CN115381950A

CN115381950A - Application of Rab35 or downstream regulatory molecule thereof in treating chronic pain after fracture

Info

Publication number: CN115381950A
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: 2022-11-25
Anticipated expiration: 2042-09-06
Also published as: CN115381950B

Abstract

The invention discloses application of Rab35 or downstream regulatory molecules thereof in treating chronic pain after fracture. Experiments prove that after fracture, the expression of Rab35 and Secretogranin II of the mouse spinal cord with chronic pain is increased, the injection of Rab35 Morpholino relieves mechanical pain and thermal pain caused after fracture of the mouse, and Rab35 relieves mechanical pain and thermal pain caused after fracture of the mouse through Secretogranin II. Based on the above research, the present invention provides a new method for treating chronic pain after fracture.

Description

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

Background

With the rapid development of industries such as China, buildings, transportation and the like and the aggravation of aging of population, the occurrence of industrial accidents, building injuries, traffic injuries and senile osteoporosis fracture is increased. According to statistics, the number of people suffering from traumatic fracture per year in China exceeds 440 ten thousand, and the number of people suffering from osteoporotic fracture per year in the world is about 900 ten thousand. The chronic pain after fracture is a chronic pain state secondary to the fracture, has serious symptoms and high morbidity, and has no effective treatment measure. It has been reported that the incidence of chronic pain after fracture of ankle and wrist joints is as high as 61.7%, and the incidence of chronic pain after fracture of tibia is 55.1%. Serious pain not only hinders early rehabilitation and skeletal muscle function recovery of patients, but also increases the risk of occurrence of cardiovascular and cerebrovascular accidents, depression, acute Post-traumatic cognitive dysfunction (Post-traumatic cognitive dysfunction) and chronic Complex regional pain syndrome (Complex regional pain syndrome), seriously affects the quality of life of patients, and increases the burden on families and socioeconomic performance of patients, thus becoming an important public health problem to be solved urgently. The mechanism of chronic pain after fracture is not clear, and the method is one of the research hotspots in the pain field at present.

Research shows that Rab GTPases can be used as a molecular switch to switch between a GTP-bound active form and a GDP-bound inactive form, can regulate the endocytosis and axonal transport of neuronal cells, and are involved in the pathological process of various neurological diseases, such as Alzheimer's Disease (AD), parkinson's Disease (PD) and the like, but the Rab GTPases are not researched in chronic pain after fracture. The research centers on the Rab GTP enzyme family protein Rab35 for regulating and controlling membrane vesicle transport by means of vivo-Morpholino interference, behavioral determination and the like, and researches the effect of the Rab GTP enzyme family protein Rab35 in chronic pain after fracture.

Disclosure of Invention

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

Further, the inhibitor includes an agent that reduces the level of the mRNA or protein of Rab35 or its downstream regulatory molecule, or reduces the activity of the 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, micro RNA, antisense nucleic acid or a construct thereof.

Further, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

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

According to another aspect of the invention, the invention also provides the application of the inhibitor of functional expression of Rab35 or the downstream regulatory molecule thereof in preparing the medicine for preventing or treating chronic pain after fracture.

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

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

Drawings

Fig. 1 is a graph showing the results of spinal cord Rab35 and Secretogranin II expression in chronic pain mice after fracture, in which a: an immunoblot; b: and (6) a histogram. * P < 0.001, compared to S group;

fig. 2 is a graph showing the results of mechanical pain and thermal pain after Rab35 Morpholino injection is administered to relieve fracture in mice, wherein a: mechanical paw withdrawal response threshold (MWF (%))); b: heat-shrinkable foot latency (TWL); * P<0 .001； ^&&& P<0 .001；

FIG. 3 shows a graph of the results of Secretogranin II expression, in which A: immunoblot; b: a histogram; * P<0 .001； ^&&& P<0 .001。

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations.

