Application of dendrobine combined with lactobacillus paracasei

Abstract

The invention discloses application of dendrobine combined with lactobacillus paracasei, and particularly relates to application of dendrobine combined with lactobacillus paracasei in preparation of products for preventing and / or relieving learning and memory ability damage induced by high-sugar diet. Experiments prove that the combination of the dendrobine and the lactobacillus paracasei increases the variety diversity of intestinal flora, improves the glucose and lipid metabolism disorder of rats induced by high glucose, improves the abnormal blood fat and blood glucose, can prevent and / or improve the influence of the high glucose on the learning and memory ability of the rats, and has a good synergistic effect compared with the single use of the dendrobine or the lactobacillus paracasei. The combined use of dendrobine and lactobacillus paracasei also avoids the problem that the influence effect on learning and memory ability is not obvious when the dendrobine and lactobacillus paracasei are independently used, and the dendrobine and lactobacillus paracasei composition is effective and safe and has very high clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of dendrobium alkaloids combined with Lactobacillus paracasei, and especially relates to the use of dendrobium alkaloids combined with Lactobacillus paracasei in preparing a product for preventing and / or alleviating learning and memory impairment induced by a high-sugar diet. Background Art

[0002] A high-sugar diet is a common dietary pattern in modern society. However, long-term high-sugar intake can lead to decreased learning and memory abilities, which has become a public health concern. Numerous studies have confirmed that a high-sugar diet can cause metabolic disorders, inducing conditions such as obesity, diabetes, non-alcoholic fatty liver disease, and enteritis. It can also exacerbate the progression of myopia, promote the development and growth of various tumors, and even be linked to adverse health effects such as impaired brain memory. It can also gradually weaken brain function, cause memory loss, and increase the risk of Alzheimer's disease. Therefore, the learning and memory problems associated with a high-sugar diet warrant attention and resolution.

[0003] Existing solutions primarily rely on medication to improve learning and memory impairment caused by a high-sugar diet. For example, antioxidants and neuroprotectants are used to mitigate damage to nerve cells. Common antioxidants include vitamin C, vitamin E, glutathione, coenzyme Q10, and alpha-lipoic acid. These antioxidants neutralize harmful oxidants in the body, protecting nerve cells from oxidative damage, thereby contributing to their protection and repair. Specific amino acids, peptides, brain protein hydrolysates, edaravone, flupentixol, and riluzole protect damaged nerves from further damage and promote nerve regeneration. Furthermore, probiotics are used to regulate intestinal flora and improve lipid metabolism. For example, Yanfei Li et al. found that dendrobium alkaloids can inhibit memory impairment caused by lipopolysaccharide (LPS). While probiotic treatments have fewer side effects, their effectiveness in improving learning and memory is often less pronounced. Therefore, finding new preventive and treatment options that effectively improve learning and memory impairment caused by high sugar intake, reduce medication side effects, and improve overall health is an urgent need.

[0004] A high-sugar diet not only disrupts the dynamic balance of the intestinal flora but is also associated with disorders of glucose and lipid metabolism, leading to a range of health issues such as cognitive impairment. Dendrobium has the effects of lowering blood sugar and regulating immunity, but little research has examined the mechanisms by which dendrobium improves cognitive impairment through intestinal flora. Lactobacillus paracasei promotes the balance of the human microbiome and enzymes. Dendrobium is an alkaloid extracted from the medicinal and edible Dendrobium officinale. It is a pyrrolizidine derivative alkaloid with multiple physiological activities, including antioxidant, anti-inflammatory, and neuronal cell protection. Lactobacillus paracasei (Lactobacillus casei), a member of the Lactobacillus casei group (LCG), is a common candidate probiotic strain. Studies have shown that Lactobacillus paracasei has been proven to have functions such as regulating intestinal flora and improving lipid metabolism. It can delay age-related cognitive dysfunction in aging mice through anti-inflammatory, antioxidant and gut-brain axis regulation. It also plays a good regulatory role in inflammatory-related diseases. It has an auxiliary therapeutic effect on type 2 diabetes and shows potential in the auxiliary treatment of cancer.

[0005] After searching, there is no prior art that uses dendrobium alkaloids and Lactobacillus paracasei in combination to improve the problem of decreased learning and memory ability caused by a high-sugar diet. Summary of the Invention

[0006] In order to achieve the above-mentioned purpose, the technical solution is as follows: The present invention protects the use of dendrobium alkaloids combined with Lactobacillus paracasei in preparing a product for preventing or alleviating learning and memory impairment induced by a high-sugar diet.

[0007] The present invention also protects the use of dendrobium alkaloids combined with Lactobacillus paracasei in preparing a product for treating microglial cell damage caused by high sugar.

[0008] Furthermore, the mass ratio of the Lactobacillus paracasei to dendrobium is 2-10:1.

[0009] Preferably, the Lactobacillus paracasei is HH-LP58.

[0010] Preferably, the concentration of Lactobacillus paracasei is 8-10 µg / µL, and the concentration of dendrobium alkaloids is 3-5 µg / µL.

