Use of a capsule of mukit in the preparation of a medicament for treating neuroinflammation and improving neurogenesis

Abstract

The application discloses application of a Muxie capsule in preparation of a medicine for treating nerve inflammation and improving neurogenesis, and belongs to the technical field of medicines. The Muxie capsule can improve the abundance of beneficial bacteria, inhibit pathogenic bacteria, reshape the microecological balance of the intestines, reduce the levels of serum proinflammatory factors IL-1beta and TNF-alpha, increase the level of an anti-inflammatory factor IL-10, inhibit abnormal excessive proliferation of nerve stem cells and neuroblasts in the hippocampal dentate gyrus region induced by LPS, restore the steady state of neurogenesis, and play a role in treating diseases such as depression, Alzheimer's disease, multiple sclerosis, Parkinson's disease or traumatic brain injury. The application expands the range of clinical application indications of the Muxie capsule, improves the clinical application value of the Muxie capsule, and provides a new drug selection for clinical treatment of diseases related to nerve inflammation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a type of wood-derived capsule in the preparation of drugs for treating neuroinflammation, improving neurogenesis, and treating nervous system diseases. Background Technology

[0002] Neurological diseases are diverse and their pathological mechanisms are complex, with neuroinflammation playing a crucial role in the development and progression of many diseases. Lipopolysaccharide (LPS)-induced inflammation models are widely used to simulate systemic or central nervous system immune responses and can be used to analyze the role of neuroinflammation in diseases such as depression, Alzheimer's disease (AD), and multiple sclerosis (MS). In patients with depression, the levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) are significantly elevated in peripheral blood and cerebrospinal fluid, and microglia are in an activated state. Systemic LPS injection into animals can induce pathological behaviors such as anhedonia, decreased activity, and social avoidance, which are highly similar to the core symptoms of human depression.

[0003] Neurogenesis primarily occurs in the subgranular layer (SGZ) of the dentate gyrus (DG) of the hippocampus in adult mammals, and this process is involved in learning, memory, and emotion regulation. LPS-induced neuroinflammation can significantly affect hippocampal neurogenesis, rapidly activating microglia, secreting large amounts of inflammatory factors, and hindering the proliferation and differentiation of neural stem cells in the subgranular layer of the hippocampus. Abnormal or excessive neurogenesis may disrupt circuit homeostasis and exacerbate the inflammatory microenvironment. Therefore, compared to simply promoting or inhibiting neurogenesis, targeting and regulating its dynamic balance has become a research hotspot in this field.

[0004] In recent years, the role of the gut microbiota-gut-brain axis in the regulation of neuroinflammation has received increasing attention. Gut microbiota dysbiosis can lead to impaired intestinal barrier function, promoting the entry of inflammatory factors into the bloodstream, and subsequently affecting central nervous system function across the blood-brain barrier. Therefore, targeting the gut microbiota has become a novel strategy for treating neuroinflammation-related diseases.

[0005] Mujie capsules, as a traditional Chinese medicine compound, are used for symptoms such as pain, stiffness, and numbness caused by kidney deficiency and blood stasis, and cold obstruction, as well as as an adjunct treatment for orthopedic-related diseases. However, no relevant research has been found on its application in treating nerve inflammation and improving neurogenesis. Summary of the Invention

[0006] Existing Mujie capsules are limited to the treatment of bone and joint diseases, lacking application in treating central nervous system inflammation-related encephalopathy. Traditional interventions for neurological diseases often involve single-target promotion or inhibition of neurogenesis, failing to achieve homeostatic regulation and lacking multi-target intervention drugs that address peripheral inflammation, hippocampal neurogenesis, and gut microbiota. This invention provides an application of Mujie capsules in the preparation of drugs for treating neuroinflammation and improving neurogenesis. It explores the interventional effects of Mujie capsules in neuroinflammation and related nervous system diseases, systematically summarizes the therapeutic effects of Mujie capsules in treating neurological diseases caused by LPS-induced neuroinflammation, elucidates its mechanism of action, and confirms that it can inhibit neuroinflammation, correct abnormal neurogenesis, and remodel gut microbiota, making it suitable for the preparation of drugs for treating neuroinflammation and improving neurogenesis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the application of *Mujie* capsules in the preparation of drugs for treating neuroinflammation and improving neurogenesis.

