Application of traditional Chinese medicine component coniferyl ferulate in treatment of neuroinflammation related diseases

Abstract

The invention relates to application of coniferyl ferulate serving as a traditional Chinese medicine component to treatment of neuroinflammation related diseases. Through a large number of researches, it is found that the natural compound CF derived from traditional Chinese medicine can promote transformation of macrophages from M1 phenotype to M2 phenotype by adjusting a JAK / STAT signal channel. According to the effect, neuroinflammation is inhibited in a dose-dependent mode, and the motor function and the activity capacity of mice after SCI are remarkably improved. The CF can effectively promote scar healing, enhance the survival rate of motor neurons, reduce the cavity area and increase the number of the motor neurons. Besides, the CF shows an excellent motor function recovery effect, which indicates that the CF has huge potential in the aspect of preventing and improving central nervous system diseases, especially spinal cord injury, as a functional food supplement with anti-neuroinflammation and neuroprotective activity, and is high in safety and suitable for long-term use; meanwhile, the invention effectively expands the types of medicines of the natural source SCI treatment active agent, and provides a new direction for the subsequent research and development of new medicines.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of the traditional Chinese medicine ingredient ferulic acid coniferyl ester in the treatment of diseases related to neuroinflammatory diseases. Background Technology

[0002] Spinal cord injury (SCI) refers to damage to the spinal cord, resulting in temporary or permanent functional impairment. It is characterized by high incidence, high medical costs, high disability rates, and an increasingly younger age of onset. SCI can be divided into traumatic and non-traumatic causes. Traumatic SCI is an acute injury to the spinal cord caused by external physical impacts (such as motor vehicle accidents, falls, sports-related injuries, etc.). In contrast, non-traumatic SCI refers to primary injury caused by acute or chronic disease processes (such as tumors, infections, degenerative intervertebral disc disease, ischemia-reperfusion injury, or vascular disease, etc.). The consequences of SCI mainly include sensory and motor dysfunction below the site of injury.

[0003] In traumatic spinal cord injury (SCI), the primary injury leads to local damage, affecting neurons and oligodendrocytes (myelinating cells of the central nervous system), disrupting blood vessels and impairing the blood-spinal barrier, thereby triggering a series of ongoing secondary injuries. Multiple factors contribute to the development of secondary injury from primary injury, including inflammatory responses, electrolyte imbalances, excitotoxicity, lipid peroxidation, and free radical damage. Inflammation is a major factor, playing a crucial role in the pathophysiology of SCI. Activated spinal microglia release inflammatory mediators, recruiting more immune cells, including lymphocytes, neutrophils, and more microglia, leading to neuronal death, axonal degeneration, and functional impairment. In the acute phase, the inflammatory response is exacerbated by the impaired blood-spinal barrier, resulting in increased swelling and further tissue damage. In the chronic phase, dense glial scarring forms, leading to permanent loss of sensory and motor function below the site of injury. Once these factors induce secondary injury and continue to worsen, irreversible damage eventually occurs, such as progressive demyelination, disruption of neuronal connections, and ultimately, loss of sensory and motor function.

[0004] Therefore, secondary injuries are more persistent and progressive than primary trauma. While inflammation may provide some protection immediately after SCI, excessive infiltration of immune cells is a major factor in neurodegeneration. These immune cells are attracted to the injury site by cytokines and chemokines secreted by activated microglia, astrocytes, and peripherally derived macrophages (PDMs). Currently, drugs used to treat SCI can be divided into two categories: neuroprotective agents, represented by minocycline and riluzole, and neuroregenerative agents, represented by VX-210. However, these drugs still face limitations in the treatment of SCI. Researching plant-derived dietary compounds as a nutritional intervention strategy during SCI is of great value and significance. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the prior art, thereby providing the application of ferulic acid coniferate in the treatment of spinal cord injury. Through extensive research, this invention has discovered that the natural compound CF derived from traditional Chinese medicine can promote the transformation of macrophages from the M1 phenotype to the M2 phenotype by regulating the JAK / STAT signaling pathway. This effect inhibits neuroinflammation in a dose-dependent manner and significantly improves motor function and activity in mice after spinal cord injury (SCI).

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0007] The first aspect of this invention provides the use of ferulic acid coniferyl ester in the preparation of medicaments for treating diseases related to neuroinflammatory diseases.

[0008] Preferably, the neuroinflammatory-related disease is selected from spinal cord injury.

[0009] Preferably, the spinal cord injury is selected from one or more of traumatic spinal cord injury and non-traumatic spinal cord injury.

[0010] The second aspect of this invention provides the use of ferulic acid coniferyl ester in the preparation of medicaments that improve physiological indicators of diseases related to neuroinflammation.

