CN115381950B

CN115381950B - Application of Rab35 or downstream regulatory molecules thereof in treating chronic pain after fracture

Info

Publication number: CN115381950B
Application number: CN202211081932.4A
Authority: CN
Inventors: 王春艳; 徐如彬; 张麟临; 李依泽; 于泳浩; 王国林; 谢克亮
Original assignee: Tianjin Medical University General Hospital
Current assignee: Tianjin Medical University General Hospital
Priority date: 2022-09-06
Filing date: 2022-09-06
Publication date: 2023-08-18
Anticipated expiration: 2042-09-06
Also published as: CN115381950A

Abstract

The invention discloses application of Rab35 or a downstream regulatory molecule thereof in treating chronic pain after fracture. Experiments prove that the expression of the spinal cord Rab35 and the Secretogranin II of the mice with chronic pain after fracture is increased, mechanical pain and thermal pain caused by the fracture of the mice are relieved by injecting Rab35 Morpholino, and mechanical pain and thermal pain caused by the fracture of the mice are relieved by Rab35 through the Secretogranin II. Based on the above research, the present invention provides a novel method for treating chronic pain after fracture.

Description

Application of Rab35 or downstream regulatory molecules thereof in treating chronic pain after fracture

Technical Field

The invention belongs to the field of biological medicine, and relates to application of Rab35 or a downstream regulatory molecule thereof in treating chronic pain after fracture.

Background

With the rapid development of industries such as China industry, construction, traffic and the like and the aging of population, the occurrence of industrial accidents, building injuries, traffic injuries and senile osteoporosis fracture is increased. The number of traumatic fracture in China per year is counted to be over 440 ten thousand, and the number of osteoporosis fracture patients per year is about 900 ten thousand worldwide. The chronic pain after fracture is a chronic pain state after fracture, and has serious symptoms and high incidence rate, and no effective treatment measures exist. The incidence of chronic pain after ankle and wrist fractures is reported to be as high as 61.7%, and the incidence of chronic pain after tibial fractures is 55.1%. Serious pain not only prevents early rehabilitation and skeletal muscle functional recovery of patients, but also increases risks of cardiovascular accidents, depression, acute Post-traumatic cognitive dysfunction (Post-traumatic cognitive dysfunction) and chronic complex regional pain syndrome (Complex regional pain syndrome) of patients, seriously influences the life quality of the patients, aggravates the household and socioeconomic burden of the patients, and becomes an important public health problem to be solved urgently. The occurrence mechanism of chronic pain after fracture is not clear, and the traditional Chinese medicine composition is one of research hotspots in the current pain field.

Studies show that Rab GTPases are used as molecular switches, are converted between an active form of GTP binding and an inactive form of GDP binding, can regulate endocytosis and axon transport of neuron cells, and are involved in pathological processes of various nerve diseases, such as Alzheimer's Disease (AD), parkinson's Disease (PD) and other diseases, but are not studied in chronic pain after fracture. The research is to study the effect of the Rab GTPase family protein Rab35 on regulating and controlling the membrane vesicle transportation in chronic pain after fracture through the means of vivo-Morpholino interference, behavioral determination and the like.

Disclosure of Invention

According to one aspect of the present invention there is provided a pharmaceutical composition comprising Rab35 or an inhibitor thereof downstream of the functional expression of the regulatory molecule.

Further, the inhibitors include agents that reduce the level of Rab35 or its downstream regulatory molecule mRNA or protein, or reduce the activity of Rab35 protein or its downstream regulatory molecule protein.

Further, the inhibitors include nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules; preferably, the inhibitor is a nucleic acid inhibitor.

Further, the nucleic acid inhibitor is selected from shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid, or constructs thereof.

Further, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

Further, the downstream regulatory molecule of Rab35 is secretoglanin II.

According to another aspect of the invention there is also provided the use of Rab35 or an inhibitor of its downstream regulatory molecule functional expression in the manufacture of a medicament for the prevention or treatment of chronic pain following fracture.

Further, the inhibitor comprises an agent that reduces the level of Rab35 or a downstream regulatory molecule thereof mRNA or protein, or reduces the activity of Rab35 protein or a downstream regulatory molecule thereof protein;

Further, the downstream regulatory molecule of Rab35 is secretoglanin II.