[0011] Compared with the prior art, the present invention has the following technical effects: This invention proposes for the first time the combined use of dendrobium and Lactobacillus paracasei in a drug for preventing or alleviating learning and memory impairment induced by a high-sugar diet. This combination increases the diversity of microbiota, helps regulate intestinal flora, and improves glucose and lipid metabolism. It not only improves learning and memory in rats with high sugar intake, but also improves abnormal blood lipids and blood sugar. It has a wide range of physiological activities. The combined use avoids the problem of insignificant therapeutic effects of probiotics alone. It is both effective and safe, and has high clinical application value. Interestingly, experiments in this invention found that when the mass ratio of Lactobacillus paracasei to dendrobium was 1:1, the two produced an antagonistic effect, and the effect on alleviating microglial damage was not as good as when the two drugs were used alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the distance and times percentage of each group of rats in the three arms, where Figure 1 A is the percentage of the distance of the three arms; Figure 1 B is the percentage of times in the three arms; Figure 2 The escape latency and escape distance of each group of rats during the water maze training period were as follows: Figure 2 A is a comparison chart of escape latency on a daily basis; Figure 2 B is a comparison chart of swimming distances per day; Figure 3 Figure 2 is the movement trajectory diagram of the Morris water maze experiment during the test period of each group of rats. Figure 3 A is the blank control group; Figure 3 B is the high glucose model group; Figure 3 C is the dendrobium alkaloid intervention group; Figure 3 D is the dendrobium alkaloids combined with Lactobacillus paracasei intervention group; Figure 3 E is the Lactobacillus paracasei intervention group; Figure 3 F is donepezil hydrochloride positive group; Figure 4 This is a comparison chart of the number of times rats in each group crossed the platform, the time they stayed in the target quadrant, and the distance to the target quadrant during the water maze test. Figure 4 A is the number of times the platform is crossed; Figure 4 B is the target quadrant residence time; Figure 4 C is the target quadrant distance ratio diagram; Figure 5 Comparison of the expression levels of neurotrophic factors and synaptic function-related genes in each group of rats. Figure 5 A. Figure 5 B is a comparison of SNAP25 and PSD95 expression levels; n = 7. Compared with group K, ##p < 0.01 indicates an extremely significant difference. Compared with group M, **p < 0.01 and ***p < 0.001 indicate an extremely significant difference. Figure 6The comparison chart of serum triglyceride, low-density lipoprotein and blood glucose levels of rats in each group is shown in Figure 2. Figure 6 A. Figure 6 B. Figure 6 C are comparison charts of triglyceride level, low-density lipoprotein level, and blood glucose level; Figure 7 This is a comparison of serum insulin levels in rats of each group; Figure 8 This is a comparison chart of the relative abundance changes of the species and genus levels of the rat intestinal flora. Figure 8 A is the Venn diagram of species-level and genus-level composition of rat intestinal microorganisms; Figure 8 B is a multi-group comparison of the top ten species of intestinal microorganisms at the species and genus levels in rats in groups K, M, and JS; Figure 9 The KEGG functional differences of intestinal microbial flora and CAZymes gene functional differences of rats in each group are shown in Figure 2. Figure 9 A is the KEGG functional difference map between group M and group K; Figure 9 B is the functional difference diagram of CAZymes genes between groups M and K; Figure 9 C is the KEGG functional difference map of JS group, M group and K group; Figure 9 D is the functional difference diagram of CAZymes genes among JS group, M group and K group; Figure 10 The partial least squares discriminant analysis and permutation test diagram of serum metabolites for pairwise comparison of three groups of samples are shown in Figure 2. Figure 10 A is the partial least squares discriminant analysis and its permutation test under the cationic mode of M and K groups, JS and M groups; Figure 10 B is the partial least squares discriminant analysis and its permutation test under the negative ion mode of groups M and K, JS and M; Figure 11 is the non-target metabolomics characteristic diagram of the serum of rats in each group, Figure 11 A is a bar graph of the number of differential metabolites among the groups; Figure 11 B is the PLS-DA analysis of differential metabolites in rat serum; Figure 12 Cluster heat map of non-target metabolites in the serum of rats in each group; Figure 13 The difference metabolite analysis diagram of serum of rats in each group, Figure 13 A. Figure 13 B are the analysis diagrams of rat serum P_value and the top ten differential metabolites in serum abundance; Figure 14 This is the KEGG pathway analysis diagram of differential metabolites in rat serum, where Figure 14 A is the KEGG enrichment map of differential metabolites in the serum of rats in the JS group and the M group; Figure 14B is the KEGG enrichment map of differential metabolites in rat serum; Figure 15 The volcano plot and MA plot of differentially expressed genes in the hippocampus transcriptome of rats in the JS group and the M group; Figure 15 A is the volcano plot of differentially expressed genes in the hippocampal transcriptome of rats in the JS group and the M group; Figure 15 B is the MA graph of differentially expressed genes in the hippocampal transcriptome of rats in the JS group and the M group; Figure 16 GO and KEGG pathway enrichment analysis of differentially expressed genes in each group of rats, Figure 16 A is the GO function enrichment analysis diagram of differentially expressed genes between JS group and M group; Figure 16 B is the KEGG pathway enrichment analysis diagram of differentially expressed genes between rats in the JS group and the M group; Figure 17 This is the PPI protein network interaction analysis diagram of differentially expressed genes; Figure 18 Comparison of gene expression levels between JS group and M group in genes related to cognitive function. Figure 18 A. Figure 18 B is a comparison of the expression levels of KRT18 and CDH13; Figure 19 This is a comparison chart of the effects of drugs on high glucose-induced microglia, Figure 19 A is the flow cytometry analysis of rats in each group after drug administration. Figure 19 B is a comparison of the effects of drugs on high glucose-induced microglia; Note: *p<0.05 vs.K & KJS; #p<0.05 vs. MJS (1:2 / 1:1 / 2:1). DETAILED DESCRIPTION

[0013] The present invention will be further described below with reference to the accompanying drawings.

[0014] Experimental drugs and sources: Dendrobium alkaloids were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with a purity of 98%. Lactobacillus paracasei HH-LP58 was purchased from Beijing Kanglihua Biotechnology Co., Ltd. with a viable count of 1 × 10 9 CFU / g.