[0011] Preferably, the neuroinflammatory-related disease is selected from spinal cord injury.

[0012] Preferably, the spinal cord injury is selected from one or more of traumatic spinal cord injury and non-traumatic spinal cord injury.

[0013] Preferably, the physiological indicators related to neuroinflammation are selected from one or more of the following: indicators related to motor function recovery, indicators related to neuronal recovery, macrophage polarization marker levels, and inflammatory factor levels.

[0014] Preferably, the motor function recovery related indicators are selected from one or more of the following: BMS score, maximum contact area, regularity index, cadence, and motor evoked potentials.

[0015] Preferably, the neuronal recovery-related indicators are selected from one or more of the following: motor neuron survival rate, cavity area, number of motor neurons, and scar healing level.

[0016] Preferably, the macrophage polarization marker is selected from one or more of iNOS, Arg-1, Iba-1, CD86, CD206, P-JAK2, and P-STAT1.

[0017] Preferably, the inflammatory factor is selected from one or more of IFN-γ, IL-1β, IL-6, MIP-1α, MIP-1β, TNF-α, IL-4, and IL-10.

[0018] A third aspect of the present invention provides a pharmaceutical composition for treating diseases related to neuroinflammation, comprising ferulate ester and a pharmaceutically acceptable carrier.

[0019] Preferably, the neuroinflammatory-related disease is selected from spinal cord injury.

[0020] Preferably, the spinal cord injury is selected from one or more of traumatic spinal cord injury and non-traumatic spinal cord injury.

[0021] Preferably, the pharmaceutically acceptable carrier is selected from one or more of fillers, disintegrants, lubricants, binders, antioxidants, antibacterial agents, flavoring agents, fragrances, and chelating agents.

[0022] Spinal cord injury (SCI) leads to neuronal degeneration, causing irreversible motor and sensory dysfunction below the site of injury. The recovery process is influenced by multiple factors, with inflammation playing a crucial role in clearing necrotic tissue and cells in the early stages of injury. Preventing the progression of secondary injury is a key treatment strategy for SCI. While some small molecule compounds, such as methylprednisolone and 21-aminosteroids, have been used as anti-inflammatory treatments, these drugs are often accompanied by significant side effects. Plant-derived bioactive compounds are considered safe and effective alternatives for preventing or reducing inflammation due to their good safety profile and nutritional value.

[0023] As a commonly used drug for treating spinal cord injury (SCI), minocycline mainly exerts its neuroprotective effects by inhibiting inflammatory factors (such as TNF-α and IL-1β), reducing microglial cell activation, and inhibiting matrix metalloproteinases (MMPs). However, the pathological process of spinal cord injury involves multiple links, including inflammation, oxidative stress, excitotoxicity, and apoptosis, and a single anti-inflammatory effect is insufficient to comprehensively block the injury cascade. Although minocycline is more lipid-soluble than other tetracyclines, its efficiency in crossing the blood-brain barrier is still relatively low. Disruption of the blood-spinal cord barrier after spinal cord injury may affect the attainment of effective drug concentrations. As a tetracycline, long-term use of minocycline may cause gastrointestinal reactions (such as nausea and vomiting), dysbiosis, liver damage, and even induce drug-resistant bacterial infections, especially in patients with spinal cord injury complicated by infection, where the risk is higher.

[0024] The core mechanism of riluzole is to inhibit voltage-gated sodium channels, reducing glutamate release and thus alleviating neuronal excitotoxicity. This mechanism is more suitable for the chronic neuronal degeneration scenario in amyotrophic lateral sclerosis (ALS). However, the excitotoxicity in the acute phase of spinal cord injury is mainly caused by the massive release of glutamate due to trauma, and the inhibitory effect of riluzole may not be sufficient to counteract the damage caused by acute high concentrations of glutamate. Furthermore, riluzole cannot directly inhibit inflammatory factors, oxidative stress, or apoptosis pathways, making it difficult to comprehensively block the cascade reaction after spinal cord injury. Additionally, riluzole has limited lipid solubility and requires specific transport proteins to enter the central nervous system. The disruption of the blood-spinal cord barrier after spinal cord injury may not significantly increase its concentration within the spinal cord, resulting in insufficient local effective drug concentration. Moreover, since spinal cord injury is often accompanied by structural damage such as axonal rupture and demyelination, riluzole can only protect neurons that are not completely damaged and has no repair effect on existing structural damage.