[0008] Furthermore, neuroinflammatory-related diseases include any one of depression, Alzheimer's disease, multiple sclerosis, Parkinson's disease, and traumatic brain injury.

[0009] Furthermore, the aforementioned Mujie capsules enhance the ratio of Firmicutes to Bacteroidetes, thereby increasing the relative abundance of beneficial and homeostatic bacteria, while simultaneously inhibiting potentially pathogenic bacteria, improving intestinal barrier function, reducing peripheral inflammatory burden, and reshaping the intestinal microecological balance, thus playing a role in treating neuroinflammation and improving neurogenesis.

[0010] Furthermore, the beneficial and homeostatic bacteria are one or more of the following: Lactobacillus, Trichophyton spp. (UCG-006), Prevotella, Isprevotella, Erysipelothrix spp., Clostridium spp., and Adlerococcus spp.; the pathogenic bacteria are one or more of the following: Gastroenterobacter spp., Desulfovibrio, Dubois spp., Staphylococcus spp., Myxospirillum spp., Helicobacter spp., and Streptococcus spp.

[0011] Furthermore, the aforementioned Mujie capsules exert their therapeutic effects on neuroinflammation and improve neurogenesis by reducing serum levels of pro-inflammatory factors interleukin-1β and tumor necrosis factor-α, while simultaneously increasing levels of the anti-inflammatory factor interleukin-10.

[0012] Furthermore, the aforementioned Mujie capsules exert therapeutic effects on neuroinflammation and improve neurogenesis by inhibiting the abnormal and excessive proliferation of LPS-induced neural stem cells and neuroblasts in the hippocampal dentate gyrus, and reducing the number of glial fibrillary acidic protein, bicorticin, and microchromosome maintenance protein 2 positive cells in the hippocampal dentate gyrus.

[0013] The beneficial effects of this invention are: By integrating behavioral science, immunofluorescence, and gut microbiota sequencing, this study systematically revealed a novel indication for Mujie capsules: anti-neuroinflammatory effects and improvement of neurogenesis. At the gut microbiota level, Mujie capsules reshape the gut microecological balance, correcting the LPS-induced decrease in the Firmicutes / Bacteroidetes ratio at the phylum level, and specifically increasing the relative abundance of beneficial / homeostatic bacteria such as Lactobacillus and Trichophyton spp. (UCG-006) at the genus level. Simultaneously, it inhibits potentially pathogenic bacteria such as Desulfovibrio and Dubois, improving intestinal barrier function and reducing peripheral inflammatory burden. At the biochemical level, Mujie capsules significantly reduce serum levels of pro-inflammatory factors IL-1β and TNF-α, while increasing levels of the anti-inflammatory factor IL-10, systematically alleviating systemic inflammation. At the neurogenesis level, this invention found that Mujie capsules can inhibit LPS-induced abnormal excessive proliferation of neural stem cells and neuroblasts in the hippocampal DG region by reducing GFAP in the hippocampal DG region. + DCX + and MCM2 + The increased number of positive cells restores neurogenesis homeostasis and exerts a neuroprotective effect. At the behavioral level, Mujie capsules can improve motor coordination, muscle strength, and exercise endurance in inflammatory model mice. These findings preliminarily reveal the mechanism by which Mujie capsules prevent and treat neuroinflammation by improving the gut microbiota at the gut-brain axis level. This provides a new drug option for the clinical treatment of neuroinflammation-related diseases (depression, Alzheimer's disease, multiple sclerosis, Parkinson's disease, traumatic brain injury, etc.), enhancing the clinical application value and market prospects of Mujie capsules. Attached Figure Description

[0014] Figure 1 The graph shows the changes in body weight of mice in the NC group, LPS model group, and Mujie capsule administration group of this invention.

[0015] Figure 2 The figure shows the effect of the present invention's Mujie capsules on the motor function of inflammatory mice; a is the rotarod test; b is the tensile test; c is the treadmill exhaustion test; **P<0.01, ***P<0.001.

[0016] Figure 3 Immunofluorescence staining (x200) and cell homogenization histogram of GFAP / MCM2 positive cells in the DG region of the hippocampus of mice in the NC group, LPS model group, and Mujie capsule administration group of this invention; **P<0.01, *P<0.05.

[0017] Figure 4 Immunofluorescence staining (x200) and cell homogenization histogram of DCX / MCM2 positive cells in the DG region of the hippocampus of mice in the NC group, LPS model group, and Mujie capsule administration group of this invention; *P<0.05.