The invention discloses a pharmaceutical composition, which comprises Rab35 or an inhibitor of the functional expression of downstream regulatory molecules thereof; the inhibitor is any substance which can reduce the stability of Rab35 or a downstream regulatory molecule gene or an expression product thereof, down-regulate the expression of Rab35 or a downstream regulatory molecule thereof, reduce the effective action time of Rab35 or a downstream regulatory molecule thereof or inhibit the transcription of Rab35 or a downstream regulatory molecule gene thereof, and the substances can be used for the invention, and can be used as substances which are useful for down-regulating the expression of Rab35 or a downstream regulatory molecule thereof, thereby being used for treating chronic pain after fracture.

As an alternative of the present invention, the inhibitor of Rab35 or its downstream regulatory molecule is an antibody specifically binding to Rab35 or its downstream regulatory molecule. 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. As long as the fragment retains the ability to bind to Rab35 or its downstream regulatory molecule proteins. The preparation of antibodies for use at the protein level is well known to those skilled in the art and any method may be used in the present invention to prepare such antibodies.

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

The nucleic acid inhibitor of the present invention, such as siRNA, can be chemically synthesized or can be prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single-stranded RNA. Nucleic acid inhibitors, such as siRNA, can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.

As an alternative of 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, and the Small hairpin RNA can inhibit gene expression through RNA interference pathway. As described above, shRNA can 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 expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. The term "shRNA expression vector" refers to some plasmids conventionally used in the art for constructing shRNA structure, and usually there are "spacer sequence" and multiple cloning sites or alternative sequences located on both sides of the "spacer sequence", so that one can insert the corresponding DNA sequence of shRNA (or analogue) into the multiple cloning sites or replace the alternative sequences thereon in a forward and reverse manner, and the RNA transcribed from the DNA sequence can form shRNA (Short Hairpin) structure. The shRNA expression vector can be completely purchased from commercial sources at present, such as some virus vectors.

As a preferred embodiment of the present 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 by the complementary sequence, the suppression of a target gene can be achieved, the antisense oligonucleotide being a ribonucleic acid or a DNA. As a preferred embodiment, the antisense oligonucleotide comprises at least one chemical modification. Antisense oligonucleotides can contain one or more Locked Nucleic Acids (LNAs). Locked nucleic acids are modified ribonucleic acids that include an additional bridge between the 2 'to 4' carbons of the ribose moiety to have a locked (locked) conformation, and thus oligonucleotides with locked nucleic acids have improved thermal stability. Alternatively, the antisense oligonucleotide may comprise Peptide Nucleic Acids (PNA), and the antisense oligonucleotide comprises a peptide-based backbone in place of the sugar-phosphate backbone. Other chemical modifications that the antisense oligonucleotides can comprise include: sugar modifications such as 2' -O-alkyl (e.g., 2' -O-methyl, 2' -O-methoxyethyl), 2' -fluoro, and 4' -thio-oxy modifications; backbone modifications such as phosphorothioate, morpholino, or phosphocarboxylic acid linkages. The antisense oligonucleotide is 7 to 50 nucleotides, preferably 10 to 40 nucleotides, more preferably 15 to 30 nucleotides, and most preferably 20 to 25 nucleotides in length.

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

The pharmaceutical composition of the invention comprises active ingredients and pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier includes (but is not limited to) diluents, binders, surfactants, humectants, adsorptive 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, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaure Gui Zhe sugar acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), 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.; disintegrating agent 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, isotonizing agents, chelating agents, pH control agents, and surfactants.

Wherein the stabilizer comprises human serum protein, L-amino acid, sugar and cellulose derivative. The L-amino acid may further include any of glycine, cysteine, and glutamic acid. Saccharides 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 sulfate, hyaluronic acid, etc. and their derivatives. The cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and sodium hydroxymethylcellulose. Surfactants include ionic or non-ionic surfactants such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, fatty acid glycerides. Additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid, and the corresponding salts (alkali metal or alkaline rare earth metal salts thereof, such as sodium, potassium, calcium, and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars and glycerol. The chelating agent comprises sodium ethylene diamine tetracetate and citric acid.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical composition 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 can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.

