Application of lactobacillus paracasei in chronic sleep deprivation injury

Abstract

This invention relates to the field of probiotics technology, specifically to the application of a particular strain of *Lactobacillus paracasei* in the preparation of a strain for the prevention, relief, or treatment of cognitive impairment and related mood disorders caused by chronic sleep deprivation. The *Lactobacillus paracasei* is... Lactobacillus paracasei BKR005. This invention's probiotic indirectly promotes endogenous butyrate production by reshaping the gut microbiota, and effectively inhibits neuroinflammation and neuronal apoptosis through its unique MCT1 repair function, providing a clear target and novel mechanism for intervening in neuropsychiatric damage driven by chronic sleep deprivation.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically to a particular strain of Lactobacillus paracasei (… Lactobacillus paracasei The application of BKR005 in the preparation of a product for the prevention, relief or treatment of cognitive impairment and related mood disorders caused by chronic sleep deprivation (CSR). Background Technology

[0002] Chronic sleep deprivation (CSR) has become a prevalent physiological and psychological stressor in modern society. Long-term sleep deprivation caused by shift work (such as nursing and driving), high-intensity academic pressure (such as students), and insomnia poses a serious threat to human health. Clinical studies and basic experiments have confirmed that CSR not only causes direct consequences such as daytime sleepiness and poor concentration, but also significantly impairs higher cognitive functions, especially learning and memory abilities. In the long term, it increases the risk of neurodegenerative diseases (such as Alzheimer's disease) and mental disorders (such as depression and anxiety) [1,2]. With the accelerating pace of life and the diversification of work patterns, cognitive and emotional impairments caused by CSR have become a prominent public health problem that urgently needs to be addressed.

[0003] However, current interventions for cognitive impairment in CSR are limited and have significant limitations. In terms of drug treatment, commonly used wakefulness agents (such as modafinil) or sedative-hypnotic drugs (such as benzodiazepines) can improve some symptoms in the short term, but they generally have problems such as dependence, addiction risk, cognitive "hangover effect" and symptom rebound after drug withdrawal, making them difficult to use as a long-term and safe intervention plan [3]. In terms of non-drug intervention, cognitive behavioral therapy (CBT-I) has been proven to be effective, but it has a high dependence on professional therapists, a long treatment cycle, and high cost, which severely limits its accessibility and popularity among the vast number of people in distress. Therefore, existing intervention strategies have significant shortcomings in terms of safety, effectiveness and universality, and there is an urgent need to develop a safe, effective, mechanism-clear and easy-to-promote innovative intervention strategy.

[0004] In recent years, the "microbiota-gut-brain axis" theory has provided a new pathological perspective for understanding and intervening in CSR-related brain dysfunction. This theory points out that the gut microbiota communicates bidirectionally with the central nervous system through multiple pathways such as the nervous, endocrine and immune systems, profoundly affecting brain function and behavior [4]. Numerous studies have shown that CSR can significantly alter the composition and function of the gut microbiota, causing dysbiosis [5]. Existing studies have indicated that this dysbiosis can disrupt the tight junctions between intestinal epithelial cells, leading to impaired intestinal barrier integrity and increased permeability (i.e., "leaky gut," manifested as elevated serum zonulin levels). Leaky gut allows bacterial lipopolysaccharide (LPS) and other endotoxins in the intestinal lumen to easily penetrate the barrier and enter the bloodstream, triggering a systemic chronic, low-grade inflammatory response. More importantly, LPS and inflammatory factors (such as TNF-α and IL-1β) in the peripheral circulation can further disrupt the integrity of the blood-brain barrier (BBB) ​​(i.e., "brain leakage," manifested as elevated serum S100β levels), allowing these harmful substances to invade the brain parenchyma [6]. In the brain, LPS activates Toll-like receptor 4 (TLR4) on the surface of microglia, initiating downstream inflammatory signaling pathways, leading to the massive release of pro-inflammatory cytokines in key brain regions such as the hippocampus, forming a persistent neuroinflammatory microenvironment, which is considered the core pathological mechanism leading to cognitive impairment [7,8].

[0005] Based on the "microbe-gut-brain axis" theory, supplementing with probiotics to reshape the gut microbiota may become a highly promising non-pharmacological strategy for intervening in cognitive impairment caused by CSR. Studies have shown that regulating the gut microbiota to balance LPS and butyrate levels may be key to intervening in neuroinflammation [9].

[0006] Although existing research has shown that certain probiotics or combinations thereof have some effect on improving sleep quality and cognition, current technologies still have several fundamental shortcomings in applying probiotics to intervene in cognitive impairment caused by cognitive disorders (CSR). First, existing research largely focuses on multi-strain probiotic preparations. While these products may produce synergistic effects, they also suffer from unclear interactions between strains, difficulty in defining active ingredients, complex manufacturing processes, and challenges in ensuring quality control and batch-to-batch consistency, severely limiting their standardized application and promotion as a precise intervention. Second, at the mechanism of action level, current probiotic research often attributes effects broadly to "anti-inflammatory" or "microbiome regulation," lacking in-depth and systematic validation of how specific strains precisely regulate upstream key metabolites (such as LPS and butyrate) and downstream specific signaling pathways. This mechanistic ambiguity keeps probiotic intervention strategies in a "black box" stage, unable to achieve truly precise targeted intervention. Finally, although some Lactobacillus strains have been reported for their neuroprotective effects, specific screening and identification of a single, highly effective Lactobacillus paracasei strain capable of exerting a clear neuroprotective effect through the aforementioned cutting-edge mechanisms in CSR models is still lacking. Lactobacillus paracasei Research on strains is still in its infancy.