[0018] Figure 5 The graph shows the comparison of serum inflammatory factor levels in mice in the NC group, LPS model group, and Mujie capsule administration group of this invention; a is the statistical graph of IL-1β content; b is the statistical graph of IL-10 content; c is the statistical graph of TNF-α content; **P<0.01, *P<0.05.

[0019] Figure 6 This is a bar chart showing the relative abundance of intestinal flora at the phylum level in mice of the NC group, LPS model group, and Mujie capsule administration group of this invention.

[0020] Figure 7 The histograms show the relative abundance of different bacterial genera in the gut microbiota of mice in the NC group, LPS model group, and Mujie capsule administration group of this invention; a represents *Bacillus*; b represents *Isobacterium*; c represents *Desulfovibrio*; d represents *Duborcella*; e represents *Isprevotella*; f represents *UCG-006* (family Trichophytonceae); g represents *Lactobacillus*; h represents *Erysipelothrix*; i represents *Clostridium*; j represents *Prevotella*; *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0021] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0022] Research on Mujie capsules in the treatment of neuroinflammation and improvement of neurogenesis.

[0023] Example 1 The establishment of a mouse model of chronic inflammation and the administration method include the following steps: (1) Preparation of solutions required for model construction: ① LPS solution preparation: Take 0.5 mg of LPS powder, dissolve it in 10 mL of sterile physiological saline (0.9% NaCl), vortex at 400 rpm for 1 min, sonicate at 200 W for 1 min to prepare an LPS stock solution with a concentration of 0.05 mg / mL, filter it through a 0.22 μm filter membrane for sterilization, and set aside for later use. ② 0.5% Sodium Carboxymethyl Cellulose (CMC-Na) Solution: Weigh 1g of CMC-Na powder and put it into a beaker containing 160mL of distilled water. Place the beaker in a magnetic stirrer, adjust the water bath temperature to 70℃, stir at 600rpm for 15min until CMC-Na dissolves, adjust the pH to 6.8, and bring the volume to 200mL. Store at 4℃.

[0024] ③ Drug preparation: Weigh 220mg of drug powder and dissolve it in 1mL of 0.5% CMC-Na solution (2.2g / kg).

[0025] (2) Establishment of a chronic inflammation model in mice: Eight-week-old SPF-grade C57BL / 6J wild-type male mice were acclimatized for 6 days at a temperature maintained at 22±2℃, with alternating light and dark cycles, and free access to food and water. LPS 0.25 mg / kg (injection volume calculated based on mouse body weight, once daily) was injected intraperitoneally for 14 consecutive days to establish a chronic inflammation model. The blank control group (NC) was injected with an equal volume of physiological saline. The presence or absence of significant delayed weight gain or even weight loss in the mice was observed. (3) Drug administration method after establishing a mouse chronic inflammation model: After the model was established, the mice were randomly divided into a drug administration group (Mujie) and a model group (LPS). Each mouse was observed. When the condition was good, the mice were given a suspension of the contents of Mujie capsules by gavage. The drug administration group was given 2.2g / kg, while the model group and the blank group were given an equal volume of 0.5% CMC-Na solution by gavage for 4 weeks.

[0026] Example 2 The pharmacodynamic evaluation of Mujie capsules in preventing chronic inflammation included the following steps: (1) Weight monitoring and analysis: The first weight was recorded on the first day of modeling, and then every two days thereafter. After the modeling was completed, medication was administered for four weeks, with weight recorded every two days until the medication was finished. Figure 1 As shown, after LPS modeling, the weight gain of mice in the model group was significantly slowed down or even decreased; after administration of Mujie capsules, the weight of mice showed an overall upward trend, and by the end of administration, it was close to the level of the blank group.