Drawings

Fig. 1 shows the results of spinal cord Rab35 and Secretogranin II expression in post-fracture chronic pain mice, wherein a: immunoblotting; b: histogram statistics. * P < 0.001 compared to S group;

figure 2 is a graph of results of Rab35 Morpholino injection in alleviating mechanical and thermal pain resulting from fracture in mice, wherein a: mechanical footstrike response threshold (MWF (%)); b: thermal foot latency (TWL); * P:<0 .001； ^&&& P<0 .001；

fig. 3 shows a graph of the secretoglanin II expression results, wherein a: immunoblotting; b: a histogram; * P:<0 .001； ^&&& P<0 .001。

Detailed Description

The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. The experimental procedure, without specific conditions noted in the examples, is generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring HarborLaboratory Press, 1989) or as recommended by the manufacturer.

The invention discloses a pharmaceutical composition, which comprises Rab35 or an inhibitor of downstream regulatory molecule functional expression thereof; by inhibitors is meant any substance that reduces the stability of the gene or expression product of Rab35 or its downstream regulatory molecule, down-regulates the expression of Rab35 or its downstream regulatory molecule, reduces the effective duration of action of Rab35 or its downstream regulatory molecule, or inhibits the transcription of the gene of Rab35 or its downstream regulatory molecule, which can be used in the present invention as a substance useful for down-regulating the expression of Rab35 or its downstream regulatory molecule and thus can be used in the treatment of chronic pain after fracture.

As an alternative of the invention, the inhibitor of Rab35 or a downstream regulatory molecule thereof is an antibody that specifically binds to Rab35 or a downstream regulatory molecule thereof. The specific antibody comprises a monoclonal antibody and a polyclonal antibody; the invention includes not only intact antibody molecules but also any fragments or modifications of antibodies, e.g., chimeric antibodies, scFv, fab, F (ab') 2, fv, etc. Provided that the fragment is capable of retaining the ability to bind to Rab35 or its downstream regulatory molecule protein. The preparation of antibodies for protein levels is well known to those skilled in the art and any method may be used in the present invention to prepare the antibodies.

As a preferred mode of the invention, the inhibitor of Rab35 or a downstream regulatory molecule thereof is a small interfering RNA molecule specific for Rab35 or a downstream regulatory molecule thereof. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule capable of degrading a specific mRNA targeting the mRNA of homologous complementary sequence, which is the RNA interference (RNA interference) process. The small interfering RNA can be prepared in the form of a double-stranded nucleic acid comprising a sense strand and an antisense strand, which form a double strand only under hybridization conditions. A double stranded RNA complex can be prepared from the sense strand and the antisense strand separated from each other. Thus, for example, the complementary sense and antisense strands are chemically synthesized, and can be subsequently hybridized by annealing to produce a synthetic double stranded RNA complex.

The nucleic acid inhibitors of the invention, such as siRNA, may be chemically synthesized or prepared by transcription of an expression cassette in a recombinant nucleic acid structure into single stranded RNA. Nucleic acid inhibitors such as siRNA can be delivered into cells by use of an appropriate transfection reagent, or can also be delivered into cells using a variety of techniques known in the art.

As an alternative to the present invention, the inhibitor of Rab35 or its downstream regulatory molecule may also be a "Small hairpin RNA (shRNA)", which is a non-coding Small RNA molecule capable of forming a hairpin structure, which is capable of inhibiting the expression of a gene by RNA interference pathway. As described above, shRNA may be expressed from a double stranded DNA template. The double stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then ligated to a promoter for expression in vitro or in vivo. shRNA can be cleaved into small interfering RNA molecules by the action of DICER enzyme in eukaryotic cells, thereby entering the RNAi pathway. "shRNA expression vector" refers to a number of plasmids conventionally used in the art to construct shRNA structures, typically having a "spacer" and multiple cloning sites or alternative sequences flanking the "spacer" such that one can insert the corresponding DNA sequence of the shRNA (or analog) into the multiple cloning site or alternative sequences thereon in a forward and reverse manner, and the RNA transcribed from the DNA sequence can form a shRNA (Short Hairpin) structure. The "shRNA expression vectors" are now fully commercially available, for example, as some viral vectors.