[0015] Example 1 Dendrobium alkaloids combined with Lactobacillus paracasei prevent or alleviate high sugar-induced impairment of learning and memory abilities Experimental animal grouping and treatment Forty-two SPF male SD rats, 1 month old, weighing 120-150 g, were provided by the Department of Experimental Animal Science of Kunming Medical University. The rats were adaptively fed with standard diet for one week and randomly divided into 6 groups (n=7 / group), as follows: (1) blank control group (K): fed with standard diet; (2) high glucose model group (M): fed with standard diet and gavage with 100 mg / kg / d sucralose; (3) dendrobium alkaloid intervention group (S): fed with standard diet and gavage with 100 mg / kg / d sucralose and 5 mg / kg / d dendrobium alkaloid; (4) dendrobium alkaloid combined with Lactobacillus paracasei intervention group (JS): fed with standard feed, gavage of 100 mg / kg / d sucralose, 2.5 mg / kg / d dendrobine, and 25 mg / kg / d Lactobacillus paracasei; (5) Lactobacillus paracasei intervention group (J): fed with standard feed, gavage of 100 mg / kg / d sucralose, and 50 mg / kg / d Lactobacillus casei; (6) Donepezil hydrochloride positive group (Y): fed with standard feed, gavage of 100 mg / kg / d sucralose, and 1 mg / kg / d donepezil hydrochloride; the gavage administration experiment lasted for 12 weeks.

[0016] 1.1 Y-maze experiment After successfully establishing a high-sugar rat model, the rats were fasted and subjected to a Y-maze training and testing experiment. The Y-maze apparatus consists of three arms of equal length: a novel arm (wrong arm), an initial arm, and a food arm. Adjacent arms are angled 120 degrees, with a removable partition in the center of each arm to seal off the corresponding area. During training, the novel arm was blocked by a partition, and food was placed in the food arm. Rats in each group were placed in the initial arm and allowed to move freely in the initial and food arms for 5 minutes. By repeating this test and receiving food rewards, the rats learned and memorized the correct route. During testing, the partition in the novel arm was removed, and the rats entered the initial arm and were allowed to move freely in all three arms for 5 minutes. The time each rat spent in each arm, the distance traveled, and the number of times each rat crossed each arm within 5 minutes were recorded. After each experiment, the experimental apparatus was wiped and cleaned with 75% ethanol and wiped clean with a paper towel to minimize the animals' olfactory cue recognition.

[0017] 1.2 Water maze experiment After the Y-maze test, rats were subjected to the Morris water maze test. The Morris water maze apparatus consists of a training pool and a video and tracking device. The training pool is divided into four quadrants, each labeled as a cue. Warm water (24 ± 1°C) is filled in the pool, and a hidden escape platform (10 cm diameter × 40 cm height) is placed in the third quadrant, 1 cm below the water surface. The Morris water maze test consists of a location cruising phase and a spatial exploration phase. The location cruising phase lasts for five days, with each rat entering the water four times daily, once in each quadrant. At the start of the test, rats were placed in the pool facing the platform, with a timer set for 90 seconds. A video camera was used to record the time it took for each rat to find the platform, the distance it swam to the platform, and the number of times it swam across the platform. Rats that failed to find the platform within 90 seconds were manually guided to swim to the platform and remained on the escape platform for 10 seconds. On the sixth day, the platform was removed, and a spatial exploration phase was performed in the third quadrant. The distance the rats swam in the third quadrant within 90 seconds, the average time they spent in the third quadrant, and the number of times they crossed the hidden platform were recorded.

[0018] 1.3 Serum biochemical index determination After the behavioral experiment, blood samples were collected from anesthetized rats using blood collection tubes without anticoagulants, placed at 4°C for 1 hour, and then centrifuged at 3000 rpm for 10 minutes. Serum samples were used to measure GLU, TC, TG, LDL-C, and HDL-C.

[0019] 1.4 Non-targeted metabolomics sequencing analysis of serum metabolites Untargeted metabolomics was commissioned by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. After sample collection and preprocessing, sequencing, and LC-MS-based data library construction and comparison, the searched data were uploaded to the Meiji Cloud Platform for analysis. Principal component analysis (PCA) and orthogonal least squares discriminant analysis (OPLS-DA) were performed on the processed data using the R language package (Version 1.6.2). Differential metabolites were screened using the VIP of metabolites in the OPLS-DA analysis and the fold change and p-value in the univariate analysis. Metabolites with a VIP > 1 and a p < 0.05 were considered significantly differentially expressed. Metabolic pathway annotation of the differentially expressed metabolites was performed by comparing them to KEGG data. Pathway enrichment analysis was performed using Python software.

[0020] 1.5 Transcriptomic Sequencing Analysis Four rats each were selected from the control group (K), the high-glucose model group (M), and the dendrobium and Lactobacillus paracasei intervention group (JS). The rats were euthanized by overdose anesthesia and brain tissue was isolated. The hippocampus was removed and stored in liquid nitrogen for 1 hour before being transferred to a -80°C freezer. The tissue was then sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing. RNA was extracted using a Trizol kit, analyzed by agarose gel electrophoresis, and an RNA-seq library was constructed. Sequencing was performed on the NovaSeq X Plus platform. Sequencing results were obtained by Shanghai Meiji Biotechnology Co., Ltd. The core of transcriptomic sequencing analysis technology is the analysis of the significance of gene expression differences. GO and KEGG functional enrichment analyses were performed on the differentially expressed genes using clusterProfile software. PPI protein interaction network analysis was also performed on the differentially expressed genes using Cytoscape software to identify key genes.

