Application of dunaliella in preparation of medicine for promoting nerve regeneration after peripheral nerve injury

Abstract

The invention provides an application of Dubosiella Newyorkensis in preparation of a medicine for promoting nerve regeneration after peripheral nerve injury. The Dubosiella Newyorkensis can be prepared into an oral liquid preparation. Experiments show that the bacterial strain can improve the barrier function of the intestinal tract after injury, significantly down-regulate the expression of proinflammatory factors, and activate regeneration promoting signal channels such as p-Akt / Akt and beta-catenin, thereby effectively promoting nerve axon regeneration and motor function recovery. The invention provides a brand new microbial therapy strategy for repairing the peripheral nerve injury.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to application of Dubosiella in preparation of a drug for promoting nerve regeneration after peripheral nerve injury. BACKGROUND

[0002] Sciatic nerve injury is a common type of peripheral nerve injury, which can be caused by trauma, compression or surgery, and often leads to sensory and motor dysfunction. Although there are currently various means such as surgical repair, electrical stimulation and drug treatment, the problems of slow nerve regeneration, incomplete functional recovery and large individual differences in curative effect have not been effectively solved.

[0003] In recent years, the concept of gut-nerve axis has gradually attracted attention. A large number of studies have shown that gut flora has a potential regulatory effect on peripheral nerve regeneration by regulating immune response, producing metabolites (such as short-chain fatty acids) and affecting neurotrophic factor expression. Therefore, it is necessary to develop a drug for promoting nerve regeneration after peripheral nerve injury. SUMMARY

[0004] The application aims to provide application of Dubosiella in preparation of a drug for promoting nerve regeneration after peripheral nerve injury. Dubosiella newyorkensis screened through 16S rRNA sequencing is found to be able to improve the intestinal microenvironment and promote growth of sciatic nerve after injury through short-term mechanism research and long-term nerve function research. The regulation of Dubosiella newyorkensis in body homeostasis is expected to provide a new research direction for the drug for treating peripheral nerve injury.

[0005] To solve the above technical problems, the application adopts the following technical solutions: In a first aspect of the application, application of Dubosiella in preparation of a drug for promoting nerve regeneration after peripheral nerve injury is provided.

[0006] Further, the Dubosiella is NYU-BL-A4, the preservation number is ATCC: TSD-64, and is purchased from Oligo (Shanghai) Life Science Co., Ltd. Further, the drug further comprises a pharmaceutically acceptable adjuvant and carrier.

[0007] Further, the adjuvant comprises at least one of a filler, a disintegrant, a binder, a lubricant, a sweetener.

[0008] Further, the dosage form of the drug comprises at least one of a granule, a tablet, a pill, a capsule, an injection or a dispersant.

[0009] In a second aspect of the present application, a medicament for promoting nerve regeneration after peripheral nerve injury is provided, comprising an effective dose of Du's bacillus as an active ingredient, and a pharmaceutically acceptable carrier.

[0010] Further, the concentration of live Du's bacillus in the medicament is 1×10 8 CFU / mL to 1×10 10 CFU / mL.

[0011] Further, the medicament further comprises at least one additional active ingredient selected from a neurotrophic factor, an anti-inflammatory drug or an antioxidant.

[0012] Further, the peripheral nerve injury is sciatic nerve injury, common peroneal nerve injury or radial nerve injury.

[0013] Further, the medicament promotes nerve regeneration by regulating at least one of the Akt, β-catenin and NF-κB signaling pathways, and / or by down-regulating the expression of pro-inflammatory factors TNF-α, IL-6 and IL-1β and up-regulating the expression of anti-inflammatory factor IL-10.

[0014] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages: The application provides a Du's bacillus for use in the preparation of a medicament for promoting nerve regeneration after peripheral nerve injury, which has the following advantages: 1. Effectively improving intestinal barrier function: By colon alizarin blue staining and immunohistochemical staining of tight junction proteins (ZO-1, Claudin-1), it is confirmed that the Du's bacillus can significantly improve the increased intestinal permeability caused by sciatic nerve injury, repair the damaged intestinal mucosal barrier, and lay a structural foundation for the function through the "gut-brain axis".

