieves static mechanical induced pain caused by inflammatory pain, neuropathic pain and chemotherapy pain. Meanwhile, experiments show that the expression of a mechanical sensitivity ion channel TACAN in a PRKN/TACAN passage on a cell membrane is reduced by up-regulating the expression of the PRKN protein of a primary sensory neuron in the dorsal root ganglion by the HEL, so that static mechanical allodynia is remarkably inhibited. The invention discovers for the first time that HEL has the effect of relieving static mechanical induced pain and a deep mechanism, and the HEL is nontoxic and safe in source, has potential application value in preparing the medicine for preventing, treating and relieving chronic pain, and has great clinical significance.

Description

Application of hen egg white lysozyme in relieving chronic pain

Technical Field

The invention relates to a new application of hen egg white lysozyme, in particular to an application of hen egg white lysozyme in relieving chronic pain.

Background

Pain is a complex, clinically common symptom and disease that manifests subjective emotional sensations. The international society for pain research in 1979 defined pain as an unpleasant subjective feeling and emotional experience associated with tissue damage or potential tissue damage. In 2016, the pain definition was updated to: pain is a painful experience caused by actual or potential tissue damage at a sensory, emotional, cognitive, and social level. The classification of pain is complex, and according to the classification of duration, pain can be divided into acute pain and chronic pain, and chronic pain is mainly divided into the following 7 major classes: chronic primary pain, chronic cancer pain, chronic postoperative pain and post-traumatic pain, neuropathic pain, chronic head and maxillofacial pain, chronic visceral pain and chronic skeletal muscle pain.

Clinical studies have shown that mechanical allodynia (Baron, R., Neuropathic pain: a clinical therapeutic rather than Handb Exp Pharmacol, 2009(194): p.3-30) is a pain caused by harmless mechanical stimuli secondary to inflammation or nerve injury. At the cellular tissue level, mechanical touch-induced pain is mainly caused by sensitized peripheral sensory neurons in dorsal root neurons or by 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 brush sweeping. Human psychological studies have shown that static touch-induced pain is subjectively related to the perception of steady pressure or punctate irritation on the skin surface, whereas dynamic touch-induced pain is related to light touch irritation (e.g. wind blowing, dressing). Clinical studies have shown that the quality of life is severely affected by the development of allodynia in many patients following inflammation or nerve injury. Therefore, the search for potential substances to treat clinical mechanical touch-induced pain is imminent.

Hen Egg Lysozyme (HEL) is a globular protein of about 14kDa in size containing 129 amino acid residues, which has strong antibacterial properties and the ability to hydrolyze cell wall components of gram-positive bacteria. HEL has been shown to participate in humoral and cellular immunity by promoting production of related antibodies and increasing activity of immune cells, thereby enhancing its antibacterial effect (Murakami, F., T. Sasaki, and T. Sugahara, Lysozyme peptides immunization by human-human hybridoma and human bacterial cells. Cytotechnology, 1997.24 (2): p. 177-82). HEL can rapidly increase the level of body anti-inflammatory factors by inhibiting the release of inflammatory cytokines such as TNF-alpha and IL-1 beta, and can also slowly influence the expression of proinflammatory related genes in macrophages by inhibiting c-Jun N-terminal kinase (JNK) signal pathways, thereby regulating the proinflammatory and anti-inflammatory balance state (Tagashira, A., K. Nishi, and T. Sugahara, Lysozyme from egg white tissue lipids isolated polypeptide-induced system inflammation in microorganism, Cytosechnology, 2019.71 (2): p. 497-506), and finally achieving the antibacterial and anti-inflammatory effects. In addition to being involved in humoral immunity, HEL is also involved in the regulation of cellular immunity, exerting its characteristic cellular effects in viral infections, tumors and inflammatory bowel disease. Although HEL plays an important role in immune processes such as body antibiosis and anti-inflammation, whether HEL is involved in regulation of body pathological pain is still unclear.

Disclosure of Invention

Experiments show that HEL remarkably and selectively relieves inflammatory pain, neuropathic pain and static mechanical induced pain induced by chemotherapy drugs, provides a new idea for treating chronic pain, and proves the potential application value of hen egg white lysozyme in preparing medicines for treating, preventing and relieving chronic pain.

PRKN, as an E3 ubiquitinated protein ligase, is involved in the regulation of cell ion channel function and structure, as well as in the regulation of the mitochondrial autophagy process (Quinn, P.M.J., et al., PINK1/Parkin signalling in neuro-synthesis and neuroinflammation. Acta Neuropathol Commun, 2020.8 (1): p.189). Research has shown that PRKN is widely distributed in the nervous system and has a close relationship with the occurrence and development of various neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease (Stichel, C.C., et al, Parkin expression in the adult mouse damage. Eur J Neurosci, 2000.12 (12): p. 4181-94). Also, PRKN expression is decreased in primary sensory neurons of patients with static touch-induced pain caused by diabetes (Yamashita, A., et al, Dysrelation of p53 and Parkin index Mitochondrial Dysfunction and Leads to the Diabetic Neuropathic pain Panel. Neuroscience, 2019.416: p. 9-19). TACAN (TMEM120A) is used as a mechanical sensitivity ion channel to participate in the conduction of mechanical pain signals, and the ion channel for conditionally knocking TACAN in peripheral sensory neurons can obviously improve the mechanical pain threshold of mice.

Animal modeling experiments are carried out, and experiments show that the expression of a PRKN protein of a primary sensory neuron in a Dorsal Root Ganglion (DRG) is up-regulated by HEL to cause the reduction of the expression of a mechanosensitive ion channel TACAN in a PRKN/TACAN channel on a cell membrane, so that inflammatory pain, neuropathic pain and chemotherapy drug-induced static mechanical allodynia are remarkably and selectively relieved.