[0007] In summary, existing technologies have significant gaps in providing single probiotic strains with well-defined mechanisms, significant efficacy, and ease of standardization for precise targeted intervention of the core pathological mechanisms of CSR. More importantly, current research generally focuses on regulating the gut microbiota to increase the "signal output end" of beneficial metabolites such as butyrate, while seriously neglecting the potential functional impairment of the brain as the "signal receiver" under chronic stress. There are indications that neuroinflammation itself can impair the function of key transport proteins on the blood-brain barrier and brain parenchymal cells (such as microglia and neurons), such as MCT1, which is responsible for transporting monocarboxylate salts like butyrate. This "transporter damage" constitutes a fundamental bottleneck in current probiotic therapies: even if the gut produces sufficient butyrate, if the key signal receiving proteins in the brain are shut down or damaged, these beneficial molecules cannot enter target cells to exert their anti-inflammatory and neuroprotective effects. Therefore, there is an urgent need in this field for a novel intervention strategy that can not only regulate gut metabolism but also specifically repair key transport proteins in the central nervous system—a key technical challenge that existing technologies have failed to address.

[0008] References [1] Gan, Li. Effects of sleep deprivation on spatial learning and memory, as well as oxidative stress and inflammatory response in the brain and liver of mice [D]. Chongqing: Chongqing Medical University, 2018. [2] Yin Hao. Effects and mechanisms of Orexin and its receptor on learning and memory in chronic sleep-deprived mice [D]. Jinan: Shandong University, 2020. [3] Anderson G, Maes M. Role of sleep deprivation in immune-relateddisease risk and outcomes[J]. Communications Biology, 2021, 4(1):1304. [4] Wang Z, Chen W H, Li S X, et al. Gut microbiota modulates theinflammatory response and cognitive impairment induced by sleep deprivation[J]. Molecular Psychiatry, 2021, 26(11):6277-6292. [5] Poroyko V A, Carreras A, Khalyfa A, et al. Chronic sleepdisruption alters gut microbiota, induces systemic and adipose tissueinflammation and insulin resistance in mice[J]. Scientific Reports, 2016, 6:35405. [6] He Y, Wang J, Li F, et al. Blood-brain barrier disruption inducedby chronic sleep loss: low-grade inflammation may be the link[J]. Journal ofImmunology Research, 2016:4576012. [7] Wang X, Wang Z, Cao J, et al. Gut microbiota-derived metabolitesmediate the neuroprotective effect of melatonin in cognitive impairmentinduced by sleep deprivation[J]. Microbiome, 2023, 11:17. [8] Xu Huali. Study on the mechanism by which chronic sleep deprivation exacerbates brain injury in rats with ischemia-reperfusion by activating microglia [D]. Guangzhou: Guangdong Provincial People's Hospital, 2025. [9] Zheng Y, Zhang L, Bonfili L, et al. Probiotics supplementationattenuates inflammation and oxidative stress induced by chronic sleeprestriction[J]. Nutrients, 2023, 15(6):1518. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies in intervening in damage caused by chronic sleep deprivation, particularly cognitive decline and related mood disorders, by providing a *Lactobacillus paracasei* (… Lactobacillus paracasei Application of BKR005 in the prevention, relief or treatment of damage caused by chronic sleep deprivation.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows: The use of a type of Lactobacillus paracasei in the preparation of products for the prevention, relief or treatment of damage caused by chronic sleep deprivation.

[0011] This strain is *Lactobacillus paracasei* ( Lactobacillus paracasei BKR005, this strain was deposited on November 28, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36823.

[0012] The impairments include cognitive impairment, anxiety-like behavior, or increased aggressive behavior.

[0013] The Lactobacillus paracasei is used to prepare drugs or functional foods that repair or upregulate the expression of monocarboxylic acid transporter 1 (MCT1) in hippocampal tissue and regulate the interaction between the LPS / TLR4 / NF-κB signaling pathway and the MCT1 / HDAC3 signaling pathway.

[0014] Furthermore, it exerts its effects by antagonistically regulating the interaction between the lipopolysaccharide (LPS) / Toll-like receptor 4 (TLR4) / nuclear factor-κB (NF-κB) pro-inflammatory signaling pathway and the monocarboxylic acid transporter 1 (MCT1) / histone deacetylase 3 (HDAC3) anti-inflammatory signaling pathway.

[0015] The repair or upregulation of hippocampal MCT1 expression restored the transport of endogenous butyrate from the gut to the brain, thereby mediating the inhibition of HDAC3 activity.

[0016] The application is in the preparation of medicines or functional foods for the prevention, relief or treatment of damage caused by chronic sleep deprivation.

[0017] An formulation for the prevention, relief or treatment of damage caused by chronic sleep deprivation, wherein the active ingredient comprises the aforementioned Lactobacillus paracasei BKR005.

[0018] The probiotic preparation includes culture precipitate containing the strain or its metabolites, bacterial suspension, fermentation supernatant, lyophilized powder, and lyophilized and inactivated bacterial powder.

[0019] The culture precipitate was obtained by centrifuging Lactobacillus paracasei BKR005 at 4000 rpm for 10 minutes after anaerobic culture in MRS medium at 37°C.

[0020] The bacterial suspension is prepared by resuspending the culture precipitate in sterile physiological saline.

[0021] The freeze-dried powder is prepared by mixing the culture precipitate with a freeze-drying protectant and then freeze-drying it at low temperature.

[0022] The formulation is Lactobacillus paracasei BKR005 and pharmaceutically or food-acceptable carriers or excipients.

[0023] The formulation may be further supplemented with prebiotics or stabilizers. Prebiotics may include xylooligosaccharides, inulin, and fructooligosaccharides; stabilizers may include maltodextrin.

[0024] The dosage form of the preparation is capsules, tablets, powders, granules, or oral liquid preparations.