As a preferred embodiment of the invention, the inhibitor of Rab35 or a downstream regulatory molecule thereof is an antisense oligonucleotide. The antisense oligonucleotide has a sequence complementary to a target sequence, and inhibition of a target gene can be achieved by the complementary sequence, the antisense oligonucleotide being ribonucleic acid or DNA. As a preferred embodiment, the antisense oligonucleotide comprises at least one chemical modification. Antisense oligonucleotides can comprise one or more locked nucleic acids (LNAs, locked nucleicacids). Locked nucleic acids are modified ribonucleic acids that contain additional bridging bonds between the 2 'to 4' carbons of the ribose moiety to have a locked (locked) morphology, and thus oligonucleotides with locked nucleic acids have improved thermal stability. Alternatively, the antisense oligonucleotide may comprise a peptide nucleic acid (PNA, peptide nucleic acids) and the antisense oligonucleotide comprises a peptide-based backbone instead of a sugar-phosphate backbone. Other chemical modifications that antisense oligonucleotides can contain include: sugar modifications such as 2' -O-alkyl (e.g., 2' -O-methyl, 2' -O-methoxyethyl), 2' -fluoro, and 4' -thioxy modifications; backbone modifications such as phosphorothioate, morpholino, or phosphocarboxylate linkages. The antisense oligonucleotide is 7 to 50 nucleotides in length, preferably 10 to 40 nucleotides, more preferably 15 to 30 nucleotides, and most preferably 20 to 25 nucleotides.

In a specific embodiment of the invention, the inhibitor of Rab35 or its downstream regulatory molecule is a reagent used in the Morpholino technology after an improvement of the antisense oligonucleotide technology.

The pharmaceutical compositions of the present invention comprise a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carrier includes, but is not limited to, diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, disintegrants.

Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and its salts, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose and the like; surfactants such as polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride of stearic acid, cetyl alcohol, etc.; wetting agents such as glycerin, starch, and the like; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauryl saccharate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powdery), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrants such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH controlling agents, surfactants, and the like.

Wherein the stabilizer comprises human serum protein, L-amino acid, sugar and cellulose derivative. The L-amino acid may also include any one of glycine, cysteine and glutamic acid. Sugars include monosaccharides such as glucose, mannose, galactose, fructose, and the like; sugar alcohols such as mannitol, cellosolve, xylitol, and the like; disaccharides such as sucrose, maltose, lactose, and the like; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfide, hyaluronic acid, and the like, and derivatives thereof. Cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose and sodium hydroxymethyl cellulose. Surfactants include ionic or nonionic surfactants such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, fatty acid glycerides. The additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and the corresponding salts (their alkali or alkaline rare earth metal salts, such as sodium, potassium, calcium and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars and glycerol. The chelating agent comprises sodium ethylenediamine tetraacetate and citric acid.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or by an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical compositions of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients.

The dosage of the pharmaceutical composition of the present invention may be adjusted during the course of treatment according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

Example Rab35 use in chronic pain after fracture

1. Experimental procedure

(1) Experimental grouping: 18 healthy male C57BL6 mice, 2 months old, were offered by the laboratory animal center of the national academy of medical sciences of Beijing, china. The random number table method was used to divide into 3 groups (n=6):

sham surgery group (S group): after the mice are anesthetized, preparing skin, sterilizing, spreading towel, cutting skin, pulling out soft tissues, preventing tibia fracture, and suturing wounds layer by layer;

tibial fracture chronic pain model (TF group): a model is manufactured according to the following method;

rab35 Morpholino+tibial fracture chronic pain model (RMo+TF group): 24 hours before the preparation of the tibia fracture chronic pain model, the preparation was injected through L5-6 intervertebral space puncture, and Rab35 Morpholino 10 μl was injected. Rab35 morphling: 5'-TCCACTTCCCGAACAAACAGCCGGA-3' (Gene Tools, philomath, OR, USA) was diluted to 1 mM stock according to the instructions, and stored in aliquots.

(2) Mouse tibia fracture chronic pain model: healthy adult male C57BL/6 mice (purchased from the laboratory animal center of the national institute of military medical science of the free army of China) were selected, anesthetized by inhalation with sevoflurane (anesthesia induction 3%; anesthesia maintenance 1.5%), the lateral recumbent position of the animals was fixed on an operating table, conventionally prepared, sterilized, spread with towels, the skin was cut under the right knee of the mice, soft tissues were peeled off layer by layer, the tibia was exposed, a 0.7mm stainless steel needle was inserted into the bone marrow cavity, the middle section of the tibia was broken with forceps after fixation, and the wounds were sutured after the surgery.

(3) Intrathecal administration: the method comprises the steps of inhaling sevoflurane (3% of anesthesia induction; 1.5% of anesthesia maintenance) for anesthesia, prone lying, preparing skin, sterilizing, vertically and slowly injecting from the intervertebral space by using a microinjector, directly connecting a 30G needle head by using the microinjector, injecting Rab35 Morpholino through L5-6 intervertebral space puncture, and specifically knocking down Rab35 expression.