Based on the experimental result of the invention, the invention provides the following technical scheme:

application of Hen Egg Lysozyme (HEL) in preparing medicine for preventing, treating or relieving chronic pain is provided.

In the application, in the medicine for preventing, treating or relieving chronic pain in unit dose, the content of the hen egg white lysozyme is effective amount for treatment.

In the above application, the medicament is used for human or animal bodies. The animal body includes a white mouse and the like. When the medicament is applied to mice, the effective dose of the hen egg white lysozyme is 400mg/Kg of mice per time.

In the above application, the chronic pain is static mechanical allodynia.

In the above application, the chronic pain includes inflammatory pain, neuropathic pain or pain caused by chemotherapeutic drugs.

Furthermore, the invention also provides a medicament for preventing, treating or relieving chronic pain, wherein the effective component of the medicament comprises chicken egg white lysozyme. The medicine can use chicken egg white lysozyme as single effective component, and can also be compounded with other effective components.

In the medicine, the content of the hen egg white lysozyme in the unit dose medicine is effective amount for treatment.

The medicine can be used for human medicine or animal medicine, and the animal can be various animals.

In the medicine, when the medicine is used for mice, the dosage of the hen egg white lysozyme is 400mg/Kg of mice each time.

In the above drugs, the chronic pain includes inflammatory pain, neuropathic pain or pain caused by chemotherapeutic drugs.

The dosage forms of the above medicines are tablet, capsule, granule, oral liquid, dripping pill, aerosol or injection.

The chicken protein lysozyme used in the invention is derived from food, has nontoxic and safe sources, may not have tolerance and has small side effect, and animal chronic pain analgesia experiments prove that the chicken protein lysozyme has strong analgesic effect on chronic pain such as inflammatory pain, neuropathic pain, chemotherapy pain and the like, so that the chicken protein lysozyme has the characteristics of strong effect and low toxicity, and the action mechanism of the chicken protein lysozyme is different from clinically common opioid or aspirin analgesic drugs, and has small addiction.

The invention discovers that the HEL has the effect of relieving static induced pain for the first time and provides a theoretical basis for the treatment of chronic pain by a deep mechanism of the HEL, proves that the HEL has potential application value for preparing the medicine for preventing, treating and relieving chronic pain, and has great clinical significance.

The invention has the beneficial effects that:

1. the hen egg white lysozyme is an extract from food, has potential clinical treatment value, obviously expands the safe treatment window, may not have tolerance, addiction and side effect, and the action mechanism of the hen egg white lysozyme is possibly different from clinically common opioid or aspirin analgesics.

2. The hen egg white lysozyme can be derived from the egg white of a natural food, and the raw materials are common, so that the development of related industries can be promoted.

3. The invention proves that the hen egg white lysozyme has the effect of intervening static mechanical induced pain caused by chronic pain for the first time, and the chronic pain comprises inflammatory pain, neuropathic pain and pain caused by chemotherapy drugs, and can be used for preparing the treatment drugs for the chronic pain.

4. The chicken egg white lysozyme causes the expression of a mechanical sensitivity ion channel TACAN in a PRKN/TACAN passage on a cell membrane to be reduced by up-regulating the expression of PRKN protein of a primary sensory neuron in dorsal root ganglion, thereby obviously inhibiting static mechanical trigger induced pain.

Drawings

FIG. 1 is a diagram of the modeling method and design process of a mouse.

And (3) performing administration intervention 7 days before the molding, wherein the administration mode is intragastric administration, the dose is 400mg/Kg, and the administration is performed once a day until the behavioural test is finished after the molding is started. The inflammatory pain model (CFA model) was administered for a total of 13 days and behavioural tests were performed on the first, third and fifth days of the model. Neuropathic pain (SNL model) was administered for 18 days and behavioural tests were performed on days four, seven and ten of the model. Chemotherapy pain (VCR model) was administered for 18 days and behavioural tests were performed on days four, seven and ten of the model. The behavioral test is mechanical threshold determination, and the mechanical foot withdrawal threshold of the mouse is measured by using an up-down method, which is briefly described as follows: the method comprises the following steps of putting a mouse into an organic glass quilt cover, placing the mouse on a 10 x 10 mm wire mesh grid plate, stimulating the position in the rear sole of the mouse by using Von Frey wires with gradually increased strength for 6-8 s at the stimulation interval of 5 min, judging the mouse to have positive reaction when the mouse withdraws the foot or licks the paw immediately in the testing time or when the mouse removes fibers, and counting the foot lifting reaction caused by physical activity as the positive reaction. The upper limit of the test times of each mouse is 5 times, the mouse is considered to be sensitive under the stimulation intensity if the positive reaction is more than or equal to 3 times in 5 times, the mouse is considered to be insensitive if the negative reaction is more than or equal to 3 times, and the lowest sensitive stimulation intensity of the mouse is recorded as the mechanical pain threshold of the mouse.

FIG. 2 is a graph of the relief effect of HEL on inflammatory pain.

The concrete molding method of the CFA model comprises the following steps: 2% isoflurane was inhaled to anesthetized mice, and after sterilization with 0.5% iodophor, 20 μ l of Complete Freund's Adjuvant (Complete, CFA) was injected into the center of the posterior plantar aspect with a 1 ml syringe, and molding was successful due to plantar swelling for subsequent experiments.

The abscissa of the graph represents the time day for the behavioural test after moulding, and the ordinate represents the threshold g causing mechanical sensitivity. The triangle is HEL treatment group, feeding 400mg/Kg chicken protein lysozyme for 13 consecutive days for intragastric administration, and performing CFA modeling on the unilateral hind paw, the square is CFA model group, the saline water with the same volume of intragastric administration and the CFA modeling are performed on the unilateral hind paw, the circle is control group, and the saline water with the same volume of intragastric administration are administered. Behavioral test data were analyzed using the two-way expected measurers ANOVA fallen by Bonferroni post hoc test method,P< 0.05 was considered statistically significant.