[0021] 1.6 Real-time quantitative polymerase chain reaction (RT-qPCR) (detecting the expression of SNP25 and PSD95, key genes for learning and memory) 1) RNA Extraction: Weigh 0.1 g of frozen brain tissue sample and add 1 mL of Trizol RNA Extraction Solution. Incubate at 4°C for 10 min, shake, and centrifuge at 12,000 rpm at 4°C for 10 min. Transfer the supernatant to a new EP tube. Add 100 μL of chloroform substitute, shake for 15 seconds, and incubate at 4°C for 10 min. Centrifuge at 12,000 rpm at 4°C for 15 min. Transfer the upper colorless aqueous phase to a new EP tube. Add 500 μL of pre-chilled isopropanol solution, mix by inversion, and incubate at 4°C for 10 min to precipitate RNA. Add 1 mL of 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 min, and discard the supernatant. Repeat this procedure twice, then air-dry at room temperature.

[0022] 2) mRNA reverse transcription and DNA amplification: Add 20-50 μL of DEPC enzyme-free water to the extracted RNA and measure the RNA concentration using a microplate reader; reverse transcribe the RNA using the reverse transcription kit; amplify the synthesized cDNA using the 2× Universal BlueSYBR green master mix kit; use GAPDH as an internal reference and 2- The Ct method was used to calculate the relative gene expression in real-time fluorescence quantification.

[0023] 1.7 Enzyme-linked immunosorbent assay to determine rat insulin levels After the behavioral experiment, rats were euthanized by overdose of anesthesia and serum was collected. Serum insulin (INS) levels were measured using a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The sample content was calculated based on the standard curve. Specific procedures were performed according to the kit instructions.

[0024] 1.8 Analysis of intestinal flora metagenomic sequencing data After euthanasia, the entire intestinal tissue was removed under aseptic techniques. The desired intestinal segments were cut, the intestinal surface was cleaned, and the intestinal contents were removed under aseptic techniques. Samples of 0.5–1 g were collected from each rat and placed into sterile centrifuge tubes. The samples were frozen in liquid nitrogen and stored at −80°C for high-throughput DNA sequencing analysis. DNA from the microbial communities was extracted using the EZNA® soil DNA kit (Omega Bio-tek, Norcross, GA, US) according to the manufacturer's instructions. PCR amplification of the sample libraries was performed using the NEXTFLEX® Rapid DNA-Seq (Bioo Scientific, USA). Metagenomic sequencing was performed using the Illumina NovaSeq™ X Plus (Illumina, USA) platform.

[0025] Data quality control and gene prediction: The obtained template DNA fragment sequences were sheared and aligned using fastp (https: / / github.com / OpenGene / fastp, version 0.20.0). High-quality sequences were retained and contaminant sequences were removed. The quality-controlled sequences were assembled using MEGAHIT (https: / / github.com / voutcn / megahit, version 1.1.2) and translated into amino acid sequences based on the length of the nucleic acid fragments. The quality-controlled sequences were clustered using CD-HIT (http: / / weizhongli-lab.org / cd-hit / , version 4.7) to generate non-redundant gene sets.

[0026] Gene abundance calculation: SOAPaligner (https: / / github.com / ShujiaHuang / SOAPaligner, version soap2.21 release) was used to align the quality-controlled sequences with non-redundant genes to obtain gene abundance information in the corresponding samples.

[0027] Species and Function Annotation: Diamond (https: / / github.com / bbuchfink / diamond, version 2.0.13) was used to align the non-redundant gene set sequences with the NR database and the KEGG functional database to obtain NR annotations and KEGG functional annotations, respectively. HMMER (http: / / hmmer.org / , version 3.1b2) was used to align the non-redundant gene set sequences with the CAZymes database to obtain gene abundances corresponding to active enzymes.

[0028] Data processing and analysis Experimental data were analyzed using SPSS (25.0) software. Differences between samples were analyzed using one-way analysis of variance (ANOVA) with Tukey's post hoc multiple comparisons. All experimental data are expressed as mean ± standard deviation (SD). Histograms were plotted using Graphpad Prism (8.3.0). Compared with the control group (K), #p < 0.05 indicates a significant difference, and ##p < 0.01 indicates an extremely significant difference. Compared with the high glucose model group (M), *p < 0.05 indicates a significant difference, and **p < 0.01 indicates an extremely significant difference.

[0029] Here are the results:

[0030] 1. Y-maze results

[0031] The effects of dendrobium alkaloids combined with Lactobacillus paracasei on the working memory ability of high-sugar rats were studied using a Y-maze. Figure 1 As shown in the figure, compared with group K, the distance and number of explorations in the novel arm of group M were significantly reduced (p < 0.05), indicating that the short-term learning and memory ability and spatial exploration ability of rats in group M were impaired. Compared with group M, the distance and number of explorations in the novel arm of rats in group JS were significantly increased (p < 0.05), and the increase in the distance and number of explorations in the novel arm of rats in group JS was more obvious than that in groups S and J, indicating that the short-term learning and memory ability of rats in high-sugar diet was restored after the intervention of dendrobium alkaloids combined with Lactobacillus paracasei.

[0032] 2. Water Maze Results The effects of dendrobium alkaloids combined with Lactobacillus paracasei on the spatial, learning and memory abilities of high-sugar rats were studied using a water maze. Figure 2 As shown in the figure, the escape latency of rats in each group decreased day by day. On the third day, the escape latency of rats in group M was significantly higher than that of rats in group K. Compared with group M, the time and swimming distance of rats in group JS were significantly shortened, and the shortening was more obvious than that of rats in groups S and J. After the platform was removed on the sixth day, the trajectory of rats during the spatial exploration of Morris water maze was shown in the figure. Figure 3As shown in the figure, compared with group K, the movement routes of rats in group M to find the hidden platform were significantly increased. Compared with group M, the distance of rats in group JS to swim to the platform was significantly shorter, and the distance was shorter than that of the trajectory diagrams of groups S and J. Figure 4 As shown in the figure, compared with group K, the number of times the rats in group M crossed the platform, the time they stayed in the target quadrant, and the proportion of the distance they walked were significantly reduced (p < 0.05). Compared with group M, the number of times the rats in group JS crossed the platform, the time they stayed in the target quadrant, and the proportion of the distance they walked were significantly increased (p < 0.05), and the increase was more obvious than that in groups S and J, indicating that dendrobium alkaloids combined with Lactobacillus paracasei improved the learning and spatial memory abilities of rats under high glucose conditions.

