Application of lactobacillus paracasei IOB413 fermented ginseng metagen in cerebral arterial thrombosis

Abstract

The invention provides an application of a lactobacillus paracasei IOB413 fermented ginseng metagen in cerebral arterial thrombosis, and belongs to the technical field of microorganisms. The lactobacillus paracasei IOB413 fermented ginseng postbiotics can specifically act on cerebral vascular generation and neural restoration in cerebral arterial thrombosis, can promote expression of VEGF and CD34 in an ischemic penumbra region of brain tissue, increases the capillary density to induce cerebral vascular generation, and recovers blood supply in an ischemic region; in addition, PI3K / AKT / mTOR signal channels can be up-regulated, P-PI3K, P-AKT and P-mTOR expressions can be increased, the HIF-1alpha level can be adjusted, cell proliferation can be regulated to assist vascularization, nerve protection and nerve remodeling effects can be achieved, and brain tissue repair can be assisted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to application of a Paracaseolyticum IOB413 fermentation of ginseng probiotics in ischemic stroke. BACKGROUND

[0002] Stroke is a neurological disease caused by cerebral infarction or hemorrhage leading to insufficient cerebral circulation. After stroke, neurological defects occur, including balance problems, hemiplegia, loss of sensation and vibration, paralysis, reduced reflexes, ptosis, visual impairment, memory impairment, aphasia, and disability. According to the pathological changes of cerebral vessels, stroke is divided into ischemic stroke (Cerebral Ischemic Stroke, CIS) and hemorrhagic stroke (intracerebral hemorrhage and subarachnoid hemorrhage). Ischemic stroke is caused by stenosis or occlusion of cerebral vessels, leading to ischemic and anoxic injury of the brain tissue around the vessels, causing dysfunction of brain cells and related symptoms. Ischemic stroke is a pathological cascade reaction of multiple tissues and cells caused by cerebral vascular occlusion, mainly including energy deficiency, excitatory amino acid toxicity, oxidative stress damage, intracellular calcium overload, and inflammatory cytokine damage.

[0003] Vascular regeneration is the core link of the brain's intrinsic repair mechanism after stroke. It plays an indispensable multiple role in saving ischemic tissue, promoting nerve repair, and ultimately functional recovery by reconstructing blood flow, transporting nutrients, secreting neurotrophic factors, supporting neurogenesis and synapse formation, promoting inflammation resolution, and reconstructing the neurovascular unit. Vascular endothelial growth factor (VEGF) is an important factor in blood vessel development and is one of the important regulatory factors affecting brain remodeling after ischemic stroke. The core function of VEGF is to promote vascular endothelial cell proliferation, migration, and survival, increase vascular permeability, and induce neovascularization (angiogenesis). VEGF also has the effect of protecting degenerating motor neurons in vitro and in rodent models.

[0004] Current treatment for ischemic stroke is mainly divided into two stages. The first stage is thrombolytic therapy for ischemic stroke, including drug thrombolysis and mechanical thrombectomy, aiming to eliminate vascular occlusion or change stenotic vascular space to achieve the effect of unobstructed blood flow. The second stage is ischemia-reperfusion injury treatment, mainly divided into non-drug therapy, such as cognitive function training, acupuncture, etc., and drug therapy, such as oxygen free radical scavengers, NMDA (N-Methyl-D-Aspartate Receptor Antagonist) receptor antagonists, calcium ion antagonists, etc. The existing scheme focuses too much on local blood vessels and neurons, ignoring the persistent destruction of systemic inflammation and metabolic disorders mediated by the "gut-brain axis" to neural repair. SUMMARY

[0005] In order to solve the above-mentioned defects and deficiencies existing in the prior art, the application aims to provide an application of a ginseng metaplasma fermented by Paracaseiclovetus IOB413 in the treatment of ischemic stroke.

[0006] Paracaseiclovetus IOB413 (Lactobacillus paracasei IOB413) Lacticaseibacillus paracasei ) is independently screened from natural fermented sourdough in Tianjin residents' homes. The strain has been preserved and the physical and chemical indicators have been detected. On March 19, 2021, the bacterial identification test was conducted in China Food Fermentation Industry Research Institute Co., Ltd. The colony is white, round, surface wet, opaque, and the edge of the colony is neat. It is classified and named as Paracaseiclovetus (Lactobacillus paracasei). Lacticaseibacillus paracasei On June 29, 2018, it was preserved in the General Microbiological Center of China Microorganism Strain Preservation Management Committee (CGMCC) located at No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 16022.