[0015] 2. Precise regulation of inflammatory immune response: As shown in the qPCR results of G, Figure 3 The strain can significantly down-regulate the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6 and IL-1β in the local sciatic nerve, and up-regulate the expression of anti-inflammatory factor IL-10, successfully converting the inflammatory microenvironment after injury from a pro-inflammatory state to an anti-inflammatory repair state, and creating favorable conditions for nerve regeneration.

[0016] 3. Dual activation of key signaling pathways for nerve regeneration: Western blot experimental evidence ( Figure 3F) indicates that this intervention method can significantly increase the p-Akt / Akt ratio in neural tissue and upregulate β-catenin protein expression, directly activating intracellular signaling pathways that promote neuronal survival and axonal growth; at the same time, it effectively inhibits the activation of the NF-κB signaling pathway, thus synergistically promoting regeneration from multiple aspects.

[0017] 4. Significantly promotes the recovery of neural structure and function: Long-term animal experiments have confirmed its remarkable repair effects from multiple dimensions. Sensory function recovery: thermal pain test ( Figure 4 B) showed a significant improvement in pain sensitivity. Motor function recovery: Footprint analysis ( Figure 4 C) indicates that the gait is close to normal. Electrophysiological function recovery: nerve conduction velocity test ( Figure 4 D) demonstrates a significant improvement in neural electrical signal transmission capacity. Structural regeneration is confirmed by SCG10 immunofluorescence staining ( Figure 4 E) Visually demonstrates the large number and long distance of axonal regeneration. Target organ protection: HE staining of gastrocnemius muscle ( Figure 4 F) shows that muscle atrophy has been significantly alleviated.

[0018] 5. The formulation is safe and reliable and easy to use: The oral liquid formulation uses PBS as a carrier, has simple components, high biocompatibility, does not require the addition of chemical stabilizers, has a simple preparation process, is suitable for long-term administration, and has good prospects for clinical translation. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Experimental procedure and intestinal permeability staining results: (A) Experimental procedure diagram; (B) Colon albedo blue staining and immunohistochemical staining of ZO-1 and Claudin-1; (C) Statistical graph of colon albedo blue staining and immunohistochemical staining results of ZO-1 and Claudin-1.

[0021] Figure 2 16S rRNA sequencing results of intestinal contents at different time points after sciatic nerve transection in mice: (A) PCoA analysis; (B) Stacked diagram of species composition at the phylum and genus levels; (C) ACE index and Shannon index in Alpha diversity difference analysis; (D) LDA score.

[0022] Figure 3Short-term experimental results of gavage administration of Dubosiella newyorkensis after sciatic nerve transection in mice: (A) Experimental flowchart; (B) 16S rRNA sequencing verification of the effect after ABX intestinal cleansing, which is a stacked diagram of species composition at the phylum level; (C) 16S rRNA sequencing verification of the effect after ABX intestinal cleansing, which is a heatmap of species at the family level; (D) Alixin blue staining and ZO-1 and Claudin-1 immunohistochemical staining of the colon on the fourth day after sciatic nerve injury in mice; (E) Statistical graph of the results of Alixin blue staining and immunohistochemical staining; (F) Western blot detection of p-Akt, Akt, β-catenin and NF-κB levels after sciatic nerve harvesting; (G) qPCR detection of the expression of inflammatory factors: TNF-α, IL-6, IL-1β and IL-10.