FIG. 3 is a graph of the relief effect of HEL on neuropathic pain.

The specific modeling method of the SNL model comprises the following steps: 2% isoflurane was inhaled to anesthetize mice, the shaver shaved, sterilized with 0.5% iodophor to prepare skin, the skin was incised at the left side of the spinal cord stage of L4-S1, a 1-2 cm surgical incision was opened, L5 and L6 nerves were found, the nerves were ligated with a number 4-0 silk thread, the muscles and skin were sutured layer by layer, and molding was completed for subsequent experiments.

The abscissa of the graph represents the time day for the behavioural test after moulding, and the ordinate represents the threshold g causing mechanical sensitivity. The triangle is HEL treatment group, the stomach is perfused with 400mg/Kg of chicken egg white lysozyme for 18 days continuously, the SNL molding is carried out on one side, the square is SNL model group, the saline water with the same volume of the stomach is perfused and the SNL molding is carried out on one side, the circle is control group, and the saline water with the same volume of the stomach is perfused. Behavioral test data were analyzed using the two-way expected measurers ANOVA fallen by Bonferroni post hoc test method,P< 0.05 was considered statistically significant.

FIG. 4 is a graph of the pain relief effect of HEL on chemotherapeutic agents.

The specific model making method of the VCR model comprises the following steps: appropriate amount of vincristine was dissolved in physiological saline to make the concentration 0.1 mg/kg, and was injected into the abdominal cavity continuously for 7 days to establish a model of chemotherapy pain, which was used in the subsequent experiments.

The abscissa of the graph represents the time day for the behavioural test after moulding, and the ordinate represents the threshold g causing mechanical sensitivity. Triangle is HEL treatment group, continuous 18 days give 400mg/Kg chicken protein lysozyme intragastric, and carry on VCR model, the square is VCR model group, intragastric equal volume saline and carry on VCR model alone, the circle is contrast group, intragastric equal volume saline. Behavioral test data were analyzed using the two-way expected measurers ANOVA fallen by Bonferroni post hoc test method,P< 0.05 was considered statistically significant.

FIG. 5 is a diagram of RNA-seq screening HEL differential expression genes for relieving inflammatory pain.

The graphical representation shows the sequencing results of the transcriptome after modeling, and the CFA model was obtained on ice on day 5 after modeling according to the scheme shown in FIG. 1. Each group of DRG was loaded in enzyme-free EP tubes and sent to Hangzhou Union Biotechnology GmbH for RNA-seq. The gene heatmap is shown, and the control group, the CFA model group and the HEL treatment group are sequentially arranged from left to right, and the group difference change of 7 genes is arranged from top to bottom. That is, after CFA molding, 7 genes were down-regulated in the CFA group, and after HEL dry-up, the levels of the genes in the CFA + HEL group increased. The lighter the color indicates a change in level, the lighter the color indicates a greater difference.

FIG. 6 is a graph of variation in differentially expressed genes detected by qPCR.

In the embodiment shown in FIG. 1, the material was taken on ice 5 days after CFA molding. Each set of DRGs was loaded in enzyme-free EP tubes for conventional qPCR. qPCR results are shown in the figure, with abscissa representing the group-specific differential genes, circle as control, square as CFA model (CFA + Veh), triangle as HEL treatment (CFA + HEL), and ordinate representing mRNA expression level. Data analysis was performed using One-way ANOVA closed by Tukey's multiple complexes test methodP < 0.05，**P < 0.01，***P < 0.001。

FIG. 7 is a graph showing the amount of protein level changes in each component of TACAN following Western Blotting to detect PRKN knockdown and HEL intervention.

The graph shows the protein level changes of TACAN in each fraction following intrathecal injection of PRKN-siRNA, and the dry gastric lavage of HEL. FIGS. 7A-C show the relative expression of TACAN in DRG tissue membrane protein, cytosolic protein and total protein, respectively, with protein WB band above the statistical chart, TACAN expression above the statistical chart, and reference protein expression below the statistical chart, wherein TfR is the internal reference of membrane protein, and β -actin is the internal reference of cytosolic protein and total protein. The statistical chart is statistics of the relative expression level of TACAN, the abscissa represents different groups, the circle represents a control group (control), the square represents a knockdown group (PRKN-siRNA), the triangle represents a treatment group (PRKN-siRNA + HEL), and the ordinate represents the relative expression level of TACAN. Data analysis was performed using the One-way ANOVA closed by Tukey's multiple complexes test method,P< 0.05 was considered statistically significant.

FIG. 8 is a graph of HEL modulating static mechanical allodynia via the PRKN/TACAN pathway.

The abscissa represents the group, the circle represents the control group (control), the square represents the over-expression group (TACAN-over), the triangle represents the treatment group (TACAN-over + HEL), the diamond represents the re-intervention group (TACAN-over + HEL + PRKN-siRNA), and the ordinate represents the mechanical threshold size g of each group. Behavioral test data were analyzed using the two-way replicated measures ANOVA fallen by Bonferroni post hoc test method,P< 0.05 was considered statistically significant.

FIG. 9 is a graph of the change in TACAN in membrane proteins following TACAN overexpression virus, HEL, PRKN-siRNA intervention.

The upper part of the statistical chart is a protein WB strip, the upper part is the expression quantity of TACAN, and the lower part is the expression quantity of TfR internal reference protein. The statistical chart is statistics of the relative TfR expression of TACAN, the abscissa represents each group, the circle represents a control group (control), the square represents an overexpression group (TACAN-over), the triangle represents a treatment group (TACAN-over + HEL), the diamond represents a re-intervention group (TACAN-over + HEL + PRKN-siRNA), and the ordinate represents the relative expression of TACAN. Data analysis was performed using the One-way ANOVA closed by Tukey's multiple complexes test method,P< 0.05 was considered statistically significant.

Detailed Description

The invention is further described below with reference to the figures and examples.