[0025] Coniferyl ferulate (CF) is found in a variety of medicinal and edible plants, including Angelica sinensis (Oliv.) Diels, Ligusticum chuanxiong Hort., and Coreopsis longstem ngula, and is widely distributed and abundant in natural sources. CF possesses various pharmacological activities, such as vasodilatory, antibacterial, anticancer, and antioxidant effects. Previous studies have shown that CF, at appropriate doses, has a significant anti-inflammatory effect on the central nervous system. In a chronic unpredictable stress model, CF not only reduced depressive-like behavior but also improved the ultrastructure of the colonic mucosa and alleviated colonic inflammation. This was achieved by inhibiting stress-induced increases in IL-6, IL-1β, and TNF-α levels while promoting the release of the anti-inflammatory cytokine IL-10. However, the anti-inflammatory effects of CF in neuroinflammatory injuries, especially spinal cord injuries, and its underlying mechanisms remain unclear.

[0026] This invention aims to explore the anti-inflammatory effects of CF in the context of SCI (spasmodic inflammatory disease) and elucidate its mechanism, with a particular focus on the role of microglial polarization in regulating neuroinflammation. In summary, through extensive research, this invention has found that the natural compound CF derived from traditional Chinese medicine can promote the transformation of macrophages from the M1 phenotype to the M2 phenotype by regulating the JAK / STAT signaling pathway. This effect inhibits neuroinflammation in a dose-dependent manner and significantly improves motor function and activity in mice after SCI. CF can effectively promote scar healing, enhance motor neuron survival, reduce cavity area, and increase the number of motor neurons. Furthermore, CF exhibits excellent motor function recovery effects, indicating that as a functional food supplement with anti-neuroinflammatory and neuroprotective activities, it has great potential in preventing and improving central nervous system diseases, especially spinal cord injuries. As a plant extract, ferulic acid coniferyl ester is mainly derived from medicinal and edible plants (such as angelica and chuanxiong), possessing typical medicinal and edible properties. Its toxicity is generally lower than that of small molecule chemically synthesized drugs such as minocycline and riluzole. It can be metabolized in the liver into components such as coniferyl alcohol, exhibiting excellent safety and suitability for long-term use. At the same time, this invention effectively expands the types of drugs with natural SCI therapeutic activity, providing a new direction for subsequent new drug development. Attached Figure Description

[0027] Figure 1 This is a schematic diagram showing the BMS score results of different groups of SCI mice at different time points.

[0028] Figure 2 This is a schematic diagram showing the MEP test results of different groups of SCI mice.

[0029] Figure 3 This is a schematic diagram showing the results of the maximum contact area analysis for SCI mice in different groups.

[0030] Figure 4 This is a schematic diagram showing the results of regularity index analysis in different groups of SCI mice.

[0031] Figure 5 This is a schematic diagram showing the gait frequency analysis results of SCI mice in different groups.

[0032] Figure 6 This is a schematic diagram showing the Nissl staining analysis results of different groups of SCI mice.

[0033] Figure 7 This is a schematic diagram showing the ChAT staining analysis results of different groups of SCI mice.

[0034] Figure 8 This is a schematic diagram showing the results of GFAP immunostaining analysis in different groups of SCI mice.

[0035] Figure 9This is a schematic diagram showing the results of immunofluorescence assay for detecting the levels of relevant biomarkers (Iba-1, iNOS, Arg-1) in the spinal cord of SCI mice.

[0036] Figure 10 This is a schematic diagram showing the results of quantitative analysis of relevant biomarkers (iNOS, Arg-1) in the spinal cord of SCI mice using immunofluorescence assay.

[0037] Figure 11 This is a schematic diagram showing the results of qRT-PCR detection of RNA expression levels of relevant markers (Arg-1, iNOS, CD86, CD206) in the spinal cord of SCI mice.

[0038] Figure 12 This is a schematic diagram showing the results of Western blot analysis of the expression of phosphorylated JAK2 (P-JAK2) and phosphorylated STAT1 (P-STAT1) in different groups of SCI mice.

[0039] Figure 13 This is a schematic diagram showing the results of qRT-PCR detection of phosphorylated JAK2 (P-JAK 2) and phosphorylated STAT1 (P-STAT1) expression in different groups of SCI mice.

[0040] Figure 14 This is a schematic diagram showing the results of Luminex system analysis of the levels of inflammation-related cytokines in different groups of SCI mice.

[0041] Figure 15 This diagram illustrates the results of quantitative analysis of inflammation-related cytokine levels in different groups of SCI mice. Detailed Implementation

[0042] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0043] Unless otherwise specified, the experimental animals used in this invention (adult female C57BL / 6 mice (6-8 weeks old, weighing 17-22g)) were purchased from Beijing Huafukang Medical Experimental Animal Center and housed in a sterile environment at the animal facility of Jinan University; CF (purity ≥98%) was purchased from Chengdu Ruifende Biotechnology Co., Ltd.; and all other reagents used were commercially available. For animal experiments, the relevant procedures and methods complied with the requirements of Jinan University's experimental animal and medical ethics guidelines and were performed in accordance with their regulations. The experimental methods used in this invention, such as cell biology experiments, molecular biology experiments, and animal experiments, are all conventional methods and techniques in the field.