[0025] Compared with the prior art, the advantages of the present invention are: 1. This invention is the first to reveal and target the repair of the brain's MCT1 transporter protein. The greatest innovation of this invention lies in its transcendence of the traditional single-dimensional approach of probiotics in regulating the gut. It is the first to discover and demonstrate that *Lactobacillus paracasei* BKR005 possesses the unique ability to specifically repair and upregulate hippocampal MCT1 expression. Compared to existing therapies with unclear mechanisms or single targets, this invention elucidates the precise action network of the strain at the molecular pathway level, providing a solid theoretical basis for intervention strategies.

[0026] 2. Antagonistic Regulation of Pro-inflammatory and Anti-inflammatory Pathway Interactions. Building upon the core function of MCT1 repair, this invention synergistically achieves bidirectional intervention on two key signaling pathways: it inhibits the activation of the "LPS / TLR4 / NF-κB" pro-inflammatory pathway while unlocking the "MCT1 / HDAC3" anti-inflammatory pathway through MCT1 repair. This strategy of blocking upstream pro-inflammatory signals while simultaneously unblocking downstream anti-inflammatory pathways achieves a synergistic effect greater than the sum of its parts (1+1>2).

[0027] 3. Validated through animal experiments and in vitro cell experiments, the strain described in this invention not only significantly improves multidimensional cognitive impairment caused by chronic sleep deprivation, including spatial learning and memory, and recognition memory, but also effectively alleviates accompanying emotional disorders such as anxiety and increased aggression. Its efficacy is supported by clear physiological mechanisms, including repairing the gut-brain barrier, inhibiting peripheral systemic inflammation, and effectively inhibiting microglial overactivation and neuronal apoptosis at the central nervous system level.

[0028] 4. *Lactobacillus paracasei* is a widely used and safe strain in the food industry, with a recognized safety profile. Using it as a single strain avoids the potential antagonistic effects and uncertainties between strains in compound probiotic preparations. The probiotic intervention program provided by this invention has no side effects like traditional drugs and can be used as a long-term preventative or adjunctive treatment. It has significant health value and broad application prospects for a large population suffering from sleep deprivation due to factors such as shift work, academic pressure, and disrupted lifestyles. Detailed Implementation

[0029] 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.

[0030] This invention utilizes *Lactobacillus paracasei* BKR005 to precisely address cognitive impairment caused by chronic sleep deprivation (CSR). Its core technology overcomes the fundamental deficiency of existing intervention strategies that focus solely on "signal generation" while neglecting "signal reception." Chronic sleep deprivation not only leads to gut microbiota dysbiosis and peripheral inflammation, but more importantly, it directly damages monocarboxylic acid transporter 1 (MCT1), responsible for transporting energy and signaling molecules such as butyrate, in the hippocampus of the brain, causing its expression to be downregulated. This "blocked intrabrain pathway" prevents butyrate from effectively entering neurons and microglia to exert its anti-inflammatory effects, even if the gut can produce it.

[0031] The most important and innovative mechanism of action of *Lactobacillus paracasei* BKR005 proposed in this invention is its ability to specifically repair and upregulate damaged MCT1 expression in hippocampal tissue. This repair function opens a key pathway for subsequent anti-inflammatory mechanisms. Secondly, this strain can remodel the gut microbiota, indirectly promoting the production of endogenous butyrate. Finally, the repaired MCT1 and the increased butyrate work synergistically to effectively inhibit HDAC3 activity, thereby antagonistically blocking the LPS-activated TLR4 / NF-κB pro-inflammatory pathway, inhibiting neuroinflammation and neuronal apoptosis at the source (e.g., reducing cleaved caspase-3 levels), thus effectively alleviating cognitive decline during CSR, regulating immune and inflammatory states, and improving overall central nervous system health.

[0032] The *Lactobacillus paracasei* BKR005 proposed in this invention not only improves learning and memory decline caused by cognitive impairment (CSR) (as shown in water maze and new object recognition experiments), but also effectively repairs damaged intestinal and blood-brain barriers (manifested as a reduction in serum zonulin and S100β levels). Furthermore, this probiotic significantly reduces serum LPS and peripheral pro-inflammatory cytokines (such as TNF-α and IL-1β) levels, while simultaneously increasing the content of the beneficial metabolite butyrate in the gut.

[0033] This invention significantly improves chronic sleep deprivation-induced cognitive impairment by regulating key metabolites in the gut-brain axis and influencing the regulation of central nervous system signaling pathways.

[0034] The strain BKR005 involved in the following examples is a strain of Lactobacillus paracasei identified in this invention. Lactobacillus paracasei It was deposited in 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36823.

[0035] The culture media involved in the following examples are as follows: MRS medium (g / L): peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, sodium acetate 5g / L, diammonium hydrogen citrate 2g / L, magnesium sulfate 0.1g / L, manganese sulfate 0.05g / L, Tween 80 1mL / L.

[0036] The following examples illustrate the method for preparing probiotic suspensions: BKR005 strain, stored in glycerol tubes at -80℃, was inoculated into MRS liquid medium and activated under anaerobic conditions at 37℃ for 24 hours. The activated bacterial culture was then transferred to fresh MRS medium at a 2% inoculation rate and cultured anaerobically at 37℃ until the end of the logarithmic growth phase (OD50).600 =1.5-2.0). The fermentation broth was centrifuged at 4℃ and 4000 rpm for 10 minutes, and the supernatant was discarded to recover the bacterial cells. The cells were washed twice with sterile physiological saline and finally resuspended to 1×10⁻⁶. 10 CFU / mL, store at 4℃ for later use.

[0037] The experimental animals involved in the following examples are: Healthy male 8-week-old SPF-grade C57BL / 6J mice, weighing 22-25g, were housed individually in a barrier environment with a temperature of 22±2℃, humidity of 50-60%, and a 12-hour light / dark cycle (lighting time 7:00-19:00). The mice had free access to standard feed and water.