In the following examples, all the components are contained by mass% unless otherwise specified.

Example 1 study of the relief Effect of HEL on inflammatory pain

1.1 materials and reagents:

hen egg white lysozyme, Sigma, cat # 62971-10g-F, was diluted with 0.9% physiological saline and administered at a dose of 400 mg/Kg. Complete Freund's Adjuvant (CFA), Sigma Inc., cat # F5881-10 ML.

1.2 animals

C57BL/6 mice, male, 6-8 weeks, were supplied by the laboratory animal center, university of Zhongshan. The animals are randomly distributed, raised in cages, freely eaten and drunk water, the indoor temperature is controlled to be 24 +/-1 ℃, the indoor relative humidity is 50-60%, and the circulating illumination is carried out for 12 hours in the day and 12 hours in the night according to the biological rhythm of the animals. The experimental process is operated according to the national relevant animal experiment regulations, the pain of experimental animals is reduced as much as possible, and the mice are used for formal experiments after being adapted to a behavioristics testing laboratory for 3 days.

1.3 methods

15 mice were randomly divided by body weight into a control group (control), a CFA model group (CFA + veh), and an HEL-treated group (CFA + HEL), and the experiment was divided into 3 groups of 5 mice each. As shown in figure 1, each group of mice performed the corresponding intervention as follows:

1) the HEL treatment group starts to administrate 400mg/Kg HEL solution into the stomach every day 7 days in advance, 20 mul of complete Freund's adjuvant is injected into the left hind paw after 7 days, and then the administration of 400mg/Kg HEL solution into the stomach is continued;

2) injecting 20 mul of complete Freund adjuvant into the left hind paw of the CFA model group, and perfusing normal saline with the same volume as the stomach;

3) the control group was gavaged with saline of the same volume.

And (3) performing static mechanical threshold measurement on days 1, 3 and 5 after the CFA model is molded, wherein the specific measurement method comprises the following steps: the mechanical foot withdrawal threshold of the mouse is measured by using an up-down method, the mouse is placed in an organic glass quilt cover and is placed on a 10 x 10 mm wire mesh grid plate, the position in the hindpaw of the mouse is stimulated by using Von Frey wires with increasing intensity for 6-8 s, the stimulation interval is 5 min, the foot withdrawal or paw licking reaction of the mouse immediately appears in the testing time or when the fibers are removed is regarded as positive reaction, the foot lifting reaction caused by physical activity is not counted as positive reaction, and the threshold reflects the chronic pain condition caused by inflammation. The upper limit of the test times of each mouse is 5 times, if the positive reaction is more than or equal to 3 times in 5 times, the mouse is considered to be sensitive under the stimulation intensity, if the negative reaction is more than or equal to 3 times, the mouse is considered to be insensitive, and the lowest sensitive stimulation intensity of the mouse is recorded as the mechanical pain threshold of the mouse.

1.4 results

The mechanical thresholds of the mice in different time periods of each group are shown in fig. 2, and the results show that the mechanical thresholds of the mice are improved in 1 st, 3 rd and 5 th days after HEL is given under the CFA model, and the statistical significance is achievedP＜0.001，**P＜0.01，***PLess than 0.05, indicating that HEL has obvious analgesic effect on inflammatory pain.

Example 2 study of the relief Effect of HEL on neuropathic pain

2.1 materials and reagents:

hen egg white lysozyme, Sigma, cat # 62971-10g-F, was diluted with 0.9% physiological saline and administered at a dose of 400 mg/Kg.

2.2 animals

C57BL/6 mice, male, 6-8 weeks, were supplied by the laboratory animal center, university of Zhongshan. The animals are randomly distributed, raised in cages, freely eaten and drunk water, the indoor temperature is controlled to be 24 +/-1 ℃, the indoor relative humidity is 50-60%, and the circulating illumination is carried out for 12 hours in the day and 12 hours in the night according to the biological rhythm of the animals. The experimental process is operated according to the national relevant animal experiment regulations, the pain of experimental animals is reduced as much as possible, and the mice are used for formal experiments after being adapted to a behavioristics testing laboratory for 3 days.

2.3 method

15 mice were randomly divided into a control group (control), an SNL model group (SNL + veh), and an HEL treatment group (SNL + HEL) by body weight, and the experiment was divided into 3 groups in total, each group consisting of 5 mice. As shown in figure 1, each group of mice performed the corresponding intervention as follows:

1) the HEL treatment group starts to administrate 400mg/Kg HEL solution to the stomach every day 7 days in advance, ligates the left L5/L6 nerve 7 days later, and continues to administrate 400mg/Kg HEL solution to the stomach every day later;

2) ligating the left L5/L6 nerve of the SNL model group, and perfusing the stomach with normal saline with the same volume;

3) the control group was gavaged with saline of the same volume.

And (3) performing behavioral determination on days 4, 7 and 10 after the SNL model is molded, wherein the specific determination method comprises the following steps: the mechanical foot withdrawal threshold of the mouse is measured by using an up-down method, the mouse is placed in an organic glass quilt cover and is placed on a 10 x 10 mm wire mesh grid plate, the position in the hindpaw of the mouse is stimulated by using Von Frey wires with increasing intensity for 6-8 s, the stimulation interval is 5 min, the foot withdrawal or paw licking reaction of the mouse immediately appears in the testing time or when the fibers are removed is regarded as positive reaction, the foot lifting reaction caused by physical activity is not counted as positive reaction, and the threshold reflects the chronic pain condition caused by nerve injury. The upper limit of the test times of each mouse is 5 times, the mouse is considered to be sensitive under the stimulation intensity if the positive reaction is more than or equal to 3 times in 5 times, the mouse is considered to be insensitive if the negative reaction is more than or equal to 3 times, and the lowest sensitive stimulation intensity of the mouse is recorded as the mechanical pain threshold of the mouse.