[0007] One of the purposes of the application is to provide an application of a ginseng metaplasma fermented by Paracaseiclovetus IOB413 in the preparation of a drug for treating ischemic stroke. The preservation number of Paracaseiclovetus IOB413 is CGMCC No. 16022.

[0008] Preferably, the preparation method of the ginseng metaplasma fermented by Paracaseiclovetus IOB413 comprises the following steps: Inoculate the Paracaseiclovetus IOB413 strain in the cryopreservation tube into the slant medium and place it in 34-38℃ for 22-26h. Inoculate the slant strain into the liquid culture medium, and culture at 34-38℃ in a closed state for 18-22h to obtain the fermentation seed liquid. Inoculate the fermentation seed liquid into the ginseng solid-state fermentation medium for solid-state fermentation, then inactivate, dry and crush the fermented raw materials to obtain the ginseng metaplasma fermented by Paracaseiclovetus IOB413.

[0009] Preferably, the solid-state fermentation has a material-water ratio of 1:1.0-1:1.5, an inoculation amount of 5-10%, a fermentation time of 24-48h at 34-38℃, a sterilization temperature of 85-100℃, and a drying moisture content controlled at ≤12%.

[0010] Preferably, the ischemic stroke includes hemiplegia, forelimb flexion, walking imbalance, abnormal sensory function, paralysis, reduced reflex, eyelid ptosis, visual impairment, eye shrinkage and darkening caused by brain tissue ischemia, memory impairment, aphasia, disability, brain tissue edema, brain tissue cell vacuolar degeneration, cell swelling and necrosis, and inflammatory cell infiltration.

[0011] The second object of the present application is to provide an application of the Paracaseicilactobacillus IOB413 fermented ginseng metaplasma in preparing an antioxidant health food or microecological preparation.

[0012] The beneficial effects of the present application are: The present application relates to the application of Paracaseicilactobacillus IOB413 fermented ginseng metaplasma in ischemic stroke. The strain is isolated from naturally fermented sourdough in Tianjin residents' homes, has been preserved in the China General Microbiological Culture Collection Center (CGMCC No. 16022), and is identified as lactobacillus with clear biological characteristics, and the acute oral toxicity test shows that it is actually non-toxic. The Paracaseicilactobacillus IOB413 fermented ginseng metaplasma is prepared by using ginseng as a solid fermentation substrate, and is fermented, inactivated, dried, and crushed, and can be used for targeted treatment of ischemic stroke.

[0013] The metaplasma can significantly improve the neurological behavior of model animals, reduce the forelimb flexion and walking imbalance, reduce the serum MDA and NO levels, increase the SOD and GSH activities to resist oxidation, and inhibit the release of inflammatory factors such as IL-1β, TNF-α, and IL-6; it can also reduce the brain tissue water content and cerebral infarction volume, improve the blood coagulation function to prevent thrombosis, reduce the brain tissue cell degeneration and necrosis and inflammatory cell infiltration, and reduce the neural cell apoptosis rate.

[0014] In addition, the metaplasma can promote the expression of VEGF and CD34 in the ischemic penumbra of brain tissue, increase the microvessel density to induce angiogenesis, restore blood supply in the ischemic area, and up-regulate the PI3K / AKT / mTOR signaling pathway, increase the expression of P-PI3K, P-AKT, and P-mTOR, and regulate the level of HIF-1α, which can regulate cell proliferation, induce angiogenesis, and also play a role in nerve protection and neural remodeling, providing multi-dimensional help for the relief of ischemic stroke and brain tissue repair. BRIEF DESCRIPTION OF DRAWINGS