[0038] Experimental results were statistically analyzed using GraphPad Prism 9.5 and are expressed as mean ± standard deviation (mean ± SD). Differences between groups were assessed using one-way ANOVA, and post-hoc analyses were performed using Tukey's multiple comparison test. ###p represents a highly significant difference compared to the normal control group (p < 0.001); ##p represents a highly significant difference compared to the normal control group (p < 0.01); #p represents a significant difference compared to the normal control group (p < 0.05). p represents a highly significant difference compared to the model group (p < 0.001). p represents a highly significant difference compared to the model group (p < 0.01). p indicates a significant difference compared to the model group (p < 0.05).

[0039] Example 1: Effects of Lactobacillus paracasei BKR005 on cognitive behavior and gut-brain barrier in CSR mice Experimental objective: This embodiment aims to verify the effect of Lactobacillus paracasei BKR005 on improving learning and memory impairment induced by chronic sleep deprivation (CSR) in mice, and to evaluate its protective effect on intestinal and blood-brain barrier permeability. Simultaneously, the sleep deprivation effect of the CSR model was objectively verified using EEG / EMG monitoring.

[0040] Test method: 1. Animals and Grouping: Thirty-six healthy male C57BL / 6J mice, weighing 22-25g, were randomly divided into three groups of 12 mice each after one week of acclimatization feeding, as follows: Group 1: Normal control group (CON): Feeded under a normal 12h / 12h light-dark cycle, and given 200 μL of sterile saline by gavage daily for 8 weeks as a pre-supplementation.

[0041] Group 2: Model Group (CSR): The patient was treated with a chronic sleep deprivation model and was given 200 μL of sterile saline by gavage daily.

[0042] Group 3: Probiotic intervention group (CSR+BKR005): The probiotics were treated using the CSR model, and simultaneously administered 200 μL of Lactobacillus paracasei BKR005 bacterial suspension (containing 2×10⁻⁶ bacteria) via gavage daily. 9 CFU).

[0043] 2. Intervention plan: From day 1 to day 28 of the experiment, the CSR+BKR005 group was given 200 μL of Lactobacillus paracasei BKR005 bacterial suspension (containing 2×10⁻⁶ bacteria) by gavage daily. 9 CFU); the CON group and the CSR group were given an equal volume of sterile saline by gavage daily.

[0044] CSR Model Establishment: Chronic sleep deprivation was performed using an automated rotating rod method. From day 15 to day 28 (14 days in total), mice in the CSR and CSR+BKR005 groups underwent 14 consecutive days of chronic sleep deprivation. An automated sleep deprivation system (Pinnacle Technology Inc., USA) was used, with a rotating rod at 2 rpm during the mice's normal sleep period (i.e., the light period from 7:00 AM to 7:00 PM). Mice in the CON group were placed in the same environment, but the rotating rod was not rotated. To objectively verify the effect of sleep deprivation, all mice underwent EEG / EMG electrode implantation surgery (using Pinnacle prefabricated electrode caps according to standard protocol) before the CSR model was established (day 14). Monitoring began one week after surgery. EEG / EMG recorded sleep-wake cycles (NREM, REM, and percentage of wakefulness were analyzed using Sirenia Sleep software), confirming a significant reduction in sleep time in the CSR group (>70% deprivation rate).

[0045] 3. Behavioral tests: Behavioral tests began the day after the CSR ended (i.e., day 29 of the experiment).

[0046] Morris Water Maze (MWM Test): Starting on day 29 of the experiment, a 5-day positioning and navigation training program was conducted (days 29-33), followed by a space exploration experiment on day 34. The platform was removed, and the number of times the platform was traversed within 60 seconds and the percentage of time spent in the target quadrant were recorded.

[0047] Novel Object Recognition (NOR Test): Conducted on day 35 of the experiment, this test consisted of three phases: adaptation, training, and testing. Mice were first allowed 10 minutes to adapt to the environment in a test chamber (40 cm × 40 cm × 40 cm). During the training phase, two identical objects, A1 and A2, were placed in the chamber, and the mice were allowed to explore them for 10 minutes. One hour later, the testing phase began, and A2 was replaced with a new object B. The mice's exploration time for object B within 5 minutes was recorded (T0). new ) and old object A1 (T old The time frame is calculated, and the identification index (DI = T) is determined. new / (T new + T old () × 100%).

[0048] 4. Sample collection and testing: After the behavioral tests, mice were fasted for 4 hours and anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). Blood was collected via cardiac puncture, and the blood samples were centrifuged at 3000g for 15 minutes at 4°C. The supernatant was collected as serum and stored at -80°C. The concentrations of the corresponding indicators in the serum were detected using commercially available mouse Zonulin and S100β ELISA kits (both purchased from Wuhan Elite Biotechnology Co., Ltd.), strictly following the instructions.

[0049] Experimental results: 1. Lactobacillus paracasei BKR005 improves spatial learning and memory abilities in CSR mice. As shown in Table 1, in the spatial exploration experiment of MWM, compared with the CON group, the CSR model group mice showed a significant reduction in the number of times they traversed the original platform location and the percentage of time spent in the target quadrant (###p<0.001), indicating that CSR severely impaired the mice's spatial memory retention ability. However, after BKR005 intervention, both of these indicators in the CSR+BKR005 group mice significantly improved ( p<0.001. In the NOR test, the recognition index (DI) of CSR group mice was significantly lower than that of CON group (p<0.01), indicating impaired recognition memory ability. BKR005 intervention significantly improved the DI value of CSR mice (p<0.01). (p<0.01), confirming that BKR005 can effectively improve cognitive impairment caused by CSR.

[0050] EEG / EMG monitoring showed that NREM and REM sleep time were significantly reduced in the CSR group (>70% deprivation, ###p<0.001), while there was no significant effect in the BKR005 group, confirming the effectiveness of the model.

[0051] Table 1 Comparison of behavioral test results of mice in each group

[0052] Note: Data are expressed as mean ± standard deviation (n=12). Compared with the normal control group (CON), ##p<0.01, ###p<0.001; compared with the model group (CSR), p<0.01, p<0.001.