[0044] Representative results from biological experiments were selected from replicates and presented in the contextual figures. Data were displayed as mean ± SD and mean ± SEM as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 8.0 or SPSS 22.0 software. Standard medical statistical methods such as t-tests, chi-square tests, and ANOVA were used to compare differences in means between two or more groups. p < 0.05 was considered statistically significant, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0045] Example 1

[0046] First, an animal model of spinal cord injury is constructed. The specific steps are as follows:

[0047] (1) Remove the hair from the back of the mouse to expose the skin on the back of the mouse.

[0048] (2) Anesthesia: Deep anesthesia was achieved by intraperitoneal injection of 0.3% pentobarbital at a dose of 37.5 mg / kg. Stable breathing, significantly weakened limb muscle strength, and disappearance of pain reflex and corneal reflex indicated successful anesthesia.

[0049] (3) Exposure of the spinal cord: Disinfect the skin by wiping twice with iodine-soaked cotton balls; determine the level of the 11th-12th thoracic vertebrae (T11-T12) and make a 2.5cm longitudinal incision in the midline of the back at the T11-T12 level. Bluntly dissect the paraspinal muscles, remove the spinous process and lamina with bone forceps, and thoroughly expose the spinal cord at the T11-T12 level. Perform laminectomy at the T11-T12 vertebral body level, taking care to avoid additional tissue damage during the procedure. Then use a fixator to open and fix the spinal cord to fully expose it.

[0050] (4) The mice were secured to the impactor (New York University impactor), and the spinal cord was positioned under the impactor. Simultaneously, the impact location was confirmed by a laser beam monitored by the impactor. The degree of spinal cord injury was determined by the impact depth. In this embodiment, a 10-gram rod was dropped from a height of 6.25 mm to cause contusion damage to the exposed spinal cord. After the impact, the mice were removed from the injury device and from the fixator. Sutures were used to suture the mouse muscles and skin after adequate hemostasis. Mice in the sham surgery group underwent T11-T12 laminectomy without spinal cord impact.

[0051] (5) Close the incision and administer 2000U of gentamicin subcutaneously once a day for 3 consecutive days. Empty the artificial bladder every 8 hours until the patient can urinate on their own.

[0052] Mice with spinal cord injury were randomly divided into 5 groups (n=8 per group): sham-operated group (Sham group, only the lamina was removed, without spinal cord injury), model group (SCI group), minocycline treatment group (Injury + Mino), low-dose CF group (Injury + CF (25 mg / kg / day)), and high-dose CF group (Injury + CF (50 mg / kg / day)). The SCI group received 200 μL of PBS by gavage daily for one week after injury; the Mino group received 200 μL of Mino (50 mg / kg) intraperitoneally once daily for one week after injury. The CF group received CF dissolved in ethanol by gavage. Mice in the sham-operated group received an equal volume of physiological saline.

[0053] Damage to the structure and function of the spinal cord can lead to motor, sensory, and autonomic dysfunction below the level of injury. Therefore, behavioral analysis in SCI models is crucial for assessing the extent of injury and treatment efficacy. These analyses are used to determine the severity and location of the lesion, record post-SCI recovery, and identify the integrity of specific motor and sensory pathways that may contribute to post-SCI recovery. To assess motor function recovery in CF-treated SCI mice, BMS analysis was first performed on each group of mice. Specifically, mice were placed in an open field for 4 minutes, and two independent observers assessed hindlimb motor function in each group using a single-blind method. The average of the two hindlimb scores was recorded as the BMS score for each sample. Results showed that all treatment groups exhibited complete paraplegia (BMS score = 0) one day after SCI. However, compared to the SCI group, mice treated with CF (50 mg / kg) showed significantly improved BMS scores 2 to 8 weeks after SCI (see [link to relevant documentation]). Figure 1 ).

[0054] To confirm the above conclusions, motor evoked potential (MEP) testing was performed 8 weeks after SCI, and the specific steps are as follows:

[0055] The stimulating electrode was placed on the rostral end of the spinal cord, and the recording electrode was placed on the flexor biceps femoris muscle. A single 0.5 mA square wave stimulus was applied for 0.5 ms duration, 2 ms delay, and 1 Hz frequency. Neural conduction function in the hindlimb was represented by the amplitude (PP value), which was measured from the onset of the first response wave to its peak value.