[0015] The drawings incorporated into the specification and forming a part thereof, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the application, wherein: Figure 1 Figure 1 is a diagram of the effect of Paracaseicilactobacillus IOB413 fermented ginseng metaplasma on the neurological behavior score of ischemic stroke model rats; Figure 2 Figure 2 is a diagram of the effect of Paracaseicilactobacillus IOB413 fermented ginseng metaplasma on the serum SOD level of cerebral ischemia-reperfusion rats; Figure 3 Figure 3 is a diagram of the effect of Paracaseicilactobacillus IOB413 fermented ginseng metaplasma on the serum MDA level of cerebral ischemia-reperfusion rats; Figure 4 Figure for the influence of panaxans fermented by paracasei IOB413 on serum NO level of cerebral ischemia-reperfusion rats; Figure 5 Figure for the influence of panaxans fermented by paracasei IOB413 on serum GSH level of cerebral ischemia-reperfusion rats; Figure 6 Figure for the influence of panaxans fermented by paracasei IOB413 on serum IL-1β content of cerebral ischemia-reperfusion rats; Figure 7 Figure for the influence of panaxans fermented by paracasei IOB413 on serum TNF-α content of cerebral ischemia-reperfusion rats; Figure 8 Figure for the influence of panaxans fermented by paracasei IOB413 on serum IL-6 content of cerebral ischemia-reperfusion rats; Figure 9 Figure for the influence of panaxans fermented by paracasei IOB413 on brain tissue water content of ischemic stroke rats; Figure 10 Figure for the influence of panaxans fermented by paracasei IOB413 on cerebral infarction area of ischemic stroke rats; Figure 11 Figure for the influence of panaxans fermented by paracasei IOB413 on PT of blood coagulation function of ischemic stroke rats; Figure 12 Figure for the influence of panaxans fermented by paracasei IOB413 on TT of blood coagulation function of ischemic stroke rats; Figure 13 Figure for the influence of panaxans fermented by paracasei IOB413 on APTT of blood coagulation function of ischemic stroke rats; Figure 14 Figure for the influence of panaxans fermented by paracasei IOB413 on FIB of blood coagulation function of ischemic stroke rats; Figure 15 Figure for the influence of panaxans fermented by paracasei IOB413 on HE staining of brain tissue pathology of ischemic stroke rats; Figure 16 Figure for the influence of panaxans fermented by paracasei IOB413 on TUNEL fluorescence value of brain tissue nerve cell apoptosis of ischemic stroke rats; Figure 17 Figure for the influence of panaxans fermented by paracasei IOB413 on microscope observation of VEGF expression of brain tissue of ischemic stroke rats; Figure 18A quantitative analysis of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on VEGF protein expression levels in brain tissue of rats with ischemic stroke. Figure 19 Microscopic observation of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on CD34 expression in brain tissue of rats with ischemic stroke; Figure 20 A quantitative analysis of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on the expression level of CD34 protein in the brain tissue of rats with ischemic stroke. Figure 21 The effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on microvessel density in brain tissue of rats with ischemic stroke. Figure 22 Western blot quantitative analysis of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on VEGF protein expression in brain tissue of rats with ischemic stroke; Figure 23 Western blot quantitative analysis of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on P-PI3K protein expression in brain tissue of rats with ischemic stroke; Figure 24 Western blot quantitative analysis of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on P-AKT protein expression in brain tissue of rats with ischemic stroke; Figure 25 Western blot quantitative analysis of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on P-mTOR protein expression in brain tissue of rats with ischemic stroke; Figure 26 This is a Western blot quantitative analysis of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on HIF-1α protein expression in brain tissue of rats with ischemic stroke.

[0016] Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracasei This strain was independently selected from naturally fermented sourdough from a resident's home in Tianjin. The strain has undergone strain preservation and physicochemical testing. On March 19, 2021, bacterial identification testing was conducted at the China National Research Institute of Food Fermentation Industries Co., Ltd. The colonies were white, round, moist, opaque, and with neat edges, and were classified and named *Lactobacillus paracasei*. Lacticaseibacillus paracasei), which was preserved in China General Microbiological Culture Collection Center (CGMCC) on June 29, 2018, and the address of the CGMCC is No. 1, Yihuan Road, Beijing, China. The preservation number of the CGMCC is CGMCC No. 16022. DETAILED DESCRIPTION

[0017] The application will be described in further detail below with reference to the drawings and embodiments. The specific embodiments described herein are intended for purposes of illustration only and are not intended to limit the application. In addition, it should be noted that in the drawings, only parts related to the application are shown. The embodiments in the application and the features in the embodiments can be combined with each other without conflict. The application will be described in further detail below with reference to the drawings and embodiments.

[0018] Example 1: Preparation of Paracasei IOB413 fermented ginseng postbiotic (1) Preparation of ginseng solid-state fermentation medium: first, clean the ginseng, then soak the ginseng in water according to the solid-liquid ratio of 1:1.5, and then sterilize at 115°C for 20 min to obtain the ginseng solid-state fermentation medium; (2) Strain activation: inoculate the strain in the slant medium, and culture at 37°C in a closed and static state. The culture time is 24 h, and the activated seed solution is obtained. Inoculate a fresh slant strain in the liquid medium, and culture at 37°C in a closed and static state. The culture time is 20 h, and the fermentation seed solution is obtained; (3) Solid-state fermentation: inoculate the fermentation seed solution in the ginseng solid-state fermentation medium for fermentation, the inoculation amount is 5%, the culture temperature is 37°C, and the culture time is 24 h.