[0023] Figure 4 Long-term experimental results of gavage administration of *Dubosiella newyorkensis* after sciatic nerve transection in mice: (A) Experimental flowchart; (B) Results of thermal pain experiments in mice at 2, 4, 6, and 8 weeks after sciatic nerve injury; (C) Footprint analysis and statistical results in mice at 2, 4, 6, and 8 weeks after sciatic nerve injury; (D) Electrophysiological experiments and statistical results in mice at 8 weeks after sciatic nerve injury; (E) SCG10 immunofluorescence staining and statistical results of sciatic nerve tissue taken from mice at 8 weeks; (F) HE staining and statistical results of gastrocnemius muscle taken from mice at 8 weeks, and statistical results of wet weight ratio and average muscle fiber area. Detailed Implementation

[0024] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0025] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0026] As used herein, the term "Dubosiella newyorkensis" refers to a strictly anaerobic, Gram-positive bacterium belonging to the family Erysipelotrichaceae in the phylum Bacillota, strain numbered ATCCTSD-64, found in the mouse intestine. Dubosiella newyorkensis is commercially available and can be cultured and propagated using methods well-known to those skilled in the art. In embodiments of the invention, after centrifugation to remove the culture medium, Dubosiella newyorkensis is resuspended in sterile PBS and administered to mice by gavage at a final concentration of 10⁸–10¹. 0 CFU / day, 10 preferred 9 CFU / day. The bacterial strains used in these embodiments were purchased from Unicore (Shanghai) Life Science Co., Ltd.

[0027] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0028] The present application will now be described in detail with reference to embodiments and experimental data.

[0029] Example 1: Establishment of an animal model and observation of intestinal changes I. Experimental Objective To observe the effect of sciatic nerve injury on intestinal barrier function.

[0030] II. Experimental Methods A peripheral nerve injury model was established using 8-week-old male C57BL / 6 mice by performing a left sciatic nerve transection. Colon tissue was harvested from the mice on postoperative days 1, 4, and 7.

[0031] III. Experimental Results like Figure 1 As shown in Figure A, immunostaining and alcinocyanine blue staining of colon tissue revealed that, compared with the sham-operated group, the intestinal barrier permeability increased and tight junction protein expression was downregulated in the model group mice after nerve transection. Figure 1 B and Figure 1 The staining and statistical results of C indicate that sciatic nerve injury can affect intestinal barrier function.

[0032] Example 2: Changes in Gut Microbiota Structure and Screening for Dubosiella newyorkensis I. Experimental Objective We analyzed the dynamic changes in gut microbiota after sciatic nerve injury and screened out key strains related to injury repair.

[0033] II. Experimental Methods Intestinal contents of mice from each group in Example 1 were collected at different time points (days 1, 4, 7, 14, and 28 after surgery), sent to a professional company for 16S rRNA sequencing, and the data were analyzed using the Omicsmart cloud computing platform of Kiddio.

[0034] III. Experimental Results like Figure 2 PCoA analysis of A showed that there were significant differences in the gut microbiota structure between the model group and the sham-operated group of mice. Figure 2 Species composition stacking diagram of B and Figure 2 LDA analysis of D further showed that the relative abundance of the genus *Dubosiella* was significantly upregulated at specific time points after injury (e.g., day 4). Through differential analysis and correlation screening, *Dubosiella newyorkensis* (strain number ATCC TSD-64) was finally selected as a candidate strain for subsequent studies.

[0035] Example 3: Short-term mechanism experiment I. Experimental Objective To verify the short-term therapeutic effect of Dubosiella newyorkensis and to preliminarily explore its mechanism of action.

[0036] II. Experimental Methods Mice were given a four-drug regimen (metronidazole 1 g / L, neomycin 1 g / L, penicillin 1 g / L, vancomycin 0.5 g / L) for two weeks prior to surgery to clear the pre-existing intestinal flora, followed by the establishment of a sciatic nerve transection model. Starting 24 hours post-surgery, the experimental group was orally administered a PBS suspension of *Dubosiella newyorkensis* (1 × 10⁻⁶ bacteria) daily. 9 CFU / day was administered for 7 consecutive days. Sciatic nerves of mice were harvested on days 1, 4, and 7 for Western blot and qPCR experiments, and colon samples were taken for immunohistochemical staining and alcinocyanine blue staining.