[0056] The results showed that, compared with the SCI group (mean 0.67 mV) and the CF (25 mg / kg) group (mean 2.44 mV), the mean amplitude of motor evoked potentials (MEPs) in the CF (50 mg / kg) treatment group was significantly higher (mean 3.73 mV) (see [link to original text]). Figure 2 ).

[0057] Subsequently, an automated quantitative gait analysis system (CatWalk XT, Noldus) was used to comprehensively evaluate the recovery of motor function in mice after spinal cord injury using CF (CatWalk XT). Specifically, gait was assessed in each group of mice 8 weeks after spinal cord injury using the CatWalk system. Each mouse walked continuously along a 50 cm path on a glass plate, completing at least three trials. The CatWalk system automatically detected and marked each paw print, generating a series of parameters, including paw statistics, average speed, gait frequency, and gait sequence. The regularity index was used to assess walking coordination, calculated as: the number of normal gait sequence patterns multiplied by 4, then divided by the total number of paw placements. The maximum contact area refers to the maximum area of ​​contact between the paw and the walking surface.

[0058] The results showed that, in terms of maximum contact area, regularity index, and gait frequency, the CF (50 mg / kg) treatment group exhibited a larger mean maximum contact area (0.08 ± 0.003 cm²) at 8 weeks post-injury. 2 vs. 0.04±0.01cm 2 (p < 0.0001, n = 8 animals / group), higher average regularity index (66.41 ± 0.85% vs. 40.06 ± 1.5%, p < 0.0001, n = 8 animals / group), and higher average cadence (16.61 ± 0.84 vs. 7.66 ± 0.53, p < 0.001, n = 8 animals / group) (see See Figure 3-5 ).

[0059] Furthermore, Nissl staining (for labeling neurons), ChAT staining (for labeling motor neurons), and GFAP immunostaining (for labeling astrocytes) were used 8 weeks after injury to assess the neuroprotective effect of CF on SCI mice. Results showed that the mean lesion volume was significantly reduced in the CF (50 mg / kg) treatment group compared to the SCI group, indicating that CF promoted the recovery of damaged tissue. Quantitative analysis revealed that in the Nissl staining experiment, the CF (50 mg / kg) treatment group had more surviving motor neurons in the anterior horn of the injured spinal cord compared to the SCI group; the ChAT staining experiment also showed that the CF (50 mg / kg) treatment group had more motor neurons than the SCI group; in addition, astrocyte staining also showed that CF significantly reduced the cavity area, exhibiting a dose-response relationship (see [link to relevant documentation]). Figure 6-8 These results collectively confirm that CF promotes neuroprotection and enhances neuronal function in vivo.

[0060] Example 2

[0061] Macrophages play a crucial role in the inflammatory progression following spinal cord injury (SCI) and significantly influence tissue repair activity after injury. To further investigate whether free radical fibrosis (CF) regulates macrophage polarization in SCI animals, immunofluorescence was used to analyze the levels of relevant biomarkers in the spinal cord of SCI mice. The specific steps are as follows:

[0062] (1) Each group of SCI mice was deeply anesthetized with pentobarbital, and then 30 mL of phosphate-buffered saline (PBS) was perfused through the heart, followed by perfusion of 4% paraformaldehyde (PFA).

[0063] (2) A 5 mm segment of spinal cord was removed centered on the T11 vertebra and immediately immersed in 4% PFA at 4°C overnight. The spinal cord segment was then transferred to a 15% sucrose solution until the tissue settled, and then transferred to a 30% sucrose solution, following the same procedure.

[0064] (3) After fixation, the spinal cord tissue sample was rinsed in water several times, dehydrated, embedded in paraffin, frozen, and then cut into 15μm thick slices using a Leica CM3050 S microtome.

[0065] (4) Permeabilize the sections with 0.2% Triton X-100 (Sigma-Aldrich) and then incubate overnight with the following mouse-specific primary antibodies: Iba-1 (1:1000), Arg-1 (1:500), iNOS (1:500), choline acetyltransferase (ChAT, 1:500) and glial fibrillary acidic protein (GFAP, 1:1000).

[0066] (5) Use 1:1000 of fluorescent Alexa Fluor red or green secondary antibody (Invitrog en Vector) for single and double staining, and incubate at room temperature for 2 hours.

[0067] (5) After washing with phosphate-buffered saline (PBST) containing Tween, tissue sections were mounted with DAPI-containing Vectashield slides for observation. Images were captured and analyzed using an inverted fluorescence microscope (Axio Observer A1; Carl Zeiss).

[0068] Test results as follows Figure 9-10 As shown in the figure. The results showed that, compared with other groups, the CF (50 mg / kg) treatment group had the lowest iNOS expression and the highest Arg-1 expression.