[0033] 3. Dendrobium alkaloids combined with Lactobacillus paracasei upregulate the expression of synaptic expression-related genes (SNAP25, PSD95) in rats with cognitive dysfunction To examine the effects of dendrobium combined with Lactobacillus paracasei on synaptic-related gene expression, RT-qPCR experiments were performed. The results showed that compared with group K, the expression of neurotrophic factors and synaptic function-related genes (SNAP25, PSD95) in the brain tissue of rats in group M was significantly downregulated (p < 0.05), while dendrobium combined with Lactobacillus paracasei intervention significantly upregulated the expression of these genes (p < 0.05). Figure 5 These results indicate that dendrobium alkaloids combined with Lactobacillus paracasei can improve synaptic damage caused by high glucose, and are more effective than either drug alone.

[0034] 4. Effects of dendrobium alkaloids combined with Lactobacillus paracasei on high sugar-induced glucose and lipid metabolism disorders like Figure 6 Compared with the M group, the levels of serum triglyceride (TG), low-density lipoprotein (LDL-C), and blood glucose (GLU) in the JS group were significantly reduced (p < 0.05), and the reduction was more obvious than that in the S and J groups. Figure 7 As shown in the figure, compared with group K, the serum insulin level of rats in group M was significantly decreased (p < 0.05). Compared with group M, the serum insulin level of rats in group JS was significantly increased (p < 0.05), and the increase was more obvious than that in groups S and J. This shows that dendrobium alkaloids and Lactobacillus paracasei can improve high glucose-induced glucose and lipid metabolism disorders and insulin levels in rats, and the effect is better than using either drug alone.

[0035] 5. Effects of dendrobium alkaloids combined with Lactobacillus paracasei on intestinal microorganisms in high-sugar rats

[0036] 5.1 Combination of dendrobium and Lactobacillus paracasei increases bacterial flora According to the results of species annotation, the rat intestinal microorganisms were analyzed at the species level and genus level. The Venn diagram of the rat intestinal microorganisms at the species level and genus level was constructed ( Figure 8As shown in A), the combined intervention of dendrobium alkaloids and Lactobacillus paracasei increased the diversity of intestinal microbial communities in rats. The top ten species of intestinal microorganisms at the species and genus levels in the K, M, and JS groups were compared. Figure 8 B. Lactobacillus was found to be significantly decreased in the JS group (p < 0.05), while Ruminococcus was significantly increased in the JS group (p < 0.05). Studies have shown that Lactobacillus, Ruminococcus, and other genera can affect cognitive function by affecting metabolism.

[0037] Effects of dendrobium alkaloids combined with Lactobacillus paracasei on the function of intestinal microbial flora in high-glucose rats Functional prediction of intestinal flora in rats in groups K, M, and JS Figure 9 shown. Figure 9 A and Figure 9 B shows that compared with group K, the intestinal flora of group M rats had significantly decreased functions in KEGG metabolic pathways such as galactose metabolism and propionate metabolism, while significantly increased functions in metabolic pathways such as cysteine and methionine metabolism and glycosyltransferase (GT2_Glycos_transf) (p < 0.05).

[0038] Compared with the JS group, M group and K group, Figure 9 C and Figure 9 As shown in Figure D, the JS group had significantly increased functions of galactose metabolism, sphingolipid metabolism, and glycosyltransferases (p < 0.05), indicating that these pathways and functions regulate glycolipid metabolism disorders and cognitive dysfunction.

[0039] 6. Non-targeted metabolomics analysis of rat serum

[0040] 6.1 Effects of Dendrobium alkaloids combined with Lactobacillus paracasei on serum metabolites in high-glucose rats PLA-DA analysis was performed on the sample data in the cation and anion modes, respectively. Figure 10As shown. At the same time, in order to avoid model fitting during the modeling process, the permutation test method was used to test the model. In each comparison group, whether in the cation mode or the anion mode, the random model gradually decreased with the decrease of the permutation retention, R2 and Q2, and the regression line showed an upward trend, indicating that the original model did not have overfitting and the model was robust. At the same time, the data set was collected by liquid chromatography with positive polarity (pog) and negative polarity (neg), and the differential metabolites of rat serum samples were obtained after data preprocessing. The differential metabolites that were upregulated and downregulated between the groups are shown as follows. Figure 11 As shown in A. PLS-DA analysis of differential metabolites in the serum of rats in group JS, group M, and group K is shown in Figure 11 As shown in Figure B, the differential metabolites in rat serum showed a separation trend, indicating that the characteristics of differential metabolites among rat groups were different.

[0041] The overall differences between the groups were analyzed by PCA analysis and PLS-DA. The VIP value of the metabolites in the OPLS-DA analysis (if the OPLS-DA was overfitted, the VIP value of the PLS-DA was used) and the Fold change and p-value in the univariate analysis were used to screen the differential metabolites. A total of 224 differential metabolites in the serum of rats in the JS group, M group, and K group were screened under the conditions of FC>1; VIP≥1 of the OPLS-DA model and p<0.05. The cluster analysis of rat serum metabolites was as follows: Figure 12 These differential metabolites mainly include organic acid compounds, phospholipid compounds, choline compounds, and heterocyclic compounds. These metabolites may affect the cognitive function of rats through the blood-brain barrier.