[0019] (4) Preparation of postbiotic: after fermentation, inactivate, dry, and crush to obtain the Paracasei IOB413 fermented ginseng postbiotic.

[0020] Example 2: Establishment of a rat model of stroke Sixty Sprague-Dawley SD rats (200-240g) were randomly divided into a sham group (n=12) and a middle cerebral artery ischemia-reperfusion group (n=48). Rats were fasted for 12 hours prior to surgery but allowed free access to water. They were anesthetized with an intraperitoneal injection of 350mg / kg of 10% chloral hydrate. The rats were fixed in a supine position, their necks were shaved, and disinfected with alcohol and iodine. The common carotid artery (CCA) was carefully dissected to expose the external carotid artery (ECA). The proximal end of the ECA was ligated, and the proximal end of the CCA was clamped with an arterial clamp. A small incision was made in the left CCA with ophthalmic scissors, and a suture was inserted into the internal carotid artery (ICA) until resistance was encountered, to a depth of approximately 17-18mm. The suture was then secured, the CCA clamp was released to restore blood perfusion, and the wound was sutured. After 2 hours of ischemia, the suture was removed for reperfusion. The sham group only underwent vessel dissection and exposure without further treatment. After surgery, the animals were returned to their cages and allowed free access to food. The next day, successfully operated rats were selected based on the following criteria: the presence of asymmetrical eyes (smaller and darker eyeballs at the site of cerebral ischemia), circling towards the paralyzed side, or flexion and adduction of the forelimb on the contralateral side when the tail was lifted. Rats without these symptoms were excluded. Successfully operated rats were randomly divided into a model group, a nimodipine (N-20) group, a low-dose group, and a high-dose group of ginseng post-biotic fermented with *Lactobacillus paracasei* IOB413. The sham surgery group and the model group were given an equal volume of physiological saline by gavage. The N-20 group was given nimodipine 20 mg / kg by gavage. The low-dose group was injected intraperitoneally with 0.1 g / kg of Lactobacillus paracasei IOB413 fermented ginseng post-biotic, and the high-dose group was injected intraperitoneally with 0.3 g / kg of Lactobacillus paracasei IOB413 fermented ginseng post-biotic. The treatment was administered twice daily for 7 consecutive days. Four hours after the last administration, behavioral scores were assessed. Blood was collected from the abdominal aorta after anesthesia for biochemical index testing. The animals were euthanized by exsanguination, and brain tissue was quickly removed for index determination.

[0021] Example 3: Effects of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on neurobehavioral characteristics in a rat model of ischemic stroke. Neurological function in rats was assessed 72 hours after cerebral ischemia-reperfusion. Mice scoring 1-3 using the Long scale were considered successfully modeled and ready for further experiments. The scoring criteria are shown in Table 1. Results are as follows: Figure 1As shown, the sham-operated group did not exhibit any neurological dysfunction, so the average score was 0, excluding the influence of surgery on neurological function. Compared to the sham-operated group, the model group showed neurological damage, mainly manifested as contralateral forelimb flexion, difficulty walking in a straight line, circling to the contralateral side, balance difficulties, abnormal reflexes, and a significantly higher neurological function impairment score. The different dose groups of *Lactobacillus paracasei* IOB413-fermented ginseng post-biotic showed significant improvement in neurological function and behavior compared to the model group, with significantly lower scores. *Lactobacillus paracasei* IOB413-fermented ginseng post-biotic significantly reduced neurological function impairment in stroke rats.

[0022] Table 1 Long Scoring Criteria

[0023] Example 4: Effects of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on serum biochemical parameters in rats with cerebral ischemia-reperfusion injury Immediately after behavioral scoring of the rats, blood was collected from the abdominal aorta under anesthesia at 3000 rpm. -1 After centrifugation for 10 minutes, the serum was separated, and the levels of SOD, MDA, NO and GSH in the serum were detected according to the kit instructions.