Example application of Rab35 in post-fracture Chronic pain

1. Experimental procedure

(1) Grouping experiments: the healthy male C57BL6 mice are 18 mice, 2 months old, and provided by Experimental animals center of Beijing military medical science institute, china. Using a random number table method, dividing into 3 groups (n = 6):

sham group (S group): after the mice are anesthetized, skin preparation, disinfection, tissue spreading, skin cutting, soft tissue poking, no tibia fracture, and wound layer by layer suture;

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

rab35 Morpholino + chronic pain model of tibial fracture (RMo + TF group): 24 hours before the model of the chronic pain of the tibial fracture is made, injection is carried out through L5-6 intervertebral puncture, and Rab35 Morpholino 10 mu L is injected. Rab35 Morpholino: 5'-TCCACTTCCCGAACAAACAGCCGGA-3' (Gene Tools, philograph, OR, USA), diluted to 1 mM mother liquor as specified, and stored separately.

(2) Mouse tibia fracture chronic pain model: selecting healthy adult male C57BL/6 mice (purchased from the center of laboratory animals of the national academy of military medical science and academic society), performing inhalation anesthesia by using sevoflurane (anesthesia induction is 3%; anesthesia maintenance is 1.5%), fixing the animals in the lateral decubitus position on an operating table, performing conventional skin preparation, sterilizing, spreading a towel, cutting the skin below the right knee of the mice, peeling off soft tissues layer by layer, exposing the tibia, inserting a 0.7mm stainless steel needle into the bone marrow cavity, breaking the middle section of the tibia by using an operation forceps after fixing, and suturing the wound after the operation.

(3) Intrathecal administration: the rats are subjected to inhalation anesthesia by using sevoflurane (anesthesia induction is 3 percent; anesthesia maintenance is 1.5 percent), the rats are prone, preserved, sterilized, and inserted vertically and slowly from the intervertebral space by using a micro syringe, the micro syringe is directly connected with a 30G needle head and injected by L5-6 intervertebral space puncture, rab35 Morpholino is injected, and the Rab35 expression can be specifically reduced.

(4) Behavioral experiments: mechanical paw withdrawal frequency (MWF (%)) and thermal paw withdrawal latency (TWL) were measured at 24 zxft 8978 (T0) before The model, 1, 3, 7, and 14d after The fracture model was made (T1-4), and mice were allowed to acclimate for 15 min before testing. MWF (%) was determined using BSEVF3 electronic von Frey filament (Harvard Apparatus, USA). The stimulation force of positive reaction (such as rapid paw-shortening reaction, right paw licking or hoarseness) is recorded by applying von Frey fiber vertically between 2 and 3 phalanges of the right hind paw of the rat, and the stimulation force is continuously measured for 10 times at an interval of 1 min, and the frequency of paw-shortening is MWF (%).

TWL is measured by an infrared plantar pain measuring instrument (IICT Life Science 390), the time from the time when the left hind foot contacts a hot plate to the time when any reaction of retraction, tiptoe standing, struggling, whoosing and foot licking is recorded as TWL, the TWL is measured for 3 times continuously at intervals of 5min, and the average value is taken as PWL (sec). To prevent scalding of the rat paw, the upper TWL limit was set to 20s.

(5) Western Blot: protein expression was determined by Western Blot. After the final 1 ethological determination, the rats were sacrificed, the spinal cord L4-6 segment was taken, pre-cooled histon lysate was added, and ground into a tissue homogenate. And (3) centrifuging the homogenate for 5min at 4 ℃,12000rpm, wherein the centrifugation radius is 10cm, and obtaining the supernatant, namely the total protein of the spinal cord tissue. The relative expression of Rab35, secretogranin II, GADPH was determined experimentally using Rab35 (Abcam, uk), secretogranin II (Leading Biology, usa), GADPH primary antibody (Abcam, uk) according to the manual.