[0053] 2. Lactobacillus paracasei BKR005 repairs CSR-induced gut-brain barrier damage. As shown in Table 2, ELISA results indicated that the concentrations of zonulin (a marker of intestinal barrier permeability) and S100β (a marker of blood-brain barrier permeability) in the serum of mice in the CSR model group were significantly higher than those in the CON group (p<0.001). After intervention with BKR005, the levels of zonulin and S100β in the CSR+BKR005 group were significantly reduced (…). (p<0.001), confirming that BKR005 has a significant protective effect against CSR-induced "gut leakage" and "brain leakage".

[0054] Table 2 Comparison of serum barrier marker concentrations in mice of different groups

[0055] Note: Data are expressed as mean ± standard deviation (n=12). Compared with the normal control group (CON), ###p<0.001; compared with the model group (CSR), p<0.001.

[0056] in conclusion: The results of this embodiment clearly demonstrate that *Lactobacillus paracasei* BKR005 can effectively improve spatial learning and recognition memory impairment caused by chronic sleep deprivation. Its mechanism of action involves repairing damaged intestinal and blood-brain barriers, reducing the risk of harmful substances from the gut and periphery entering the central nervous system. Furthermore, EEG / EMG monitoring further validated the reliability of the CSR model.

[0057] Example 2: Improvement of anxiety-depression-like behavior and aggression in CSR mice by Lactobacillus paracasei BKR005 Experimental objective: This embodiment aims to verify the effect of Lactobacillus paracasei BKR005 on improving the enhancement of anxiety-depression-like behavior and increased aggression induced by chronic sleep deprivation (CSR) in mice.

[0058] Test method: Animal grouping, model establishment, and intervention protocols were the same as in Example 1.

[0059] 1. Behavioral tests: Behavioral tests began the day after the CSR ended (i.e., day 29 of the experiment).

[0060] Open field test (OFT): conducted on day 29 of the experiment. Test box (50 cm × 50 cm × 40 cm), total movement distance (voluntary activity), time spent in the central area and number of entries were recorded within 5 minutes to characterize anxiety level.

[0061] Elevated Cross Maze (EPM): Conducted on day 30 of the experiment. The maze arms were 50 cm long and 15 cm high, with two open arms and two closed arms. The number of times the participants entered the open arms and the percentage of time spent in the open arms were recorded within 5 minutes to indicate their anxiety level.

[0062] Resident-Intruder Test (RIT): Conducted on day 31 of the experiment. After the test mice (residents) were placed in their cages to acclimatize for 1 hour, a weight-matched unfamiliar male intruder was introduced. Aggressive behavior was recorded over 10 minutes, mainly the number of attacks, latency, and duration of attacks.

[0063] Experimental results: Lactobacillus paracasei BKR005 improves anxiety-depression-like behavior and aggression in CSR mice. As shown in Table 3, in OFT, compared with the CON group, the total movement distance of mice in the CSR model group did not change significantly (p>0.05), indicating that the level of spontaneous activity was not significantly affected, excluding the interference of drugs or models on the basic motor ability of mice, and ensuring the validity of other behavioral indicators. However, the dwell time and number of entries in the central area were significantly reduced (p<0.001), reflecting enhanced anxiety-like behavior, that is, the mice were more inclined to avoid the open area and showed higher stress sensitivity. The dwell time and number of entries in the central area of ​​the CSR+BKR005 group were significantly restored ( (p<0.001), close to the level of the CON group, indicating that probiotic intervention effectively alleviated CSR-induced anxiety-like behavior.

[0064] In EPM, the number of open arm entries and the percentage of time spent in the CSR group were significantly lower than those in the CON group (###p<0.001), further confirming the increase in anxiety-depression-like behavior, as mice preferred closed arms for a sense of security. These indicators were significantly elevated in the CSR+BKR005 group ( (p<0.001), indicating that probiotics can reverse this avoidance behavior and restore the mice's exploratory tendencies.

[0065] In RIT, the CSR group mice showed a significant increase in the number of attacks (###p<0.001), a significant shortening of the attack latency (###p<0.001), and a significant prolongation of the attack duration (###p<0.001), indicating that CSR induced a significant increase in aggression, mimicking the emotional instability and irritability following sleep deprivation. The CSR+BKR005 group showed a significant decrease in the number of attacks ( p<0.001, significantly prolonging the attack latency period ( p<0.001, and the attack duration was significantly shortened ( (p<0.001), confirming that probiotic intervention can effectively suppress aggressive behavior and restore normal patterns of social interaction.

[0066] Table 3 Comparison of anxiety and aggression-related behavioral test results among the groups of mice

[0067] Note: Data are expressed as mean ± standard deviation (n=12). Compared with the normal control group (CON), ###p<0.001; compared with the model group (CSR), p<0.001. OFT: Open field experiment; EPM: Elevated cross maze; RIT: Inhabitant-Intruder experiment.

[0068] in conclusion: The results of this embodiment confirm that Lactobacillus paracasei BKR005 can effectively improve mood disorders caused by chronic sleep deprivation, mainly manifested in the significant inhibitory effect of BKR005 on anxiety and aggressive behavior in mice after chronic sleep deprivation.

[0069] Example 3: Regulatory effect of Lactobacillus paracasei BKR005 on intestinal metabolites and inflammatory factors in CSR mice Experimental objective: This embodiment aims to investigate whether Lactobacillus paracasei BKR005 mediates its protective effect against CSR by regulating the levels of key intestinal metabolites (LPS and butyrate) and peripheral inflammatory factors.

[0070] Test method: Animal grouping, model establishment, and intervention protocols were the same as in Example 1.

[0071] 1. Sample collection: After euthanizing the mice, the colon contents were collected and frozen at -80°C. Blood samples collected by cardiac puncture were used partly to prepare serum (same as in Example 1) and partly collected using EDTA anticoagulant tubes. The plasma was collected by centrifugation at 1500g for 10 minutes at 4°C and frozen at -80°C.