[0024] Superoxide dismutase (SOD) is an endogenous antioxidant enzyme. During ischemia, SOD activity decreases, accompanied by an increase in the concentration of malondialdehyde (MDA), a lipid peroxide in brain tissue, further exacerbating damage. The depletion of SOD and glutathione (GSH), two antioxidants, and the accumulation of MDA directly reflect the degree of lipid peroxidation in ischemic stroke. Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown, *Lactobacillus paracasei* IOB413 fermented ginseng post-biotic exhibits significant antioxidant activity, substantially reducing MDA and NO levels in rats with middle cerebral artery occlusion (MCAO) and increasing the activities of antioxidant enzymes SOD and GSH. Here, MCAO rats are used as a representative rat model in this study.

[0025] Example 5: Effects of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on serum inflammatory factors in rats with cerebral ischemia-reperfusion injury After 7 days of continuous gavage, the levels of IL-1β, TNF-α, and IL-6 inflammatory factors were detected using enzyme-linked immunosorbent assay (ELISA). The results are as follows:Figure 6 , Figure 7 and Figure 8 As shown, compared with the sham-operated group, the levels of IL-1β, TNF-α, and IL-6 inflammatory factors were significantly increased in the model group. After gavage administration of various doses of Lactobacillus paracasei fermented ginseng post-biotic, the levels of IL-1β, TNF-α, and IL-6 inflammatory factors were significantly inhibited.

[0026] Example 6: Effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on brain water content in rats with ischemic stroke The water content of brain tissue is calculated using the following formula: Brain water content (%) = (wet weight - dry weight) / wet weight * 100%.

[0027] Depend on Figure 9 It was found that after 2 hours of ischemia and reperfusion, the water content of the rat brain tissue increased significantly. The ginseng post-fermentation of Lactobacillus paracasei IOB413 in all dose groups could reduce the water content of the rat brain tissue, alleviate cerebral edema, reduce the degree of cerebral lesions induced by middle cerebral artery occlusion, and reduce the incidence of injury. However, the ginseng post-fermentation of Lactobacillus paracasei IOB413 in the low dose group only showed a decreasing trend but no difference.

[0028] Example 7: Effect of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on infarct volume in ischemic stroke The brain was cut into 2 mm thick coronal sections, soaked in physiological saline solution containing 3% TTC, stained at 37°C for 30 min in the dark, and stirred every 10 minutes to ensure full contact between the brain sections and the TTC solution. After staining, the sections were rinsed with water and then fixed with 4% paraformaldehyde solution for 2 hours. The brain sections were then photographed.

[0029] In studies related to ischemic stroke, infarct size serves as an important reference indicator, reflecting the severity of the infarction and assessing the effectiveness of drug intervention. Figure 10 It can be seen that, compared with the sham surgery group, the cerebral infarction area in the model group was significantly increased, and compared with the control group, the cerebral infarction area was significantly reduced after gavage administration of each dose of Lactobacillus paracasei IOB413 fermented ginseng.

[0030] Example 8: Effects of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on coagulation function in ischemic stroke Coagulation function was tested using a coagulation analyzer.

[0031] Thrombosis plays a crucial role in the development of ischemic stroke. Due to thrombosis, blood flow is obstructed, leading to ischemia, hypoxia, and even necrosis of brain tissue supplied by local blood vessels, resulting in various stroke symptoms. Thrombosis is also a major cause of high stroke recurrence rates. When prothrombin time (PT) and thrombin time (TT) are shortened, activated partial thromboplastin time (APTT) is decreased, and fibrinogen (FIB) levels are increased, it indicates enhanced coagulation function in the body. Conversely, prolonged PT and TT, increased APTT, and decreased FIB levels indicate decreased coagulation function. Experimental results are as follows... Figure 11 , Figure 12 , Figure 13 and Figure 14 As shown, after one week of gavage administration of *Lactobacillus paracasei* IOB413-fermented ginseng post-biotic, the PT and TT of all groups were prolonged, the APTT increased, and the FIB content decreased, with the high-dose ginseng post-biotic group showing more significant effects. This indicates that *Lactobacillus paracasei* IOB413-fermented ginseng post-biotic has the effect of improving coagulation function and promoting blood circulation, which is beneficial in preventing thrombosis and recurrence of ischemic stroke.

[0032] Example 9: Effects of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotics on brain tissue pathological sections Brain tissue was fixed with 4% paraformaldehyde, and 4μm thick paraffin sections were prepared for hematoxylin-eosin staining (HE).