(4) Behavioural experiments: mechanical paw withdrawal frequency (the frequency of The mechanical foot contraction response, MWF (%)) and heat-shrinkage foot latency (thermal foot contraction latency, TWL) were measured at 24 (T0) before model, 1, 3, 7 and 14d (T1-4) after fracture model fabrication, and mice were acclimatized for 15 min before testing. MWF (%) was measured using BSEVF3 electronic von Frey fiber (Harvard Apparatus company, usa). The stimulation force of positive reaction (such as rapid foot contraction reaction, licking of right foot or fizzing) is recorded by vertically applying stimulation between the right hind foot 2 and 3 phalanges of the rat with von Frey fiber yarn, and the measurement is continuously carried out for 10 times at intervals of 1 min, wherein the foot contraction frequency is MWF (%).

The TWL was measured with an infrared plantar pain meter (IICT Life Science, 390), the time from the contact of the left hind foot with the hot plate to the appearance of retraction, standing foot, struggling, hoarseness, and licking of any reaction was recorded as TWL, the measurement was continued 3 times, 5min intervals, and the average value was taken as PWL (sec). To prevent scalding the paw, the TWL is defined to be set at 20s.

(5) Western Blot: protein expression was determined by Western Blot. After the end of the last 1 behavioural assay, rats were sacrificed, spinal cord L4-6 segments were taken, pre-chilled cathepsin lysate was added, and ground to tissue homogenate. Centrifuging the homogenate at 4 ℃ for 5min at 12000rpm with a radius of 10cm, and obtaining the supernatant as the total protein of the spinal cord tissue. The relative expression of Rab35, secretoglannin II, GADPH was determined experimentally using Rab35 (Abcam, uk), secretoglannin II (loading Biology company, usa), GADPH primary antibody (Abcam, uk) according to the guidelines of the specification.

(6) Statistical analysis: the SPSS 18.0 statistical software is adopted for analysis, the normal distribution measurement data is expressed by mean ± standard deviation (±s), the measurement data of the random block design is compared by single factor analysis of variance, the measurement data of the repeated measurement design is compared by repeated measurement design analysis of variance, and P <0.05 is the difference and has statistical significance.

2. Experimental results

(1) The spinal cord Rab35 and secretoglobin II expression in chronically painful mice after fracture was increased.

The results are shown in FIG. 1, and after the last behavioural 14 days after fracture, mice were sacrificed to obtain spinal cord L4-6, and the results found in Western Blot: compared with normal saline group (group C), the expression of the spinal cord Rab35 and the Secretogranin II of the mice with chronic pain after fracture is increased, and the Rab35 and the Secretogranin II are proved to be involved in the chronic pain generating process of the mice after fracture.

(2) Injection of Rab35 Morpholino alleviates mechanical and thermal pain caused by fracture in mice

As a result, as shown in fig. 2, the post-operative 1, 3, 7 and 14d (T1-4) MWF (%) were significantly increased and the heat-shrink foot latency (TWL) was shortened (< 0.001) in the tibial fracture group (TF group) compared to the sham operation group (S group). These results indicate that mechanical trigger-induced pain (fig. 2A) and thermal stimulus-induced pain (fig. 2B) caused by the fracture of the tibia in mice occurred 3 days after surgery and peaked at 7-14 days, confirming that the fracture of tibia can induce chronic pain.

Compared with the TF group, the MWF (%) of the Rab35 Morpholino group (RMo+TF group) is obviously reduced, TWL is obviously prolonged (& gt, P < 0.001), and the inhibition of Rab35 expression can relieve mechanical pain and thermal pain caused by fracture of mice.

3) Rab35 relieves mechanical and thermal pain after fracture in mice through secretoglanin II

After the last behavioural 14 days after fracture, mice were sacrificed to obtain spinal cord L4-6, found in Western Blot results: the reduced expression of secretoglinin II by injection of Rab35 morpho group (rmo+tf group) compared to TF group demonstrated that Rab35 resulted in mechanical and thermal pain after fracture in mice by secretoglinin II relief (fig. 3).

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

Use of a rab35 inhibitor in the preparation of a medicament for the prevention or treatment of chronic pain following fracture; the Rab35 inhibitor is Rab35 Morpholino, and the nucleotide sequence of the Rab35 inhibitor is 5'-TCCACTTCCCGAACAAACAGCCGGA-3'.
2. The use according to claim 1, wherein the medicament comprises a pharmaceutically acceptable carrier.