2.4 results

The mechanical thresholds of the mice in different time periods of each group are shown in fig. 3, and the results show that the mechanical thresholds of the mice are improved in 4 th, 7 th and 10 th days after HEL is given under the SNL model, and the statistical significance is achievedP＜0.001，**P＜0.01，***PLess than 0.05, indicating that the HEL has obvious analgesic effect on neuropathic pain.

Example 3 study of the relief Effect of HEL on pain caused by chemotherapeutic Agents

3.1 materials and reagents:

hen egg white lysozyme, Sigma, cat # 62971-10g-F, was diluted with 0.9% physiological saline and administered at a dose of 400 mg/Kg. Vincristine (VCR) is available from Shenzhen Wanle pharmaceutical Co., Ltd, product lot number 2011V 2.

3.2 animals

C57BL/6 mice, male, 6-8 weeks, were supplied by the laboratory animal center, university of Zhongshan. The animals are randomly distributed, raised in cages, freely eaten and drunk water, the indoor temperature is controlled to be 24 +/-1 ℃, the indoor relative humidity is 50-60%, and the circulating illumination is carried out for 12 hours in the day and 12 hours in the night according to the biological rhythm of the animals. The experimental process is operated according to the national relevant animal experiment regulations, the pain of experimental animals is reduced as much as possible, and the mice are used for formal experiments after being adapted to a behavioristics testing laboratory for 3 days.

3.3 methods

15 mice were randomly divided by weight into a control group (control), a VCR model group (VCR + veh), and an HEL-treated group (VCR + HEL), and the experiment was divided into 3 groups in total, each group consisting of 5 mice. As shown in figure 1, each group of mice performed the corresponding intervention as follows:

1) the HEL treatment group was injected intraperitoneally with 0.1 mg/Kg VCR solution for 7 days and was intragastrically administered with 400mg/Kg chicken egg white lysozyme solution daily;

2) the VCR model group is injected with 0.1 mg/Kg VCR solution in the abdominal cavity and is perfused with normal saline with the same volume;

3) the control group was gavaged with saline of the same volume.

And (3) performing behavioral determination on days 4, 7 and 10 after the model of the VCR model is molded, wherein the specific determination method comprises the following steps: the mechanical foot withdrawal threshold of the mouse is measured by using an up-down method, the mouse is placed in an organic glass quilt cover and is placed on a 10 x 10 mm wire mesh grid plate, the position in the hindpaw of the mouse is stimulated by using Von Frey wires with increasing intensity for 6-8 s, the stimulation interval is 5 min, the foot withdrawal or paw licking reaction of the mouse immediately appears in the testing time or when the fibers are removed is regarded as positive reaction, the foot lifting reaction caused by physical activity is not counted as positive reaction, and the threshold reflects the chronic pain condition caused by chemotherapeutic drugs. The upper limit of the test times of each mouse is 5 times, the mouse is considered to be sensitive under the stimulation intensity if the positive reaction is more than or equal to 3 times in 5 times, the mouse is considered to be insensitive if the negative reaction is more than or equal to 3 times, and the lowest sensitive stimulation intensity of the mouse is recorded as the mechanical pain threshold of the mouse.

3.4 results

The mechanical thresholds of the mice in each group at different times are shown in fig. 4, and the results show that in the VCR model, the mechanical thresholds of the mice were increased in days 4, 7 and 10 after HEL administration, which is statistically significantP＜0.001，**P＜0.01，***PLess than 0.05, indicating that the HEL has obvious analgesic effect on chronic pain caused by the chemotherapeutic drugs.

Example 4 HEL Chronic pain-relieving action target screening

4.1 materials and reagents:

hen egg white lysozyme, Sigma, cat # 62971-10g-F, was diluted with 0.9% physiological saline and administered at a dose of 400 mg/Kg. Complete Freund's adjuvant, Sigma Co., Cat No. F5881-10 ML.

4.2 animals

C57BL/6 mice, male, 6-8 weeks, were supplied by the laboratory animal center, university of Zhongshan. The animals are randomly distributed, raised in cages, freely eaten and drunk water, the indoor temperature is controlled to be 24 +/-1 ℃, the indoor relative humidity is 50-60%, and the circulating illumination is carried out for 12 hours in the day and 12 hours in the night according to the biological rhythm of the animals. The experimental process is operated according to the national relevant animal experiment regulations, the pain of experimental animals is reduced as much as possible, and the mice are used for formal experiments after being adapted to a behavioristics testing laboratory for 3 days.

4.3 methods

36 mice were randomly divided by body weight into a control group (control), a CFA model group (CFA + veh), and an HEL-treated group (CFA + HEL), and the experiment was divided into 3 groups of 12 mice each. Each group of mice performed the corresponding intervention as follows:

1) the HEL treatment group starts to administer 400mg/Kg of chicken egg white lysozyme solution for intragastric administration every day 7 days in advance, 20 mu l of complete Freund's adjuvant is injected into the left hind paw after 7 days, and then 400mg/Kg of chicken egg white lysozyme solution is continuously administered for intragastric administration;

2) injecting 20 mul of complete Freund adjuvant into the left hind paw of the CFA model group, and perfusing normal saline with the same volume as the stomach;

3) the control group was gavaged with saline of the same volume.

On day 5 after modeling of the CFA model, mouse DRG tissues were dissected and mounted in enzyme-free EP tubes for RNA-seq and q-PCR quantitative analysis, respectively.

4.4 results

To further analyze how HEL reduces static mechanical trigger-induced pain, we performed RNA-seq analysis to screen for differential gene expression in DRG tissues. RNA-seq analysis As shown in FIG. 5, there were 7 genes differentially expressed, with the CFA model group genes down-regulated and the CFA + HEL group genes up-regulated. To confirm the changes in these 7 genes, we further performed q-PCR analysis. As shown in the data in fig. 6, 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 allodynia.