[0033] Depend on Figure 15 It was found that the sham-operated group showed normal neuronal cell morphology, normal nerve fiber bundle arrangement, and no inflammatory cell infiltration in the interstitium. Compared with the sham-operated group, the model group showed large areas of vacuolar degeneration and cell swelling and necrosis in the brain tissue of rats, accompanied by a large number of inflammatory cell infiltrations. After gavage administration of Lactobacillus paracasei IOB413 fermented ginseng as a probiotic, the rat brain tissue showed varying degrees of vacuolar degeneration and cell swelling and necrosis, accompanied by varying degrees of inflammatory cell infiltration, with a reduced incidence and severity of lesions.

[0034] Example 10: Effects of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotic on apoptosis in brain tissue of rats in various groups After adding an appropriate amount of anti-fluorescence quenching mounting medium to the paraffin sections and mounting them, they were observed under a fluorescence microscope.

[0035] TUNEL staining, short for Terminal Deoxynucleotidyl Transferase-Mediated dUTP nick-end Labeling, is a key technique for detecting apoptosis after stroke. The results directly reflect the degree of programmed cell death of neurons / glial cells in the ischemic penumbra and infarct core, and are of great significance for assessing injury mechanisms and treatment efficacy. Results are shown below. Figure 16 Compared with the sham-operated group, the apoptosis rate of nerve cells in the brain tissue of rats in the model group was significantly increased, while the apoptosis rate of nerve cells in the brain tissue of rats in each dose group was significantly decreased.

[0036] Example 11: Results of the effect of Lactobacillus paracasei IOB413 fermentation of ginseng on VEGF expression in brain tissue Neutral resin mountings were observed and photographed using an upright microscope (Olympus BX60, Tokyo, Japan). Images are as follows: Figure 17 As shown. Images were analyzed using ImagePro-Plus version 6.0 (Media Cybernetics, Silver Spring, MD, USA) image analysis software. Cells in the observation area were analyzed using Image Pro Plus image measurement and analysis software, and the mean optical density (MOD) of positive cells with yellow or brownish-yellow cytoplasm was measured as the VEGF protein expression level.

[0037] VEGF is a crucial factor in angiogenesis and a key regulator of brain remodeling following ischemic stroke. VEGF also protects against neuronal degeneration in vitro and in rodent models. Besides its role in angiogenesis, VEGF also has neurotrophic, neuroprotective, and blood-brain barrier permeability regulation functions. Figure 18 As shown, compared with the sham surgery group, the expression of VEGF in the ischemic penumbra area of ​​the brain tissue in the model group was reduced. Compared with the model group, the expression of VEGF in the low- and high-dose ginseng post-fermentation group of Lactobacillus paracasei IOB413 was increased, and angiogenesis was more obvious.

[0038] Example 12: Effects of Lactobacillus paracasei IOB413 fermentation of ginseng post-biotics on CD34 in brain tissue + expression The expression result detection method is as described in Example 11, involving taking a picture, and the image is as follows: Figure 19 As shown. CD34 +Microvessel density (MVD) analysis method: CD34 is expressed in the cell membrane and / or cytoplasm of endothelial cells. The presence of light to dark brown granular deposits within vascular endothelial cells is considered CD34 positive. MVD is counted for CD34-positive vessels under 200x magnification. + This refers to cells that express the CD34 molecule. The principle of histochemistry is that the direct target is the CD34 molecule (CD34), and the presence and quantity of CD34 in the sample are ultimately determined by the colorimetric results. + cell.

[0039] CD34 is a specific leukocyte differentiation antigen selectively expressed on the surface of human hematopoietic stem cells, progenitor cells, and vascular endothelial cells. It is also an antigen associated with newly formed small blood vessels and has been extensively studied as a highly sensitive marker of neovascular endothelial cells. Similar to VEGF, CD34 is also a regulator of angiogenesis, thereby promoting angiogenesis and protecting neural tissue after ischemic injury. Figure 20 As shown, compared with the sham-operated group, CD34 expression in the ischemic penumbra region of the brain tissue was reduced in the model group. Compared with the model group, CD34 expression in the ischemic penumbra region was significantly increased in the high-dose group of ginseng fermented with Lactobacillus paracasei IOB413, while the low-dose group showed an increase, but it was not significant. Figure 21 As shown, compared with the sham-operated group, the microvessel density in the model group was reduced. Compared with the model group, the microvessel density in the ischemic penumbra region of brain tissue was increased in the high-dose group of *Lactobacillus paracasei* IOB413-fermented ginseng post-biotic group, while the low-dose group showed an increase, but it was not significant. This indicates that *Lactobacillus paracasei* IOB413-fermented ginseng post-biotic can promote CD34 expression, increase the number of CD34-positive cells in blood vessels, induce cerebral angiogenesis, thereby restoring and improving blood supply to the ischemic area, and has a neuroprotective effect.