(6) Statistical analysis: SPSS 18.0 statistical software is adopted for analysis, normally distributed metering data is represented by mean +/-standard deviation (+/-s), single-factor variance analysis is adopted for metering data comparison designed for random block, variance analysis designed for repeated measurement is adopted for metering data comparison designed for repeated measurement, and P <0.05 has statistical significance for difference.

2. Results of the experiment

(1) Chronic pain mice after fracture have increased spinal cord Rab35 and Secretogranin II expression.

Results as shown in fig. 1, after 14 days of fracture, after the last ethology, the spinal cords L4-6 were taken from the sacrificed mice and found in the Western Blot results: the expression of Rab35 and Secretogranin II in the spinal cord of mice with chronic pain after fracture was increased compared to the saline group (group C), demonstrating that Rab35 and Secretogranin II are involved in the process of chronic pain in mice after fracture.

(2) Rab35 morphine injection relieves mechanical pain and thermal pain caused by fracture of mice

Results as shown in fig. 2, the tibial fracture group (TF group) mice significantly increased post-surgery 1, 3, 7 and 14d (T1-4) MWF (%) and shortened heat-shrinkable foot latency (TWL) (. About.p < 0.001) compared to the sham group (S group). These results indicate that mechanical touch-induced pain (fig. 2A) and thermal stimulation-induced pain (fig. 2B) caused by tibial fracture in mice occurred 3 days after the operation, and peaked at 7 to 14 days, confirming that the tibial fracture can induce chronic pain.

Compared with the TF group, the MWF (%) of the Rab35 Morpholino injection group (RMo + TF group) is remarkably reduced, the TWL is remarkably prolonged (& & P < 0.001), and the inhibition of the expression of Rab35 is shown to relieve mechanical pain and thermal pain caused after fracture of mice.

3) Rab35 relieves mechanical and thermal pain after fracture of mice through Secretogranin II

After 14 days post fracture, the last ethology, sacrifice mice to take spinal cord L4-6, found in Western Blot: the decrease in Secretogranin II expression of the Rab35 Morpholino group (RMo + TF group) injected compared to the TF group demonstrates that Rab35 causes mechanical and thermal pain after alleviating fracture of mice by Secretogranin II (fig. 3).

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Claims

1. A pharmaceutical composition comprising an inhibitor of Rab35 or its downstream regulatory molecule functional expression.

2. The use of claim 1 wherein the inhibitor comprises an agent that reduces the level of mRNA or protein of Rab35 or a downstream regulatory molecule thereof, or reduces the activity of Rab35 protein or a downstream regulatory molecule thereof.

3. The use of claim 1, wherein the inhibitor comprises a nucleic acid inhibitor, a protein inhibitor, a proteolytic enzyme, a protein binding molecule; preferably, the inhibitor is a nucleic acid inhibitor.

4. The use of claim 3, wherein the nucleic acid inhibitor is selected from shRNA, small interfering RNA, dsRNA, microRNA, antisense nucleic acid or a construct thereof.

5. The use of claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

6. Use according to any one of claims 1 to 5, wherein the downstream regulatory molecule of Rab35 is Secretogranin II.

The use of rab35 or an inhibitor of the functional expression of a downstream regulatory molecule thereof in the preparation of a medicament for the prevention or treatment of chronic pain following fracture;

preferably, 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;

preferably, the inhibitor comprises a nucleic acid inhibitor, a protein inhibitor, a proteolytic enzyme, a protein binding molecule; preferably, the inhibitor is a nucleic acid inhibitor.

8. The use of claim 9, wherein the nucleic acid inhibitor is selected from shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid or a construct thereof.

9. The use of claim 3, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

10. Use according to any one of claims 7 to 9, wherein the downstream regulatory molecule of Rab35 is Secretogranin II.