[0027] (2) Behavioral testing and analysis: From day 1 to day 3 after drug administration, rotarod test, pull test, and treadmill test were performed sequentially. In the rotarod test, mice were placed on an accelerating rotarod, with the rotation speed increasing from 4 rpm to 45 rpm for 6 minutes. The fall latency was recorded. Each mouse was repeated 3 times, and the average value was taken. If no fall occurred within 6 minutes, it was counted as 360 seconds. In the pull test, the mouse's forelimbs were placed on a pull bar, and its tail was pulled backward at a constant speed of 5 cm / s. The peak grip strength was recorded. Each mouse was repeated 3 times, and the median value was taken. In the treadmill test, the initial speed was set at 5.5 m / min, and the speed was increased by 3 m / min every 2 minutes. The time to exhaustion was recorded (the mouse remained on the electrically stimulated board for more than 5 seconds without being able to follow the running belt or gave up after 3 consecutive attempts to run). Figure 2 As shown, the results of three behavioral studies—rotary bar, pull, and treadmill—showed that, compared to the NC group, mice with LPS-induced neuroinflammation had significantly reduced motor coordination, limb grip strength, and exercise endurance. Intervention with Mujie capsules could reverse the above-mentioned motor function impairments and restore the mice's motor performance.

[0028] (3) Histopathological analysis: After behavioral testing, mice were anesthetized with urethane, and their brain tissue was fixed by cardiac perfusion with physiological saline and 4% paraformaldehyde (PFA). Brain tissue was then fixed in 4% PFA for 48 hours and then transferred to 0.4% PFA for preservation. Coronal brain slices (40 μm) were obtained using a vibratory microtome. After antigen retrieval with citrate buffer and serum blocking, the slices were incubated overnight at 4°C with primary antibodies against neural stem cell marker GFAP (glial fibrillary acidic protein) (1:800), immature neuron marker DCX (dicortin) (1:400), and proliferating cell marker MCM2 (minimal chromosome maintenance protein 2) (1:400), respectively. The slices were then incubated with fluorescent secondary antibodies at room temperature for 70 minutes. After mounting, the hippocampal DG region was imaged using a laser confocal microscope. Imaging parameters were standardized, and three slices were scanned continuously at a slice thickness of 1 μm. Positive cells were counted using ImageJ software (3 brain slices per mouse, 3 fields of view per slice). GFAP + Labeling neural stem cells (NSCs), MCM2 + Mark newly proliferating / activated cells (PCs), GFAP + MCM2 + Labeling activated neural stem cells (aNSCs), DCX + Labeled neuroblasts (NBs), DCX + MCM2 + Activated neuroblasts (aNBs) were labeled, and the nuclei were labeled with 4',6-diamidinyl-2-phenylindole (DAPI). For example... Figure 3 , Figure 4 As shown, the number of NSCs, PCs, aNSCs, NBs, and aNBs in the hippocampal DG region of the model group mice was significantly increased, indicating that compensatory abnormal neurogenesis occurred after LPS-induced neuroinflammation. After treatment with Mujie capsules, the above indicators decreased significantly, suggesting that Mujie capsules can inhibit the abnormal excessive proliferation of inflammation-induced neural stem cells and neuroblasts and restore neurogenesis homeostasis.

[0029] (4) Biochemical index detection: Last blood samples were collected from mice and incubated at 37℃ for 2 hours. After serum separation, the samples were centrifuged at 1500 rpm for 15 minutes, and the supernatant clear serum was aliquoted. IL-1β, TNF-α, and IL-10 levels in the last mouse blood samples were detected using an ELISA kit (Wuhan Saixiaoman Biotechnology Co., Ltd.). Throughout the experimental workflow, the measurement procedure followed the manufacturer's instructions, measuring optical density at 490 nm and calculating concentrations based on the standard curve. Figure 5 As shown, compared to the blank group, the model group had a higher level of pro-inflammatory factor IL-1. 1β, TNF Increased α secretion, anti-inflammatory factor IL 10. Decreased secretion leads to an imbalance between pro-inflammatory and anti-inflammatory homeostasis in the body; after intervention with Mujie capsules, IL... 1β, TNF α release is inhibited, while IL release is also inhibited. 10. Secretion levels have rebounded significantly.

[0030] Example 3 The gut microbiota analysis of Mujie capsules in preventing chronic inflammation included the following steps: After euthanizing mice, cecal contents were collected under aseptic conditions and frozen at -80°C. Total DNA from gut microbiota was extracted using a DNA extraction kit, and the V4 region of the bacterial 16S rRNA gene was amplified using specific primers with sequencing barcodes (primers 515F and 806R for the 16S V4 region). After the PCR products passed quality control, libraries were constructed according to standard procedures, and PE250 sequencing was performed on the Illumina platform. The raw data underwent quality control (Q30 ≥ 80%) and splicing to obtain clean tags. Amplicon sequence variant (ASV) clustering was performed based on 97% similarity for species classification and abundance analysis. Based on ASVs, the richness and diversity of the bacterial community within a single sample (Alpha diversity, where a higher Simpson index indicates higher community diversity, and a higher Chao1 index indicates a greater number of bacterial species in the sample) and the differences in bacterial community structure between samples (Beta diversity) were analyzed. Species annotation was completed using the SILVA database (v138). Effect size analysis (LEfSe) was used to screen differentially expressed bacterial species (LEfSe) with a threshold score ≥4 to evaluate the regulatory effect of Mujie capsules on LPS-induced gut microbiota dysbiosis.