[0042] 6.2 Analysis of Differential Metabolites and Metabolic Pathways in Rat Serum

[0043] like Figure 13 Among the top ten metabolites with significant differences, it was found that the levels of Ruscogenin and other metabolites in the serum of rats in the JS group were significantly increased (p < 0.05), while the levels of N-Lauroyl Arginine, N-Jasmonoylisoleucine and other metabolites were significantly decreased (p < 0.05). Figure 13 Among the top ten metabolites in terms of abundance shown in Figure B, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylcholine (LysoPC) were found to be metabolites with higher abundance in the serum of the three groups of rats, reflecting their potential roles in energy metabolism, oxidative stress, and inflammatory response.

[0044] KEGG pathway enrichment analysis of differential metabolites between JS group and M group Figure 14As shown in A, the JS group downregulated almost all metabolite enrichment pathways, including neuroactive ligand-receptor interaction, sphingolipid signaling pathway and other metabolic pathways (p < 0.05), especially upregulated the glycerophospholipid metabolism pathway (p < 0.05). KEGG enrichment pathways of differential metabolites in the serum of rats in the JS group, M group and K group are as follows Figure 14 As shown in Figure B, similarly, the glycerophospholipid metabolism pathway was the most enriched, and the sphingolipid signaling pathway also showed significant changes. These metabolisms revealed a potential association between rat intestinal flora and rat cognitive function.

[0045] 7. Transcriptomic Sequencing Analysis of Rat Hippocampus

[0046] 7.1 Effects of dendrobium alkaloids combined with Lactobacillus paracasei on gene expression in rat hippocampus A transcriptomic analysis of rat hippocampus tissue was performed. First, the dataset was filtered for differentially expressed genes (DEGs) using a P < 0.05 filter condition. Figure 15 A is the volcano plot of differentially expressed genes in the hippocampus of rats in the JS and M groups. Figure 15 As shown in B, compared with the M group, 83 differentially expressed genes were upregulated and 218 differentially expressed genes were downregulated in the hippocampus transcriptomics of the JS group rats.

[0047] 7.2 GO and KEGG pathway enrichment analysis of differentially expressed genes like Figure 16 A, The top 20 GO enrichment functional annotations of genes with significant differences in expression between the JS and M groups were analyzed. Protein binding, signaling receptor binding, etc. were significantly enriched. This indicates that these biological processes play an important role in the JS group. According to the KEGG pathway enrichment bubble diagram of the differentially expressed genes, as shown in Figure 16As shown in B, key pathways such as neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, and PI3K-Akt signaling pathway were significantly enriched.

[0048] 7.3 Key differentially expressed gene-protein network interaction analysis and RT-qPCR validation of genes (KRT18, CDH13) The PPI protein network interaction analysis was performed on the differentially expressed genes in the hippocampus of rats in the JS and M groups using the STRING database. Figure 17 As shown in the figure, core genes were screened using the MCC (Maximum Clique Centrality), MNC (Neighborhood Component Centrality), and DMNC (Maximum Neighborhood Component Centrality) algorithms. Based on the resulting PPI network interaction diagram, the hub genes identified by the DMNC algorithm intersection are DSP, CDH3, KRT8, and KRT18.

[0049] To detect the effects of dendrobium alkaloids combined with Lactobacillus paracasei on the expression levels of key differentially expressed genes, RT-qPCR experiments were performed. Figure 18 As shown in the figure, compared with group K, the expression level of KRT18 in group M was significantly decreased (p < 0.05), while the expression level of CDH13, a member of the same cadherin family as CDH3, was significantly increased (p < 0.05). Compared with group M, the expression level of CDH13 in groups S and Y was significantly decreased (p < 0.05), while that in group J was significantly increased (p < 0.05). Compared with group M, the expression level of KRT18 in group JS was significantly increased (p < 0.05), while that of CDH13 was significantly decreased (p < 0.05).

[0050] Example 2 Dendrobium alkaloids combined with Lactobacillus paracasei alleviate high glucose-induced microglial cell damage Chronic hyperglycemia can damage the brain and may lead to cognitive decline, such as memory loss and slowed thinking. Microglia are the primary immune cells in the central nervous system. They not only participate in the brain's immune response but also play a key role in learning, memory, and neuroplasticity. Research has shown that microglia also play a key role in memory regulation. They can influence the connections between neurons (synapses), which are important communication hubs for brain cells to communicate with each other and transmit brain signals. During brain development, microglia actively remove or "prune" synapses, which helps shape the circuits that enable efficient brain function and maintain normal memory. However, microglial dysfunction can also lead to cognitive impairment. Therefore, the inventors conducted experiments to investigate the effect of dendrobium alkaloids combined with Lactobacillus paracasei on the damage of HMC3 microglia induced by high glucose.

[0051] Experimental materials: cell line: HMC3 microglial cells (human), six-well plates, complete culture medium (DMEM-H + 10% FBS + 1% P / S), loading buffer, Annexin V-FITC and PI staining solution, PBS, centrifuge tubes, pipettes, etc.

[0052] Experimental methods 1. Cell Culture: HMC3 microglial cells were seeded at an appropriate density in a six-well plate. Culture medium containing 10% FBS was added to each well. Culture was continued in a 37°C, 5% CO2 incubator until the cells reached the desired density (approximately 80%).

[0053] 2. Group treatment: blank control group (group K: no treatment, serving as blank control), high glucose model group (group MM: culture medium supplemented with sucralose to a final concentration of 35 mmol / L), Lactobacillus paracasei group (group J: culture medium containing 35 mmol / L sucralose, Lactobacillus paracasei added to a fixed final concentration of 10 μg / μL, bacterial suspension concentration of 1×10 9 cfu / mL), dendrobium-treated group (S group, culture medium containing 35 mmol / L sucralose, dendrobium added to a fixed final concentration of 5 µg / µL), Lactobacillus paracasei combined with dendrobium-treated groups (MJS group: culture medium containing 35 mmol / L sucralose, and the following drug additions: MJS1:1 group: Lactobacillus paracasei to dendrobium volume ratio of 1:1 (each well of a six-well plate contained 1 mL of Lactobacillus paracasei suspension + 1 mL of dendrobium solution), MJS1:2 group: volume ratio of 1:2 (each well of a six-well plate contained 0.67 mL of Lactobacillus paracasei suspension + 1.33 mL of dendrobium solution), MJS2:1 group: volume ratio of 2:1 (each well of a six-well plate contained 1.33 mL of Lactobacillus paracasei suspension + 0.67 mL of dendrobium solution).