[0040] Example 13: Regulation of the PI3K / AKT / mTOR signaling pathway in brain tissue of mice with ischemic stroke by *Lactobacillus paracasei* IOB413 fermented with ginseng post-biotic. Western blot analysis was used to detect the expression of VEGF, HIF-1α, PI3K, AKT, P-AKT, and mTOR. The PI3K (phosphatidylinositol 3-kinase) / AKT (protein kinase B) signaling pathway is mainly involved in the regulation of various functions such as cell proliferation, differentiation, migration, and apoptosis. Vascular endothelial growth factor (VEGF) is an exocrine homodimeric glycoprotein that can promote angiogenesis and increase vascular permeability. It can activate the PI3K / AKT signaling pathway along with human growth factor (HGF), angiotensin I (Ang1), fibroblast growth factor (FGF), and insulin. Hypoxia-inducible factor-1α (HIF-1α) is a core factor regulating the hypoxic response. It can promote the transport of oxygen and glucose to tissues, reduce the energy demand of the ischemic brain by inhibiting neural activity and protein synthesis, and thus inhibit the effects of energy deficiency. HIF-1α plays an essential role in the normal development of nerve cells in the brain. In addition, HIF-1α also plays a key role in vascular development. HIF-1α can induce the expression of vascular endothelial growth factor (VEGF), which can mediate angiogenesis in ischemic brain regions.

[0041] Depend on Figure 22 It was found that, compared with the sham-operated group, VEGF protein expression was significantly decreased in the model group. Compared with the model group, VEGF protein expression was significantly increased in the low- and high-dose ginseng fermentation group using *Lactobacillus paracasei* IOB413. Figure 23 It was found that, compared with the sham-operated group, the expression level of P-PI3K (phosphorylated PI3K) protein in the model group was significantly decreased. Compared with the model group, the expression of P-PI3K protein in the low- and high-dose ginseng fermentation group of *Lactobacillus paracasei* IOB413 was significantly increased. Figure 24 It was found that, compared with the sham-operated group, the expression level of P-AKT (phosphorylated AKT) protein in the model group was significantly decreased. Compared with the model group, the expression level of P-AKT protein in the low- and high-dose ginseng fermentation group with *Lactobacillus paracasei* IOB413 was significantly increased. Figure 25 It was found that, compared with the sham-operated group, the expression level of phosphorylated mammalian target of rapamycin (P-mTOR) protein in the model group was significantly decreased. Compared with the model group, the expression level of P-mTOR protein in the low- and high-dose ginseng fermentation group with *Lactobacillus paracasei* IOB413 was significantly increased. Figure 26It was found that, compared with the sham surgery group, the HIF-1α content in the model group was significantly increased, and compared with the model group, the HIF-1α protein expression content in the low- and high-dose ginseng fermentation group of Lactobacillus paracasei IOB413 was significantly decreased.

[0042] The *Lactobacillus paracasei* strain IOB413, with the preservation number CGMCC No. 16022, was isolated from naturally fermented sourdough from Tianjin residents. It was identified as belonging to the *Lactobacillus* genus, and its acute oral toxicity test showed it to be practically non-toxic. The preparation of this metabiotic requires activation of the strain to obtain a secondary seed culture, which is then inoculated onto ginseng solid-state fermentation medium for fermentation. Subsequent inactivation, drying, and pulverization are then performed to obtain the final product. Key parameters such as the material-to-water ratio, inoculum size, and temperature are all clearly controllable within a defined range during fermentation to ensure stable product quality.

[0043] The post-fermentation of ginseng by Lactobacillus paracasei IOB413 has shown significant effects in improving cerebral angiogenesis in ischemic stroke. It can promote the expression of vascular endothelial growth factor (VEGF) and CD34 in the ischemic penumbra of brain tissue. VEGF can promote the proliferation and migration of vascular endothelial cells to induce angiogenesis, while CD34, as a marker of angiogenesis, can increase microvascular density. The two work synergistically to restore blood supply to the ischemic area and provide basic blood flow and nutritional support for brain tissue repair. Meanwhile, the post-biotic fermented with ginseng by Lactobacillus paracasei IOB413 has a prominent effect on neurorepair. The post-biotic fermented with ginseng by Lactobacillus paracasei IOB413 can upregulate the PI3K / AKT / mTOR signaling pathway, increase the expression of phosphorylated PI3K (P-PI3K), phosphorylated AKT (P-AKT), and phosphorylated mTOR (P-mTOR), and regulate the level of hypoxia-inducible factor-1α (HIF-1α). It can assist angiogenesis by regulating cell proliferation, exert neuroprotective effects, reduce nerve cell apoptosis, promote nerve remodeling, improve the neurological function and behavior of model animals, and alleviate obstacles such as forelimb flexion and walking imbalance. It can help alleviate ischemic stroke and repair brain tissue in multiple dimensions.