Example 5 study of the potential molecular mechanisms of PRKN in HEL inhibition of static mechanical allodynia

5.1 materials and reagents:

hen egg white lysozyme, Sigma, cat # 62971-10g-F, was diluted with 0.9% physiological saline and administered at a dose of 400 mg/Kg. TARGETplus Mouse Park2 siRNA, Dharmacon, cat # L-065413-01-0010. BCA protein quantification kit, Thermo, cat # 23227. Minute TM plasma membrane protein and cell fraction isolation kit, Invent/English, cat # SM-005-50T. 10% TGX Rapid gel preparation kit, Bio-rad, cat # 1610183. Anti-TMEM120A antibodyy, Bioss Inc., cat # bs-19952R. Beta-actin, abcam, cat # ab 8226. BSA, sigma, cat # B2064-100G. Ultrasensitive luminescence assay kit, yazyme, cat # 01592050. Uratan, McLin, cat # U820333-100 g. RIPA lysate, pecan corporation, cat # P0013B.

5.2 instruments and devices

Gel electrophoresis electrotransfer system, brand model Bio-RAD 1658033; ultrasonic cytoclasis instrument, model number SONICS VCX 130; a microplate reader, brand model Bio-Rad iMark; a full-automatic chemiluminescence image analysis system, brand model Tanon-5200; ultrarefrigerated centrifuge, brand name Thermo fresh 021.

5.3 animals

C57BL/6 mice, male, 6-8 weeks, were supplied by the laboratory animal center, university of Zhongshan. The animals are randomly distributed, raised in cages, freely eaten and drunk water, the indoor temperature is controlled to be 24 +/-1 ℃, the indoor relative humidity is 50-60%, and the circulating illumination is carried out for 12 hours in the day and 12 hours in the night according to the biological rhythm of the animals. The experimental process is operated according to the national relevant animal experiment regulations, the pain of experimental animals is reduced as much as possible, and the mice are used for formal experiments after being adapted to a behavioristics testing laboratory for 3 days.

5.4 methods

18 mice were randomly divided into a control group (control), a knockdown group (PRKN-siRNA + veh), and a treatment group (PRKN-siRNA + HEL) by body weight, and the experiment was divided into 3 groups in total, each group consisting of 6 mice. Each group of mice performed the corresponding intervention as follows:

1) the treatment group starts to administrate 400mg/Kg HEL solution to the stomach every day 7 days in advance, 10 mu l PRKN-siRNA is injected in a sheath after 7 days, and then the administration of 400mg/Kg HEL solution to the stomach is continued;

2) injecting 10 mul PRKN-siRNA into the knocking-down group intrathecally, and perfusing normal saline with the same volume as the stomach;

3) the control group was gavaged with saline of the same volume.

Mouse DRG tissues were dissected and dissolved in cold RIPA buffer 48h after intrathecal injection of 10. mu.l PRKN-siRNA. After the proteins of each component are separated by gel electrophoresis, the PVDF membrane is incubated with TACAN (Rabbit, 1:1000, Novus), TfR (Mouse,1:1000, Abcam) and beta-actin (Mouse,1: 2000, Affinity) primary antibodies at 4 ℃ overnight. And (3) incubating the next day with a secondary antibody Rabbit, 1:10000 and Mouse,1: 10000 for 1h at normal temperature, and detecting the immune complex.

Intrathecal injection of PRKN-siRNA method: the synthesized PRKN-siRNA is centrifuged, the powder is centrifuged to the bottom of the tube, 20 mu l of 5% glucose solution is added into every 5 nmol of siRNA, 1 mu g/mu l of Rvg-9R solution is added according to the proportion of 2.5 mu g of PRKN-siRNA to 7.5 mu g of Rvg-9R, the mixture is mixed evenly, and the mixture is incubated for 10-15 min at room temperature. The experimental mouse is inhaled with isoflurane of 2 percent for anesthesia, after skin preparation and disinfection, the L6 spinous process is exposed, the lower cavity of arachnoid with the gap of L5/L6 is punctured by a needle, the tail is whipped to mark the needle to enter the correct space, the needle is pulled out, a section of PE-10 tube of about 15cm is clamped by a pair of bending forceps and is slowly inserted along a small hole punctured by the needle, the PR-10 tube is slowly pushed forward by about 1 cm, if cerebrospinal fluid flows out in the PE-10 tube, the tube placement is indicated to be correct, a 20 mu L micro-injector is used for sucking 10 mu L of prepared PRKN-siRNA solution, the PRKN-siRNA solution is pushed into the PE-10 tube and is injected into a sheath, the PRKN-siRNA solution stays for 10 minutes, the PE-10 tube is pulled out, hemostasis is realized, muscles and skin are sutured layer by layer, and the experimental mouse is fed under the normal condition after the operation, and can be used for the subsequent experiment after 48 hours.

Werstern blob method:

1) reagent preparation

Glue dispensing system (Bio-Rad)

Separating glue: a Bio-Rad dispensing system, 3.5 ml of solution A, 3.5 ml of solution B, 35. mu.l of 10% APS, 3.5. mu.l of TEMED was used.

Concentrating the glue: using a Bio-Rad gel dispensing system, 1.5 ml of solution A, 1.5 ml of solution B, 15. mu.l of 10% APS, 3. mu.l of TEMED were dispensed.

1 × electrophoresis buffer: 3.03g of Tris Base, 14.4 g of glycine and 1 g of SDS are weighed respectively, and deionized water is added to the mixture to make the volume of the mixture reach 1L.

1 × transfer membrane buffer: 3.03g of Tris Base and 14.4 g of glycine are weighed respectively, 200 ml of methanol is added, and deionized water is added to the solution until the volume is 1L.

1 × TBST buffer: weighing 8.8g NaCl, respectively weighing 2.35 ml 20% Tween 20 solution and 10ml 1M Tris.HCl solution with pH =7.5, and adding deionized water to the solution to make the volume of the solution constant to 1L.