[0072] 2. Indicator Testing: Detection of short-chain fatty acids (SCFAs): Take 50 mg of colon contents, add phosphoric acid aqueous solution and internal standard (2-ethylbutyric acid), vortex mix, and extract with diethyl ether. Take the supernatant and use a gas chromatography-mass spectrometry (GC-MS, Agilent, USA) equipped with a DB-FFAP capillary column to quantitatively determine the contents of acetic acid, propionic acid, and butyric acid.

[0073] LPS detection: The concentration of LPS in serum was detected using a Limulus Amebocyte Lysate (LAL) ELISA kit. All operations were performed under pyrogen-free conditions.

[0074] Inflammatory factor detection: The concentrations of TNF-α, IL-1β, IL-6, and IL-10 in plasma were detected using a commercially available ELISA kit.

[0075] Experimental results: 1. Lactobacillus paracasei BKR005 regulates intestinal SCFAs content and serum LPS in CSR mice. As shown in Table 4, GC analysis results showed that compared with the CON group, the butyrate content in the colon contents of mice in the CSR model group was significantly decreased (p<0.01), while the serum LPS level was significantly increased (p<0.001), while the acetic acid and propionic acid contents did not change significantly. After BKR005 intervention, the butyrate content in the CSR+BKR005 group was significantly restored (p<0.01), while the serum LPS level was significantly reduced (p<0.01). (p<0.001), indicating that BKR005 can significantly promote the production of endogenous butyric acid in the gut and reduce serum LPS levels by remodeling the gut microbiota.

[0076] Table 4 Comparison of intestinal SCFAs and serum LPS levels in mice of different groups

[0077] Note: Data are expressed as mean ± standard deviation (n=12). Compared with the normal control group (CON), ##p<0.01, ###p<0.001; compared with the model group (CSR), p<0.01, p<0.001.

[0078] 2. Lactobacillus paracasei BKR005 inhibits peripheral inflammatory response in CSR mice. As shown in Table 5, the concentrations of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the plasma of mice in the CSR model group were significantly increased (###p<0.001), while the concentration of the anti-inflammatory cytokine IL-10 was significantly decreased (##p<0.01). BKR005 intervention significantly reversed these changes, namely, significantly reducing the levels of TNF-α, IL-1β, and IL-6 ( p<0.001), and significantly increased IL-10 levels ( (p<0.01), indicating that BKR005 can effectively inhibit the systemic low-grade inflammatory state induced by CSR.

[0079] Table 5 Comparison of plasma inflammatory factor concentrations in mice of different groups

[0080] Note: Data are expressed as mean ± standard deviation (n=12). Compared with the normal control group (CON), ##p<0.01, ###p<0.001; compared with the model group (CSR), p<0.01, p<0.001.

[0081] in conclusion: The results of this embodiment confirm that *Lactobacillus paracasei* BKR005 achieves bidirectional regulation of key metabolites by remodeling the gut microbiota: it increases the level of endogenous butyrate, which has anti-inflammatory effects, while decreasing the level of LPS, a major pro-inflammatory trigger. This metabolic regulation effectively inhibits the systemic low-grade inflammatory state induced by CSR.

[0082] Example 4: Molecular mechanism by which Lactobacillus paracasei BKR005 regulates the interaction between the LPS / TLR4 and MCT1 / HDAC3 pathways Experimental objective: This embodiment aims to elucidate the core mechanism by which Lactobacillus paracasei BKR005 inhibits neuroinflammation and neuronal apoptosis in the hippocampus at the molecular level, with a focus on verifying its regulatory role in the interaction of the TLR4 / NF-κB and MCT1 / HDAC3 signaling pathways.

[0083] Test method: Animal grouping, model establishment, and intervention protocols were the same as in Example 1.

[0084] 1. Sample collection and processing: After euthanizing the mice, the bilateral hippocampi were rapidly isolated on ice. One hippocampus was homogenized with RIPA lysis buffer (containing protease and phosphatase inhibitors) to extract total protein for Western blotting. The other hippocampus was fixed in 4% paraformaldehyde solution at 4°C for 48 hours, dehydrated via a sucrose gradient, embedded in OCT, and frozen sections 20 μm thick were prepared for immunofluorescence analysis.

[0085] 2. Indicator Testing: Western Blotting: After protein quantification using the BCA method, 30 μg of protein was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After blocking with 5% skim milk powder, the membrane was incubated overnight at 4°C with the following primary antibodies: TLR4, MyD88, p-p65, MCT1, HDAC3, Cleaved Caspase-3, Bax, and Bcl-2 (all purchased from Cell Signaling Technology), and the internal control GAPDH (purchased from Abcam). The following day, the membrane was incubated with HRP-labeled secondary antibody, developed using an ECL chemiluminescence kit, and analyzed for grayscale values ​​using ImageJ software.

[0086] Immunofluorescence (IF): Frozen sections were thawed, washed, permeabilized with 0.3% Triton X-100, and blocked with 5% BSA. They were then incubated overnight at 4°C with primary antibodies Iba-1 (Wako, rabbit antibody) and NeuN (Millipore, mouse antibody), respectively. The following day, they were incubated with secondary antibodies labeled with the corresponding Alexa Fluor 488 (green) and Alexa Fluor 594 (red). After DAPI staining of the nuclei, images were acquired using a confocal microscope (Leica SP8). Morphological observation (observing resting and activated states) was performed on Iba-1 positive cells in the CA1 and DG regions of the hippocampus; and NeuN positive cells were counted.