[0037] III. Experimental Results Intestinal barrier repair: such as Figure 3 D and Figure 3 As shown in Figure E, compared with the model group, the area of ​​Alcian Blue staining in the colon of mice treated with Dubosiella newyorkensis was reduced, and the expression of ZO-1 and Claudin-1 proteins was significantly restored, indicating that it can effectively improve intestinal barrier dysfunction caused by injury.

[0038] Signaling pathways and regulation of inflammatory factors: Western blot results ( Figure 3F) showed that the p-Akt / Akt ratio and β-catenin protein expression levels were significantly upregulated in the treatment group, while NF-κB expression was inhibited. qPCR results ( Figure 3 G) Further confirmation showed that the mRNA expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β was significantly decreased in the treatment group, while the expression of anti-inflammatory factor IL-10 was significantly increased.

[0039] Example 4: Long-term functional test I. Experimental Objective To evaluate the effects of long-term administration of Dubosiella newyorkensis on the structural and functional recovery of the sciatic nerve after injury.

[0040] II. Experimental Methods The animal model and dosing regimen used in Example 3 (bacterial count 1×10⁻⁶) were followed. 9 (CFU / day), with the intervention period extended to 8 weeks. After nerve transection, the two ends of the nerve were sutured to a silicone catheter. Thermal pain tests, footprint analysis, and electrophysiological tests were performed at weeks 2, 4, 6, and 8, respectively. In week 8, the sciatic nerve was harvested for SCG10 immunofluorescence staining, and the gastrocnemius muscle was harvested for HE staining.

[0041] III. Experimental Results like Figure 4 B to Figure 4 As shown in F, the Dubosiella newyorkensis treatment group exhibited comprehensive functional and structural recovery: 1. Sensory function: thermal pain test ( Figure 4 B) shows that, starting from week 4, the heat pain threshold in the treatment group was significantly higher than that in the model group.

[0042] 2. Motor function: Footprint analysis ( Figure 4 C) indicates that the gait parameters of the treated group mice were closer to normal levels.

[0043] 3. Electrophysiological function: Nerve conduction velocity test ( Figure 4 D) shows that the nerve conduction velocity recovery rate was significantly improved in the treatment group.

[0044] 4. Axonal regeneration: SCG10 immunofluorescence staining ( Figure 4 E) The results showed that the treatment group had more regenerated axons at the distal end of the injury and a longer growth distance.

[0045] 5. Target organ protection: HE staining and statistical analysis of gastrocnemius muscle ( Figure 4 F) indicates that the muscle wet weight ratio and average muscle fiber area of ​​the treated group mice were significantly greater than those of the model group, effectively alleviating muscle atrophy.

[0046] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0047] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0048] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0049] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of equivalents of the claims, this invention also intends to include them.

Claims

1. Application of Donorella in the preparation of drugs for promoting nerve regeneration after peripheral nerve injury.

2. The application as described in claim 1, characterized in that, The strain of *Dunaliella salina* has the accession number ATCC TSD-64.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients and carriers.

4. The application according to claim 1, characterized in that, The excipients include at least one of fillers, disintegrants, binders, lubricants, and sweeteners.

5. The application according to claim 1, characterized in that, The dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections, or dispersants.

6. A drug for promoting nerve regeneration after peripheral nerve injury, characterized in that, It contains an effective dose of Donorum as the active ingredient, as well as a pharmaceutically acceptable carrier.

7. The medicament according to claim 6, characterized in that, The concentration of live Donorella bacteria in the drug is 1×10⁻⁶. 8 CFU / mL to 1×10 10 CFU / mL.

8. The drug according to claim 6, characterized in that, The drug also contains at least one additional active ingredient selected from neurotrophic factors, anti-inflammatory drugs, or antioxidants.

9. The drug according to claim 6, characterized in that, The peripheral nerve injury refers to sciatic nerve injury, common peroneal nerve injury, or radial nerve injury.

10. The medicament according to claim 6, characterized in that, The drug promotes neurogenesis by regulating at least one of the Akt, β-catenin, and NF-κB signaling pathways, and / or by downregulating the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β and upregulating the expression of anti-inflammatory factor IL-10.

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