[0069] Furthermore, qRT-PCR was used to detect the RNA levels of relevant biomarkers. The specific steps are as follows:

[0070] (1) RNA was extracted from the spinal cord tissue of each group of SCI mice using an RNA extraction kit (Beyotime). Each spinal cord tissue sample was incubated in Trizol at room temperature for 5 minutes.

[0071] (2) Transfer to a purification column and centrifuge at 12000g for 30 seconds at room temperature. Then, add 600μL of wash buffer I to the column and centrifuge again at 12000g for 30 seconds at room temperature.

[0072] (3) Add 600uL of washing buffer II and centrifuge at 16000g for 2 minutes at room temperature.

[0073] (4) Elute the purified RNA into a clean 1.5 mL microcentrifuge tube with 30 μL of RNase-free water, denature at 65 °C for 5 minutes, and then cool immediately.

[0074] (5) Complementary DNA (cDNA) was synthesized using the ReverTra Ace qPCR RT kit (TOYOBO). qRT-PCR was performed using the Green Real-Time PCR Detection Kit (TOYOBO), with GAPDH as an internal control. The total reaction volume was 20 μL, containing 10 μL of 1x qRT-PCR kit. Green RT-PCR premix, 6.4 μL distilled water, 0.8 μL of each PCR forward and reverse primer (0.4 μM), and 2 μL of sample RNA. The thermal cycling conditions were as follows: 95°C for 60 seconds, followed by 40 cycles: 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 45 seconds. Each sample was tested in triplicate.

[0075] Test results as follows Figure 11 As shown in the figure, the results are consistent with the immunofluorescence assay, indicating that CF significantly promotes Arg-1 expression while inhibiting iNOS expression, and also promotes CD206 expression while inhibiting CD86 expression. These results collectively demonstrate that CF can promote the increase of M2 macrophages in the injured spinal cord while inhibiting the production of M1 macrophages.

[0076] Example 3

[0077] Existing research indicates that the inflammatory response in SCI is regulated by the JAK2 / STAT1 signaling pathway. To determine the impact of CF on this classic inflammatory pathway, Western blot analysis was performed to investigate the expression of phosphorylated JAK2 (p-JAK2) and phosphorylated STAT1 (p-STAT1) in different groups of SCI mice. The specific steps are as follows:

[0078] (1) The spinal cord tissue samples of each group of SCI mice were incubated on ice for 30 min using RIPA lysis buffer (Beyotime) containing 2 mg / mL aprotinin and 1 mM PMSF.

[0079] (2) Centrifuge the lysate at 12000g for 5 min at 4℃, take the supernatant and add loading buffer (to 1×), and incubate in a 95℃ water bath for 10 min.

[0080] (3) Take the sample prepared in step (2) and perform protein gel electrophoresis (12% separating gel concentration).

[0081] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200mA constant current transfer for 2h).

[0082] (5) After the transfer is completed, the PVDF membrane is removed and placed in a blocking solution containing 5% skim milk, and sealed at room temperature for 2 hours at 10 rpm on a vertical shaker.

[0083] (6) After the sealing is completed, clean the PVDF membrane, immerse it in the primary antibody, and incubate it overnight at 4°C and 10 rpm on a vertical shaker. After the primary antibody incubation is completed, clean the PVDF membrane, then immerse it in the secondary antibody and incubate it at room temperature and 10 rpm on a vertical shaker for 2 hours.

[0084] (8) After the secondary antibody incubation is completed, the PVDF membrane is washed and the target protein is detected using a chemiluminescence analyzer (iBrig ht™ CL1500, USA).

[0085] Test results as follows Figure 12 As shown in the figure. The results showed that, compared with the SCI group, the expression levels of P-JAK2 and P-STAT1 in the CF (50 mg / kg) treatment group were significantly lower. Subsequently, RNA levels were analyzed by qR T-PCR, and the results were consistent with those of Western blot, indicating that CF can effectively reduce the expression levels of P-JAK2 and P-STAT1, suggesting that CF inhibits JAK2 / STAT1 signaling in activated macrophages (see Figure 1). Figure 13 This, in turn, regulates the polarization of M1 / M2 macrophages.

[0086] Furthermore, the levels of inflammation-related cytokines in different groups of SCI mice were analyzed using the Luminex system, and the results are as follows: Figure 14-15As shown, different colors represent protein levels from low (blue) to high (red). Compared to the SCI group, the expression of pro-inflammatory cytokines, including IFN-γ, IL-1β, IL-6, MIP-1α, and TNF-α, was downregulated in the CF (50 mg / kg and 25 mg / kg) treatment groups. Conversely, the expression of anti-inflammatory cytokines, particularly IL-4 and IL-10, was upregulated after CF treatment. These results indicate that CF not only promotes the expression of anti-inflammatory cytokines but also inhibits the production of pro-inflammatory cytokines in SCI mice.