[0087] Experimental results: 1. Lactobacillus paracasei BKR005 inhibits CSR-induced activation of the hippocampal TLR4 / NF-κB inflammatory pathway. Western blot results (Table 6) showed that, compared with the CON group, the expression levels of TLR4, MyD88, and the phosphorylated form of key downstream proteins of the NF-κB pathway, p-p65, were significantly upregulated in the hippocampus of mice in the CSR model group (###p<0.001). This indicates that CSR triggers the classic TLR4 / NF-κB pro-inflammatory signaling cascade through LPS. After intervention with BKR005, the expression levels of these proteins were significantly inhibited ( (p<0.001), confirming that BKR005 can effectively block LPS-mediated activation of inflammatory pathways.

[0088] 2. Lactobacillus paracasei BKR005 repairs CSR-damaged hippocampal MCT1 transporter and its related MCT1 / HDAC3 inflammatory suppression pathway As shown in Table 6, compared with the CON group, the expression level of butyrate transporter MCT1 in the hippocampus of the CSR model group was significantly downregulated, while the expression level of HDAC3 protein was significantly increased (###p<0.001), indicating that CSR not only activated pro-inflammatory pathways but also disrupted the body's own anti-inflammatory defense mechanisms, suggesting that anti-inflammatory pathways were blocked. This indicates that CSR caused dual damage: it both increased inflammatory stimulation and weakened the brain's ability to use butyrate for anti-inflammatory purposes. After BKR005 intervention, MCT1 expression significantly rebounded, and the recovery of MCT1 expression provided a guarantee for the entry of gut-derived butyrate into hippocampal glial cells. At the same time, the expression of HDAC3 was significantly downregulated ( (p<0.01). This is consistent with the mechanism by which enteric butyrate enters cells via MCT1 and inhibits HDAC3 activity, indicating that BKR005 activates anti-inflammatory signals by increasing butyrate levels.

[0089] Table 6. Relative expression levels of inflammation-related signaling pathway proteins in the hippocampus of mice in each group.

[0090] Note: Data are expressed as mean ± standard deviation (n=12). Expression levels were normalized with CON group as 1. Compared with CON group, ###p<0.001; compared with CSR group, p<0.01, p<0.001.

[0091] 3. Lactobacillus paracasei BKR005 inhibits hippocampal neuroinflammation and neuronal apoptosis. As shown in Table 7, the IHC results indicated that in the CSR group, a large number of Iba-1 positive microglia exhibited activated morphology with enlarged cell bodies and reduced branching in the CA1 and DG regions of the hippocampus, and the number of NeuN positive neurons was significantly reduced (###p<0.001). In the BKR005 intervention group, most microglia were in a resting state, and the number of NeuN positive neurons significantly increased ( (p<0.001). Western blot results further confirmed that the expression of the apoptosis-executing protein Cleaved Caspase-3 and the ratio of the pro-apoptotic / anti-apoptotic protein Bax / Bcl-2 in the hippocampus of the CSR group were significantly increased (p<0.001), confirming the trend of neuronal apoptosis induced by sleep deprivation. Intervention with *Lactobacillus paracasei* BKR005 effectively inhibited the overactivation of microglia, reversed the loss of neurons to near-normal levels, and significantly reduced apoptosis markers.

[0092] Table 7. Changes in hippocampal cells and expression of apoptotic proteins in each group of mice.

[0093] Note: Data are expressed as mean ± standard deviation (n=12). White blood cell expression levels were normalized with CON group = 1. Compared with the CON group, ###p<0.001; compared with the CSR group, p<0.001.

[0094] in conclusion: This embodiment reveals the intervention of Lactobacillus paracasei BKR005 in chronic sleep deprivation injury at the molecular and cellular levels. CSR not only induces neuroinflammation by activating the LPS-TLR4-NF-κB pathway, but also disrupts the brain's own butyrate anti-inflammatory defense system by downregulating MCT1 expression. The most important finding of this embodiment is that BKR005 intervention can significantly reverse the downregulation of hippocampal MCT1 protein expression caused by CSR, restoring it to near-normal levels. This MCT1 repair function is crucial in the entire neuroprotective chain, providing the necessary pathway for gut-derived butyrate (whose level was increased after BKR005 treatment, as shown in Example 3) to enter brain cells and exert its biological functions.

[0095] This invention's probiotics act on the hippocampus by regulating the gut-brain axis: on the one hand, they inhibit pro-inflammatory pathways, specifically suppressing the activation of the LPS-TLR4-NF-κB inflammatory pathway; on the other hand, they regulate the MCT1-HDAC3 pathway, repairing MCT1 and activating the MCT1-HDAC3 pathway through the metabolite butyrate. This synergistic regulation of the two pathways effectively inhibits neuroinflammatory responses and neuronal apoptosis following chronic sleep deprivation, thereby protecting cognitive function.

[0096] Example 5: Verification of the mechanism by which intracellular active components of Lactobacillus paracasei BKR005 antagonize microglial inflammation by repairing the MCT1 / HDAC3 pathway. Experimental objective: This study aims to explore the direct molecular mechanism by which *Lactobacillus paracasei* BKR005 exerts its neuroprotective effect using an in vitro cell model. Intracellular active components of the strain were obtained through sonication to verify whether they can directly repair and upregulate MCT1 expression in microglia inhibited by lipopolysaccharide (LPS), and further clarify whether this effect depends on the inhibition of MCT1 transporter protein and its downstream HDAC3 activity. This experiment establishes the causal relationship between the repair of MCT1 function and the central anti-inflammatory effects of BKR005 components by introducing specific inhibitors and mimics.

[0097] Test method: 1. Preparation of supernatant from BKR005 ultrasonically disrupted material (BKR-lysate): Lactobacillus paracasei BKR005 was cultured on MRS medium to the end of the logarithmic growth phase, and the bacterial cells were collected by centrifugation at 4000 g for 10 minutes. The cells were washed twice with sterile phosphate-buffered saline (PBS), and then resuspended in serum-free DMEM medium, adjusting the concentration to 1×10⁻⁶. 8 CFU / mL. Cell disruption was performed using an ultrasonic cell disruptor on ice (300W, 3 seconds operation, 5 seconds interval, total 15 minutes). The disrupted solution was centrifuged at 10000 g at 4°C for 10 minutes. The supernatant was collected and filtered through a 0.22 μm filter to obtain sterile BKR005 lysate, which was stored at -80°C for later use.