[0031] like Figure 6 As shown, the gut microbiota structure of the blank group and the model group was similar; after administration of Mujie capsules, the relative abundance of Firmicutes increased significantly, while the abundance of Bacteroidetes, Actinobacteriota, and Desulfobacterota decreased simultaneously, and the proportion of other trace bacteria changed only slightly, indicating that Mujie capsules can reshape the phylum-level gut microbiota structure. Figure 7As shown, compared with the control group, LPS modeling significantly increased the abundance of *Aerococcus*, *Allobaculum*, *Desulfovibrio*, and *Dubosiella*. After administration of Mujie capsules, the abundance of the above-mentioned pro-inflammatory bacterial genera decreased, while the abundance of beneficial bacterial genera such as *Alloprevotella*, *Lachnospiraceae*, *Lactobacillus*, *Erysipelotrichaceae*, *Clostridia vadinBB60*, and *Prevotella* increased significantly. Overall results indicate that LPS stimulation disrupts intestinal homeostasis and induces dysbiosis; Mujie capsules can regulate the composition of phylum and genus-level flora, inhibit the proliferation of harmful bacteria, enrich probiotics, and effectively repair LPS-induced intestinal flora imbalance.

Claims

1. The application of a type of wood-derived capsule in the preparation of drugs for treating neuroinflammation and improving neurogenesis.

2. The application of the *Mujie* capsule according to claim 1 in the preparation of drugs for treating neuroinflammation and improving neurogenesis, characterized in that, Neuroinflammatory diseases include any one of depression, Alzheimer's disease, multiple sclerosis, Parkinson's disease, and traumatic brain injury.

3. The application of the *Mujie* capsule according to claim 1 in the preparation of drugs for treating neuroinflammation and improving neurogenesis, characterized in that, The aforementioned Mujie capsules enhance the relative abundance of beneficial and homeostatic bacteria by increasing the ratio of Firmicutes to Bacteroidetes, while simultaneously inhibiting potentially pathogenic bacteria, improving intestinal barrier function, reducing peripheral inflammatory burden, and reshaping the intestinal microecological balance, thereby playing a role in treating neuroinflammation and improving neurogenesis.

4. The application of the *Mujie* capsule according to claim 3 in the preparation of drugs for treating neuroinflammation and improving neurogenesis, characterized in that, Beneficial and homeostatic bacteria include one or more of the following: Lactobacillus, Trichophyton spp. (UCG-006), Prevotella, Isprevotella, Erysipelothrix spp., Clostridium spp., and Adlerococcus spp.; pathogenic bacteria include one or more of the following: Gastroenterobacter spp., Desulfovibrio, Dubois spp., Staphylococcus spp., Myxospirillum spp., Helicobacter spp., and Streptococcus spp.

5. The application of the *Mujie* capsule according to claim 1 in the preparation of drugs for treating neuroinflammation and improving neurogenesis, characterized in that, The aforementioned Mujie capsules exert their therapeutic effects on neuroinflammation and improve neurogenesis by lowering serum levels of pro-inflammatory factors interleukin-1β and tumor necrosis factor-α, while simultaneously increasing levels of the anti-inflammatory factor interleukin-10.

6. The application of the *Mujie* capsule according to claim 1 in the preparation of drugs for treating neuroinflammation and improving neurogenesis, characterized in that, The described Mujie capsules exert their therapeutic effects on neuroinflammation and improve neurogenesis by inhibiting the abnormal and excessive proliferation of neural stem cells and neuroblasts in the hippocampal dentate gyrus induced by lipopolysaccharide, reducing the number of glial fibrillary acidic protein, bicortin, and microchromosome maintenance protein 2 positive cells in the hippocampal dentate gyrus, and restoring neurogenesis homeostasis.

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