WB confining liquid: skimmed milk powder or BSA 0.5g was dissolved in 10ml TBST solution.

WB primary anti-diluent: BSA 0.5g was dissolved in 10ml TBST solution and primary antibody (TACAN, Bioss, Rabbit, 1:1000; β -actin, Abcam, Mouse,1: 2000; TfR, Abcam, Mouse,1: 1000) was added according to the antibody instructions.

WB secondary antibody diluent: 0.5G of skimmed milk powder or BSA is dissolved in 10ml of TBST solution, and a secondary antibody (G-M, KPL, 074-1506, 1:10000; G-Rb, KRL, 074-1506, 1:10000) corresponding to a primary antibody source is added and mixed uniformly for later use.

2) Tissue protein extraction

And (3) total protein extraction: anesthetizing the mouse with 10% urethane (0.3-0.4 ml), perfusing the mouse with 0.01M PBS, quickly taking out the mouse DRG tissue on an ice bag, removing residual blood clots in cold PBS solution, adding a proper amount of RIPA lysate containing protease inhibitor, ultrasonically breaking on ice, and centrifuging at 14800 rpm at 4 ℃ for 20 min. Collecting supernatant, packaging, and storing at-80 deg.C.

Extracting membrane protein and cytoplasmic protein: membrane proteins were extracted according to standard procedures of the membrane protein extraction kit. Anaesthetizing a mouse with 10% urethane (0.3-0.4 ml), perfusing the mouse with 0.01M PBS, quickly taking out mouse DRG tissue on an ice bag, adding 200 mu l A liquid, grinding for 1 min by a plastic rod, adding 300 mu l A liquid, culturing on ice for 10 min, centrifuging at 14000 rpm for 30 min in a 4 ℃ centrifuge, mixing uniformly by vortex, centrifuging at 3000 rpm for 1 min, taking supernatant in another EP tube, centrifuging at 14000 rpm for 30 min in the 4 ℃ centrifuge, sucking supernatant for preservation, taking cytoplasmic protein, adding 200 mu l B liquid, mixing uniformly by vortex, centrifuging at 10000 rpm for 5-10 min in the 4 ℃ centrifuge, transferring the supernatant to another new EP tube, adding 1.6 ml ice bath PBS, centrifuging at 14000 rpm for 30 min, discarding supernatant, leaving precipitate, taking membrane protein, adding a proper amount of RIPA lysate, subpackaging and preserving for later use.

3) BCA kit for determining protein concentration

According to the number of samples, according to the liquid A: liquid B = 50: 1, preparing a proper amount of BCA working solution. The standard protein of a series of concentration gradients was prepared according to the protocol of the kit instructions. The standard protein and the sample protein are added into a 96-well plate in sequence, 200 mul of BCA working solution is added into each well, the mixture is slowly shaken at 37 ℃ for 30 min, and the protein concentration is measured on a microplate reader.

4) Gel electrophoresis and membrane transfer

Adding the protein sample into the loading buffer solution according to the ratio of 4:1, and boiling for 6 min at 100 ℃. Adding protein into the gel hole, running for 30-40 min under 80V constant pressure, and running for a proper amount of time under 100V constant pressure according to the molecular weight of the protein. After electrophoresis, the gel and the PVDF membrane are placed in a membrane transferring clamp, and are transferred for a proper time by constant current of 300 mA according to the molecular weight of the protein.

5) Immune response

Blocking for 1h at room temperature with slow shaking, removing blocking solution, adding primary antibody, and incubating overnight at 4 deg.C. Primary antibody was recovered and the membrane was washed 3 times with TBST for 10 min each time. Then adding a secondary antibody, and after incubation for 1h with slow shaking at room temperature, washing the membrane for 3 times by TBST, 10 min each time.

6) Exposure method

And (3) taking the same amount of the luminous liquid A and the same amount of the luminous liquid B in a dark room, uniformly adding the luminous liquid on the film, shooting a strip by using a CCD camera, shooting a Marker under the white light condition after the clear strip is seen, and storing the picture. Images were analyzed using a Tanon GIS.

5.5 results

TACAN is involved in the transduction of mechanical pain signals as a mechanically sensitive ion channel, and conditional knockout of TACAN in peripheral sensory neurons can significantly improve the mechanical threshold of mice. Therefore, PRKN-siRNA is injected intrathecally to carry out HEL dry prognosis, and the expression change of each component protein TACAN is detected. Western blotting results showed that PRKN-siRNA increased the membrane expression of TACAN but decreased its cytoplasmic accumulation in DRG tissues compared to the control group, as shown in FIGS. 7A-B, and that TACAN expression on cell membranes decreased and cytoplasmic accumulation increased after HEL intervention, returning to levels comparable to those in the normal group. Also, as shown in fig. 7C, PRKN-siRNA did not alter total TACAN expression in DRG neurons, nor did he affect total TACAN expression changes after HEL intervention. The above data indicate that HEL increases transport of TACAN membranes by down-regulating PRKN, thereby alleviating static mechanical allodynia.

Example 6 HEL study to alleviate static mechanical allodynia via the PRKN/TACAN pathway

6.1 materials and reagents:

hen egg white lysozyme, Sigma, cat # 62971-10g-F, diluted in 0.9% saline, was administered at a dose of 400 mg/Kg. TARGETplus Mouse Park2 siRNA, Dharmacon, cat # L-065413-01-0010. TACAN overexpression Virus Raav-hSyn-TMEM120A-2A EGFP-WPRE-PA, Kingmi, Cat No. PT 4858.

6.1 animals

C57BL/6 mice, male, 6-8 weeks, were supplied by the laboratory animal center, university of Zhongshan. The animals are randomly distributed, raised in cages, freely eaten and drunk water, the indoor temperature is controlled to be 24 +/-1 ℃, the indoor relative humidity is 50-60%, and the circulating illumination is carried out for 12 hours in the day and 12 hours in the night according to the biological rhythm of the animals. The experimental process is operated according to the national relevant animal experiment regulations, the pain of experimental animals is reduced as much as possible, and the mice are used for formal experiments after being adapted to a behavioristics testing laboratory for 3 days.