[0098] 2. Cell Culture and Grouping: The BV2 immortalized microglia were cultured in DMEM medium containing 10% fetal bovine serum. After cell adhesion and reaching 80% confluence, the cells were randomly divided into the following 6 groups, with 6 biological replicates per group: Group 1: Control group: Administered with standard culture medium.

[0099] Group 2: LPS model group (LPS): Add 1 μg / mL LPS and treat for 6 hours.

[0100] Group 3: BKR005 component intervention group (LPS+BKR-lysate): First, pretreat with BKR-lysate for 2 hours, then add 1 μg / mL LPS and co-culture for 6 hours.

[0101] Group 4: MCT1 inhibition and blocking group (LPS+BKR-lysate+AZD3965): 10 μM of the MCT1 specific inhibitor AZD3965 was added during BKR-lysate pretreatment.

[0102] Group 5: HDAC3 activation blocking group (LPS+BKR-lysate+ITSA-1): 50 μM HDAC3 activator ITSA-1 was added during BKR-lysate pretreatment.

[0103] Group 6: HDAC3 inhibition simulation group (LPS+RGFP966): Pretreated with 10 μM HDAC3 specific inhibitor RGFP966 for 2 hours, then added LPS.

[0104] 3. Indicator Testing: Inflammatory factor detection: Cell culture supernatant from each group was collected, and the concentrations of pro-inflammatory factors (TNF-α, IL-6) and anti-inflammatory factors (IL-10) were determined using enzyme-linked immunosorbent assay (ELISA).

[0105] Pathway protein detection: Total cellular protein was extracted, and the protein expression levels of MCT1 and phosphorylated NF-κB p65 (p-p65) were detected by Western blotting. The level of histone H3K27 acetylation (H3K27ac) was also detected, with GAPDH as an internal control.

[0106] Experimental results: 1. Direct repair effect of BKR005 component on MCT1 expression: As shown in Table 8, LPS treatment significantly inhibited the protein expression level of MCT1 in BV2 cells (p<0.001). BKR-lysate intervention significantly reversed this inhibitory effect and upregulated MCT1 expression (p<0.001). This result demonstrates that the active components within BKR005 have the ability to directly repair the function of microglia transport proteins, rather than simply acting through intestinal metabolites.

[0107] 2. The BKR005 component inhibits the inflammatory response through the MCT1-HDAC3 pathway: Experiments showed that BKR-lysate significantly reduced LPS-induced TNF-α and IL-6 release, increased the level of the anti-inflammatory factor IL-10 (p<0.001), inhibited p-p65 protein phosphorylation, and upregulated H3K27ac levels. However, upon the addition of the MCT1 inhibitor AZD3965, the anti-inflammatory effect of BKR-lysate was greatly weakened (p<0.001), and the upregulation of H3K27ac disappeared. Similarly, the HDAC3 activator ITSA-1 could reverse the anti-inflammatory effect of BKR-lysate. The anti-inflammatory effect of using the HDAC3 inhibitor RGFP966 alone was highly consistent with that of the BKR-lysate group.

[0108] Table 8 Effects of BKR005 component on MCT1 expression and inflammatory markers in BV2 cells

[0109] Note: Data are expressed as mean ± standard deviation (n=6). Compared with the Control group: ###p<0.001; Compared with the LPS group: p<0.001; Compared with the LPS+BKR-lysate group: +++p<0.001.

[0110] in conclusion: This study confirms that the intracellular active component of *Lactobacillus paracasei* BKR005 has the function of directly repairing damaged MCT1 expression. This repair function is a prerequisite for its anti-inflammatory effect. Through the restoration of MCT1 function, the BKR005 component can further inhibit HDAC3 activity, thereby blocking the excessive activation of the NF-κB inflammatory pathway. This conclusion establishes the property of BKR005 as a transporter repair agent and provides a molecular basis for the preparation of corresponding formulations of this strain for central nervous system injury caused by chronic sleep deprivation.

Claims

1. The use of a type of Lactobacillus paracasei in the prevention, relief or treatment of damage caused by chronic sleep deprivation.

2. The application according to claim 1, characterized in that: The *Lactobacillus paracasei* is *Lactobacillus paracasei* (… Lactobacillus paracasei BKR005, this strain was deposited in 2025 at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36823.

3. The application according to claim 1, characterized in that: The impairments include cognitive impairment, anxiety-like behavior, or increased aggressive behavior.

4. The application according to any one of claims 1-3, characterized in that: The *Lactobacillus paracasei* is used in the preparation of drugs or functional foods that repair or upregulate the expression of monocarboxylic acid transporter 1 (MCT1) in hippocampal tissue and regulate the interaction between the LPS / TLR4 / NF-κB signaling pathway and the MCT1 / HDAC3 signaling pathway.

5. The application according to claim 4, characterized in that: The repair or upregulation of hippocampal MCT1 expression restored the transport of endogenous butyrate from the gut to the brain, thereby mediating the inhibition of HDAC3 activity.

6. The application according to claim 1, characterized in that: The application is in the preparation of medicines or functional foods for the prevention, relief or treatment of damage caused by chronic sleep deprivation.

7. An agent for preventing, alleviating, or treating damage caused by chronic sleep deprivation, characterized in that, Its active ingredient includes Lactobacillus paracasei as described in claim 1.

8. The formulation according to claim 7, characterized in that, The formulation also includes pharmaceutically or food-acceptable carriers, excipients, prebiotics, or stabilizers.

9. The formulation according to claim 7 or 8, characterized in that, The dosage form of the preparation is capsule, tablet, powder, granule, oral liquid preparation or inhalation preparation.

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