[0044] In addition to its core function of improving ischemic stroke, *Lactobacillus paracasei* IOB413-fermented ginseng post-biotics also possess multiple auxiliary effects. In terms of antioxidant activity, it significantly reduces serum malondialdehyde (MDA) and nitric oxide (NO) levels in model animals, increases superoxide dismutase (SOD) and glutathione (GSH) activity, alleviates oxidative stress damage induced by ischemic stroke, and reduces oxidative damage to lipids, proteins, and nucleic acids. In terms of anti-inflammatory effects, it effectively inhibits the release of inflammatory factors such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in serum, reduces inflammatory cell infiltration in brain tissue, alleviates the persistent interference of systemic inflammatory response on nerve repair, and creates a favorable microenvironment for brain tissue repair. In improving the pathological state of brain tissue and coagulation function, it can reduce the water content of brain tissue to alleviate cerebral edema, reduce the volume of cerebral infarction, and reduce the degree of degeneration and necrosis of brain tissue cells; at the same time, it can prolong prothrombin time (PT) and thrombin time (TT), increase activated partial thromboplastin time (APTT), reduce fibrinogen (FIB) content, improve coagulation function, prevent thrombosis, and reduce the risk of recurrence of ischemic stroke.

[0045] In summary, the *Lactobacillus paracasei* IOB413 strain, a post-fermenting ginseng probiotic strain, has a clearly identified source and verified safety. The preparation process parameters are controllable, and the product quality is stable. It not only restores blood supply to ischemic areas by promoting VEGF and CD34 expression, but also upregulates the PI3K / AKT / mTOR signaling pathway to achieve neuroprotection and remodeling. Its core function directly targets cerebral angiogenesis and nerve repair in ischemic stroke. Simultaneously, it possesses comprehensive auxiliary effects, including antioxidant, anti-inflammatory, improvement of brain tissue pathology, and coagulation function, providing a safe, multi-dimensional, and effective technical direction for ischemic stroke-related interventions.

Claims

1. The application of *Lactobacillus paracasei* IOB413 fermented ginseng post-biotic in the preparation of a medicine for treating ischemic stroke, characterized in that... The preservation number of the Lactobacillus paracasei IOB413 is CGMCC No. 16022.

2. The application as described in claim 1, characterized in that, The method for preparing the post-biotic of ginseng fermented with Lactobacillus paracasei IOB413 includes the following steps: The Lactobacillus paracasei IOB413 strain from the cryopreservation tube was inoculated into an agar slant culture medium and incubated at 34-38℃ for 22-26 hours; the agar slant culture was then inoculated into a liquid culture medium and incubated at 34-38℃ in a sealed container for 18-22 hours to obtain the fermentation seed liquid. The fermentation seed liquid was inoculated into ginseng solid fermentation medium for solid fermentation. The fermented raw material was then inactivated, dried, and pulverized to obtain post-biotic of ginseng fermented with Lactobacillus paracasei IOB413.

3. The application as described in claim 2, characterized in that, The solid-state fermentation process involves a material-to-water ratio of 1:1.0 to 1:1.5, an inoculum size of 5-10%, a fermentation time of 24-48 hours at 34-38℃, a sterilization temperature of 85-100℃, and a drying moisture content controlled at ≤12%.

4. The application as described in claim 1, characterized in that, Ischemic stroke includes hemiplegia, forelimb flexion, gait imbalance, sensory dysfunction, paralysis, weakened reflexes, ptosis, visual impairment, microphthalmia, memory impairment, aphasia, disability, cerebral edema, vacuolar degeneration of brain cells, cell swelling and necrosis, and inflammatory cell infiltration caused by cerebral vascular stenosis or occlusion.

5. The application of *Lactobacillus paracasei* IOB413 fermented ginseng post-biotic in the preparation of antioxidant health foods or microecological preparations, characterized in that... The preservation number of the Lactobacillus paracasei IOB413 is CGMCC No. 16022.

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