[0087] In summary, CF significantly inhibits neuroinflammation in SCI mice and improves their motor function in a dose-dependent manner. Specifically, this invention uses BMS scoring, foot error testing, and gait analysis using the CatWalk system to evaluate the pro-reparative effect of CF on SCI mice. The BMS scoring assessed various aspects of hindlimb function in mice, including mobility, joint range of motion, foot placement, gait coordination, balance, and tail movement. During an 8-week evaluation, CF significantly improved SCI-induced motor deficits. Furthermore, in gait coordination assessment, CF significantly increased regularity index, gait frequency, and maximum contact area, contributing to the recovery of hindlimb function in SCI mice. When measuring motor evoked potentials (MEPs), amplitude reflects the transmission efficiency of the motor pathway from the cerebral cortex to the muscles. Reduced or absent amplitude may indicate spinal cord or brain damage. The results show that CF significantly increased amplitude after SCI, indicating that CF can improve the transmission efficiency of motor pathway signals to near-normal levels. In general, spinal cord injury leads to changes in the spinal cord microenvironment and structural components involved in hindlimb motor function, which is detrimental to neuronal regeneration and recovery of motor function. The results of this invention show that CF significantly improves hindlimb motor ability and overall motor function, indicating that CF can enhance behavioral function in animals following T11-12 contusions.

[0088] The plasticity of spinal circuits and the intrinsic projection of the spinal cord into the lumbar spinal cord are crucial for the recovery of hindlimb motor function after injury. The death or dysfunction of spinal cord neurons can lead to interruption of signal transmission, resulting in loss of motor and sensory function. This invention, through immunofluorescence staining of neurons, found that CF (fluid-forming cells) significantly increased the number of neurons lost due to SCI (spinal cord injury). Motor neuron damage is one of the main causes of motor function loss. During SCI, motor neurons are particularly vulnerable to damage or death, leading to interruption of signal transmission, muscle weakness, or paralysis. Based on neuronal assessment, the morphology and survival status of motor neurons (cholinergic neurons) were further analyzed to confirm the above conclusions. Consistent with the neuronal staining analysis results, CF significantly enhanced the survival rate of motor neurons. Astrocytes are the most abundant type of glial cell in the central nervous system, mainly responsible for maintaining neuronal nutrition, regulating ion balance, supporting the blood-brain barrier, and playing an important protective and repairing role in neuronal injury. After SCI, astrocytes participate in the pathological process of SCI through reactive astrogenesis, proliferating and forming glial scars. This invention analyzes the area of ​​the lesion cavity by staining astrocytes. The results showed that, compared with the injury group, the CF group had a higher survival rate of spinal cord neurons and a smaller lesion cavity. This suggests that CF may play a neuroprotective role, preventing the expansion of the lesion cavity and promoting functional recovery after contusion.

[0089] Macrophages are the primary inflammatory effector cells in primary diseases. Following spontaneous intraepithelial neoplasia (SCI), a dynamic causal relationship exists between inflammation and macrophage polarization. Injury-induced inflammatory responses promote macrophage polarization towards the M1 phenotype, while M1 macrophages further exacerbate inflammation by secreting pro-inflammatory factors. M1 macrophages are typically activated by pathogens, LPS, GM-CSF, TNF-α, and IFN-γ. Macrophage polarization is a complex signaling process at the molecular level, involving multiple factors and regulated by various signaling molecules, including the JAK / STAT pathway, IRF pathway, and Notch signaling pathway. STAT proteins are found in various tissues and organs of mammals; they are transcription factors that bind to regulatory regions of DNA to control gene transcription. STAT protein activation depends on JAKs, thus forming the JAK / STAT signaling pathway. During macrophage polarization, STATs are phosphorylated by their corresponding JAKs, forming dimers that reduce their affinity for receptors, allowing them to dissociate and enter the nucleus to exert their biological effects. The results of this invention indicate that in SCI, CF can inhibit the activation of the JAK / STAT pathway by suppressing the phosphorylation levels of JAK2 and STAT1, which is related to the regulation of macrophage phenotypic polarization from M1 to M2.