6.2 methods

24 mice were randomly divided into a control group (control), an overexpression group (TACAN-over + veh), a treatment group (TACAN-over + HEL) and an intervention group (TACAN-over + HEL + PRKN-siRNA) according to body weight, and the experiment was divided into 4 groups in total, and each group had 6 mice. Each group of mice performed the corresponding intervention as follows:

1) and injecting TACAN overexpression virus in the intervention group intrathecally, continuously performing intragastric lavage on HEL every day 21 days after infection, performing intragastric injection on PRKN-siRNA 10 microliter after 7 days, performing behavioral testing and material taking 48 hours later, and continuously performing intragastric lavage on HEL until the day of material taking.

2) Injecting TACAN over-expression virus into the treatment group intrathecally, and continuously irrigating stomach HEL every day after 21 days of infection;

3) injecting TACAN over-expression virus into the over-expression group intrathecally, and irrigating normal saline with the same volume as the stomach 21 days after infection;

4) the control group was gavaged with saline of the same volume.

And (3) conducting behavioral testing 48h after 10 mu l of PRKN-siRNA is injected in the sheath, wherein the specific determination method comprises the following steps: the mechanical foot withdrawal threshold of the mouse is measured by using an up-down method, the mouse is placed in an organic glass quilt cover and is placed on a 10 x 10 mm wire mesh grid plate, the position in the hindpaw of the mouse is stimulated by using Von Frey wires with increasing intensity for 6-8 s, the stimulation interval is 5 min, the foot withdrawal or paw licking reaction of the mouse immediately appears in the testing time or when the fibers are removed is regarded as positive reaction, the foot lifting reaction caused by physical activity is not counted as positive reaction, and the threshold reflects the chronic pain condition caused by chemotherapeutic drugs. After the behavioral testing was completed, mouse DRG tissues were dissected and dissolved in cold RIPA buffer. After the sample is separated by gel electrophoresis, the PVDF membrane is incubated with primary anti-TACAN (Rabbit, 1:1000, Novus), TfR (Mouse,1:1000, Abcam) and beta-actin (Mouse,1: 2000, Affinity) at 4 ℃ overnight, and incubated with corresponding secondary antibodies the next day to detect the immune complex. The Western Blot procedure and the detailed method for intrathecal siRNA injection are as described in 5.4.

Intrathecal AAV injection methods: the purchased AAV was diluted to a suitable titer with physiological saline and mixed for use. The experimental mouse is inhaled with 2% isoflurane for anesthesia, after skin preparation and disinfection, the L6 spinous process is exposed, the lower cavity of arachnoid in the gap of L5/L6 is punctured with a needle, the tail is swung to mark that the needle enters correctly, the needle is pulled out, a section of PE-10 tube with the length of about 15cm is clamped by a pair of forceps and is slowly inserted along a small hole punctured by the needle, the PE-10 tube is slowly pushed forwards by about 1 cm, if cerebrospinal fluid flows out from the PE-10 tube, the tube is placed correctly, 10 mu L of prepared TACCAN-AAV solution is sucked by a 20 mu L injector, the prepared TACCAN-AAV solution is pushed into the PE-10 tube and is injected into a sheath, the PE-10 tube is stayed for 10 minutes, the PE-10 tube is pulled out, hemostasis is performed, muscles and skin are sutured layer by layer, the experimental mouse is fed under the conventional postoperative condition, and after 21 days, the experimental mouse can be used for the subsequent experiment.

6.3 results

The behavioral data show that the static mechanical allodynia was alleviated by HEL pre-treatment of TACAN-overexpressing mice and that PRKN-siRNA re-induced static mechanical allodynia after feeding HEL-overexpressing TACAN mice, as shown in FIG. 8. WB results also showed that HEL was able to reduce the increase in TACAN membrane expression in DRG tissues and PRKN-siRNA reversed TACAN membrane expression again, as shown in figure 9. Taken together, these data indicate that the PRKN/TACAN pathway is selectively involved in the HEL-mediated process of static mechanical allodynia.

Claims (10)

1. An application of hen egg white lysozyme in preparing the medicine for preventing, treating or relieving chronic pain is disclosed.

2. Use according to claim 1, characterized in that: the chronic pain is static mechanical allodynia.

3. Use according to claim 1, characterized in that: the chronic pain includes inflammatory pain, neuropathic pain or pain caused by chemotherapeutic drugs.

4. Use according to any one of claims 1 to 3, characterized in that: in the medicine for preventing, treating or relieving chronic pain in unit dose, the content of the hen egg white lysozyme is the effective amount for treatment.

5. Use according to any one of claims 1 to 3, characterized in that: the medicament is for use in the human or animal body.

6. Use according to claim 5, characterized in that: when the medicine is used for preventing, treating or relieving chronic pain of mice, the effective dosage of the hen egg white lysozyme is 400mg/Kg of mice each time.

7. A medicament for the prevention, treatment or alleviation of chronic pain, characterized by: the effective component comprises hen egg white lysozyme.

8. The medicament as claimed in claim 7, wherein: in the unit dose medicine, the content of the hen egg white lysozyme is effective amount for treatment.

9. The medicament of claim 7, wherein: the medicine is used for mice, and the dosage of the hen egg white lysozyme is 400mg/Kg of mice each time.

10. The medicament according to claim 7 or 8, characterized in that: the chronic pain comprises inflammatory pain, neuropathic pain or pain caused by chemotherapeutic drugs; the medicament for preventing, treating or relieving chronic pain is in the dosage form of tablets, capsules, granules, oral liquid, dripping pills, aerosol or injection.

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