[0054] 3. Cell collection and fixation: After 24 hours of treatment, trypsinize the cells and collect them into a centrifuge tube. Wash the cells twice with PBS and discard the supernatant after each centrifugation.

[0055] 4. Apoptosis staining: Add FITC and PI staining solutions respectively and stain for 30 minutes in the dark.

[0056] 5. Flow cytometry analysis: Flow cytometry was used to detect the stained cells and analyze the apoptosis rate of each group.

[0057] The results show that: Figure 19 A and Figure 19 As shown in Figure B, there was a significant difference between the model group (MM) and the blank group (K) (***p < 0.0001), indicating that a high glucose environment induced apoptosis in HMC3 microglia. The Lactobacillus paracasei combined with dendrobium (MJS) treatment groups (volume ratio of 1:2 (mass ratio of 1:1), volume ratio of 1:1 (mass ratio of 2:1), and volume ratio of 2:1 (mass ratio of 4:1)) showed significant differences compared with the model group (MM) (*p < 0.05, **p < 0.01), indicating that Lactobacillus paracasei combined with dendrobium inhibited cell apoptosis to varying degrees at different dosage ratios. The combination of Lactobacillus paracasei and dendrobium at a mass ratio of 4:1 was the most effective, while a 1:1 ratio of Lactobacillus paracasei and dendrobium was less effective than either drug alone, suggesting an antagonistic effect at a 1:1 ratio. This is consistent with the results of animal studies.

[0058] Example 3 Synergistic effect of combined use of dendrobium alkaloids and Lactobacillus paracasei The Chou-Talalay combination index method was used to calculate the synergistic coefficient (CI). CI = (D)1 / (Dx)1+(D)2 / (Dx)2+(D)1(D)2 / (Dx)1(Dx)2, where (D)1 is the concentration of dendrobium required for the combined action of dendrobium and Lactobacillus paracasei, (D)2 is the concentration of Lactobacillus paracasei required for the combined action of dendrobium and Lactobacillus paracasei, (Dx)1 is the concentration of dendrobium alone, and (Dx)2 is the concentration of Lactobacillus paracasei alone. A CI < 1 indicates a synergistic effect between the two drugs, a CI = 1 indicates an additive effect, and a CI > 1 indicates an antagonistic effect.

[0059] 3.1 To investigate the synergistic effect of the combined use of dendrobium and Lactobacillus paracasei, the duration of rats in the novel arm during the Y-maze test and the escape latency of rats during the water maze test were used as the criteria for the efficacy of the combined medication in improving learning and memory. The dosages of dendrobium alone and Lactobacillus paracasei alone required to achieve the combined medication-improving learning and memory efficacy were calculated.

[0060] Table 1 Statistics of drug synergy during the Y-maze test

[0061] Among them, there were blank control group (K), high sugar model group (M), dendrobium alkaloid intervention group (S), dendrobium alkaloid combined with Lactobacillus paracasei intervention group (JS), Lactobacillus paracasei intervention group (J); donepezil hydrochloride positive group (Y).

[0062] Improvement of spatial recognition memory effect = (mean of each intervention group - mean of group M) / mean of group M × 100%

[0063] Calculation of the combination index (CI) during the Y-maze test The improvement effect of spatial recognition memory (73.4%) in the JS group (combination of dendrobium and Lactobacillus paracasei) was selected as the target, and the doses required to achieve the same effect with either drug alone were calculated.

[0064] Dendrobium alkaloid intervention group: 5mg / kg corresponds to a 7.4% improvement in learning and memory. Assuming a linear dose-effect relationship, a 73.4% improvement in spatial recognition memory is required (D x )1=5mg / kg×73.4% / 7.4%=49.6mg / kg.

[0065] Lactobacillus paracasei intervention group: 50mg / kg corresponds to a 9.4% improvement in learning and memory. Assuming a linear dose-effect relationship, a 73.4% improvement in learning and memory is required (D x )2=50mg / kg×73.4% / 9.4%=390.4mg / kg.

[0066] In the combined medication, dendrobium alkaloids (D)1 = 2.5 mg / kg and Lactobacillus paracasei (D)2 = 25 mg / kg.

[0067] 2.5 / 49.6+25 / 390.4+(2.5×25) / (49.6×390.4)=0.113, CI<1, the combination therapy showed synergistic effect.

[0068] 3.2 Calculation of the water maze combined drug index using the same method Table 2 Statistics of drug synergy during the water maze test

[0069] Among them, there were blank control group (K), high sugar model group (M), dendrobium alkaloid intervention group (S), dendrobium alkaloid combined with Lactobacillus paracasei intervention group (JS), Lactobacillus paracasei intervention group (J); donepezil hydrochloride positive group (Y).

[0070] Improvement of learning and memory effect = (mean of intervention group - mean of M group) / mean of M group × 100%

[0071] The improvement effect of learning and memory (89.6%) in the JS group (combination medication) was selected as the target, and the doses required to achieve the same effect by using individual medications were calculated.

[0072] Dendrobium alkaloid intervention group: 5mg / kg corresponds to a 75.4% improvement in learning and memory. Assuming a linear dose-effect relationship, an 89.6% improvement in learning and memory is required (D x )1=5mg / kg×89.6% / 75.4%=5.94mg / kg.