[0090] M1 macrophages secrete inflammatory factors (such as IL-1β, TNF-α, IL-6, and IFN-γ) at the site of injury, exacerbating spinal cord injury (SCI). Conversely, M2 macrophages release IL-4, IL-10, IL-13, and neurotrophic factors, inhibiting the inflammatory response and neuronal apoptosis. Therefore, modulating macrophage phenotype to promote SCI repair is a promising therapeutic direction. Excessive macrophage activation disrupts the spinal cord microenvironment and triggers inappropriate inflammatory responses, leading to further neurological dysfunction after SCI. IL-1α and TNF-α secreted by microglia play a crucial role in activating A1 astrocytes, which do not support neuronal survival, synapse formation, or phagocytosis, but instead induce neuronal death and demyelination. Analysis of inflammation-related cytokines using the Bio-Plex system revealed that CF exhibits significant anti-inflammatory effects, indicating that CF significantly inhibits the release of IFN-γ, IL-1β, IL-6, MIP-1α, and TNF-α. IFN-γ, IL-1β, IL-6, MIP-1α, and TNF-α are key pro-inflammatory cytokines primarily produced by immune cells. They play a crucial role in SCI and other inflammatory responses by activating and maintaining M1 macrophages, thereby inhibiting M2 macrophage polarization and promoting inflammatory states.

[0091] In addition to inhibiting pro-inflammatory cytokines, this invention also found that CF significantly enhanced the release of IL-4 and IL-10. IL-10 is mainly secreted by astrocytes and microglia in the central nervous system, and exerts its anti-inflammatory effect by inhibiting the transcription of various pro-inflammatory cytokines, chemokines, and inflammatory enzymes. Numerous studies have shown that IL-10 treatment in rat SCI models can protect against secondary inflammatory damage, upregulate the expression of neurotrophic factors, and promote functional recovery. IL-4 is a pleiotropic cytokine that can induce CD4+... + T cells differentiate into TH2-like cells and promote alternative activation of myeloid cells (macrophages and microglia). In immune cells, type I IL-4 receptors activate IRS-2 and STAT6 via JAK1 and JAK3. Conversely, type II receptors signal via JAK1 and TYK2, subsequently activating STAT6. Through tyrosine phosphorylation of IRS-2, gene transcription and cell proliferation driven by PI3K, AKT (protein kinase B), and nuclear factor NF-κB are activated. The results of this invention indicate that, compared to the simple injury group, animals treated with CF showed a significant upregulation of IL-10 and IL-4 expression in their damaged spinal cords.

[0092] In summary, this invention clarifies that the natural compound CF derived from traditional Chinese medicine can promote the transformation of macrophages from the M1 phenotype to the M2 phenotype by regulating the JAK / STAT signaling pathway. This effect inhibits neuroinflammation in a dose-dependent manner and significantly improves motor function and activity in mice after spinal cord injury (SCI). CF can effectively promote scar healing, enhance motor neuron survival, reduce cavity area, and increase the number of motor neurons. Furthermore, CF exhibits excellent motor function recovery effects, indicating that as a functional food supplement with anti-neuroinflammatory and neuroprotective activities, it has great potential in preventing and improving central nervous system diseases, especially spinal cord injury, and is highly safe for long-term use. Simultaneously, this invention effectively expands the range of drugs with natural SCI therapeutic activity, providing new directions for subsequent drug development.

[0093] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. Application of ferulic acid pine ester in the preparation of drugs for treating diseases related to neuroinflammation.

2. The application according to claim 1, characterized in that, The neuroinflammatory diseases mentioned are selected from spinal cord injuries.

3. Application of ferulic acid pine ester in the preparation of drugs that improve physiological indicators of diseases related to neuroinflammation.

4. The application according to claim 3, characterized in that, The neuroinflammatory diseases mentioned are selected from spinal cord injuries.

5. The application according to claim 3, characterized in that, The physiological indicators related to neuroinflammatory diseases are selected from one or more of the following: indicators related to motor function recovery, indicators related to neuronal recovery, macrophage polarization marker levels, and inflammatory factor levels.

6. The application according to claim 5, characterized in that, The relevant indicators for motor function recovery are selected from one or more of the following: BMS score, maximum contact area, regularity index, cadence, and motor evoked potentials.

7. The application according to claim 5, characterized in that, The neuronal recovery-related indicators are selected from one or more of the following: motor neuron survival rate, cavity area, number of motor neurons, and scar healing level.

8. The application according to claim 5, characterized in that, The macrophage polarization markers are selected from one or more of iNOS, Arg-1, Iba-1, CD86, CD206, P-JAK2, and P-STA T1.

9. The application according to claim 5, characterized in that, The inflammatory factors are selected from one or more of IFN-γ, IL-1β, IL-6, MIP-1α, MIP-1β, TNF-α, IL-4, and IL-10.

10. A pharmaceutical composition for treating diseases related to neuroinflammation, characterized in that, This includes ferulate ester and pharmaceutically acceptable carriers.

Images