[0073] Lactobacillus paracasei intervention group: 50 mg / kg corresponds to a 35.9% improvement in learning and memory. Assuming a linear dose-effect relationship, to achieve an 89.6% improvement in learning and memory requires (D x )2=50mg / kg×89.6% / 35.9%=124.8mg / kg.

[0074] In the combined medication, dendrobium alkaloids (D)1 = 2.5 mg / kg and Lactobacillus paracasei (D)2 = 25 mg / kg.

[0075] Improvement of learning and memory effect by combined drug use synergy coefficient (CI)

[0076] 2.5 / 5.94+25 / 124.8+(2.5×25) / (5.94×124.8)=0.70, CI<1, the combination therapy showed synergistic effect.

[0077] 3.3 Synergistic effect of dendrobium alkaloids combined with Lactobacillus paracasei on the proliferation of HMC3 microglia in vitro

[0078] The inventors used the Chou-Talalay Combination Index method to investigate the effects of dendrobium combined with Lactobacillus paracasei on the in vitro proliferation of HMC3 microglial cells. The experiment demonstrated that when Lactobacillus paracasei was used at a concentration of 10 µg / µL and dendrobium at a concentration of 5 µg / µL, the inhibitory effect on HMC3 cell apoptosis was significantly enhanced, and the combination index (CI) was <1, indicating a synergistic effect between the two drugs at these concentrations.

[0079] Calculation steps: When Lactobacillus paracasei is used alone at a concentration of 15 µg / µL, the inhibition rate of apoptosis I Lactobacillus paracasei = 50%. When dendrobium is used alone at a concentration of 8 µg / µL, the inhibition rate of apoptosis in HMC3 microglia I dendrobium = 50%.

[0080] Combination medication 1:1 Ratio: When dendrobium alkaloids are used at a concentration of 5 µg / µL and 5 µg / µL respectively, the inhibition rate (I1:1) is 60%. To calculate the CI, assume that the dose required to achieve a 60% inhibition rate against Lactobacillus paracasei alone is Dx1, which was determined to be 18 µg / µL. The dose required to achieve a 60% inhibition rate against Lactobacillus paracasei alone is Dx2, which was determined to be 9.4 µg / µL. According to the formula CI=(D)1 / (Dx)1+(D)2 / (Dx)2+(D)1(D)2 / (Dx)1(Dx)2, at this time D1=5 µg / µL, D2=5 µg / µL, then CI=5 / 18+5 / 9.4+5*5 / 18*9.4≈0.28+0.53+0.15=0.96<1, showing a synergistic effect.

[0081] 2:1 Ratio: When Lactobacillus paracasei is used at a concentration of 10 µg / µL and dendrobium alkaloids at a concentration of 5 µg / µL, the inhibition rate (I2:1) is 75%. To calculate CI, assume that the dose of Lactobacillus paracasei alone that achieves 75% inhibition is Dx1, which has been experimentally determined to be 24 µg / µL. The dose of dendrobium alkaloids alone that achieves 75% inhibition is Dx2, which has been experimentally determined to be 20 µg / µL. According to the formula CI=(D)1 / (Dx)1+(D)2 / (Dx)2+(D)1(D)2 / (Dx)1(Dx)2, at this time D1=10 µg / µL, D2=5 µg / µL, then CI=10 / 24+5 / 20+10*5 / 24*20≈0.42+0.25+0.10=0.77<1), showing a synergistic effect, and the effect is better than when the ratio is 1:1.

[0082] 1:2 Ratio: When Lactobacillus paracasei is used at a concentration of 5 µg / µL and dendrobium alkaloids at a concentration of 10 µg / µL, the inhibition rate (I1:2) is 55%. To calculate the CI, assume that the dose of dendrobium alkaloids alone that achieves a 55% inhibition rate is Dx1, which was determined to be 15.7 µg / µL. The dose of dendrobium alkaloids alone that achieves a 55% inhibition rate is Dx2, which was determined to be 8.5 µg / µL. According to the formula CI=(D)1 / (Dx)1+(D)2 / (Dx)2+(D)1(D)2 / (Dx)1(Dx)2, at this time D1=5 µg / µL, D2=10 µg / µL, then CI=5 / 15.7+10 / 8.5+5*10 / 15.7*8.5≈0.32+1.18 +0.37=1.87>1, showing antagonistic effect, and not as effective as single administration.

[0083] In summary, the animal experiments presented here induced cognitive impairment in rats by glucolipid metabolism disorders induced by oral administration of high sugar. The combined use of dendrobium and Lactobacillus paracasei (mass ratio of 1:10) significantly improved learning and memory abilities in the rats, demonstrating a more pronounced effect than either dendrobium or Lactobacillus paracasei alone. In vitro experiments using a microglial cell line (HMC3) demonstrated that the combined use of Lactobacillus paracasei and dendrobium (mass ratio of 2-4:1) significantly inhibited cell apoptosis, demonstrating a synergistic effect.

Claims

1. Use of dendrobium alkaloids in combination with Lactobacillus paracasei in the preparation of a product for preventing or alleviating impairment of learning and memory abilities induced by a high-sugar diet.

2. The use of dendrobium alkaloids combined with Lactobacillus paracasei in the preparation of products for treating microglial damage caused by high sugar.

3. The use according to claim 1 or 2, characterized in that: The mass ratio of the lactobacillus paracasei to dendrobium alkaloids is 2-10:

1.

4. The use according to claim 3, characterized in that: The Lactobacillus paracasei is HH-LP58.

5. The use according to claim 3, characterized in that: The concentration of the Lactobacillus paracasei is 8-10 μg / μL, and the concentration of dendrobium alkaloids is 3-5 μg / μL.

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