Application of 630nm LED light source in preparation of device for regulating organism flora and metabolic steady state thereof

Abstract

The invention discloses an application of a 630nm LED light source in preparation of a device for regulating organism flora and a metabolic steady state thereof. Research finds that the community richness and diversity of intestinal bacteria of a rheumatoid arthritis patient can be remarkably recovered through irradiation of a 630 nm LED light source, photosensitive genes of microorganisms can be activated in vivo and in vitro respectively, secretion and synthesis of alanine are induced and promoted, the metabolic steady state of alanine in the intestinal flora is adjusted, and the rheumatoid arthritis can be effectively treated. Finally, the purposes of inhibiting inflammatory injury of patients and improving muscle strength are achieved. The invention provides a new method capable of regulating intestinal flora and metabolic disorder thereof, and the traditional Chinese medicine composition can replace probiotics, prebiotics and metabiotics, does not need oral administration, only needs in-vitro irradiation, is convenient to use and free of side effects, and has a wide application prospect in the medical field.

Description

Technical Field

[0001] The present invention relates to the application of a 630nm LED light source in the preparation of a device for regulating the microbial flora and its metabolic homeostasis in an organism, and belongs to the technical field of medical device manufacturing. Background Art

[0002] As the largest and most complex microbial ecosystem in the human body, changes in the composition and function of the intestinal flora are closely linked to the development and progression of numerous diseases. In recent years, with the deepening of microbiome research, people have gradually realized that the intestinal flora not only participates in the digestion and absorption of nutrients, but also plays a key role in immune regulation, metabolic balance, and neural signaling. Regulating and restoring the intestinal flora and its metabolic homeostasis through probiotics, prebiotics, or related drugs can effectively prevent and treat related diseases or health issues.

[0003] The intestinal microbiome is composed of trillions of microorganisms, including bacteria, viruses, fungi, and archaea, far outnumbering the human body's own cells. These microorganisms form a symbiotic relationship with the host, forming a "superorganism." On the one hand, the intestinal microbiome breaks down complex carbohydrates (such as dietary fiber) that the human body cannot digest, producing short-chain fatty acids (SCFAs, including acetate, propionate, and butyrate). These substances are not only the main energy source for colonic epithelial cells but also have anti-inflammatory and immunomodulatory effects. On the other hand, healthy microbiota inhibit the growth of pathogens by competing for nutrients and colonization sites, while maintaining the integrity of the intestinal epithelial barrier and preventing harmful substances from "leaking" into the bloodstream (i.e., "leaky gut"). Furthermore, the intestinal microbiome has an "educational" effect on the development and function of the immune system, helping it distinguish "self" from "non-self" and balancing pro-inflammatory and anti-inflammatory responses. Furthermore, through multiple pathways, such as the vagus nerve, neurotransmitters (such as 5-HT and GABA), and metabolites, the intestinal microbiome communicates bidirectionally with the central nervous system, influencing mood, cognition, and behavior. When the diversity of the intestinal flora decreases or the proportion of specific bacterial groups becomes unbalanced (called "dysbiosis"), these functions are disrupted, leading to a variety of pathological changes. For example, the increase of certain harmful bacteria can damage the intestinal barrier, allowing endotoxins to enter the bloodstream and triggering chronic low-grade inflammation, which is the basis of many metabolic diseases.

[0004] Metabolic homeostasis of the gut microbiota refers to a dynamic equilibrium maintained between the gut microbiota and its host. In this state, the microbiota can effectively metabolize nutrients provided by the host and produce metabolites that are beneficial to the host. Disruption of metabolic homeostasis can lead to the development of various diseases, such as diabetes, obesity, and cardiovascular disease.

[0005] The present invention provides a method for directly or indirectly regulating intestinal flora and its metabolic homeostasis by whole-body, especially abdominal illumination. The effect of illumination on flora regulation mainly depends on the light receptors carried by the flora. By irradiating the whole body or part of the body with light, the body's flora and its metabolic homeostasis can be regulated to promote disease recovery. In TNF-Tg mice, systemic or local inflammatory reactions, tissue damage, and intestinal flora disorder are manifested. After whole-body illumination treatment of the body, it can be seen that the intestinal flora and its metabolic disorder, as well as the corresponding tissue damage, such as muscle atrophy, arthritis, etc., are significantly restored. Among them, the specific metabolites of the intestinal flora are the main mediators of the therapeutic effect of illumination. In addition, the dominant strains in the intestinal flora under illumination can regulate the metabolism of the strains and the production of metabolites through the action of the light receptors of the strains, indicating that illumination is a new method that can regulate the intestinal flora and its metabolic disorder through the light receptors of the dominant strains, and can be a new way to replace probiotics, prebiotics, and postbiotics. Summary of the Invention

[0006] The purpose of the present invention is to provide a new method for regulating the body's microflora and its metabolic homeostasis, which can achieve the purpose of treating diseases related to intestinal microflora and its metabolic disorders.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention proposes the use of a 630nm LED light source in preparing a device for regulating the body's microflora and its metabolic homeostasis.

[0009] Preferably, the irradiation time of the 630nm LED light source is 30min, and the power density is 8.2mW / cm 2 , carried out in dark conditions, with an energy density of 14 J / cm 2 , twice a day, with each illumination time of 30 minutes, for 20 days.

[0010] Furthermore, the present invention also proposes the use of a 630nm LED light source in the preparation of a device for treating diseases related to intestinal flora and metabolic disorders.

[0011] Among them, preferably, the disease is rheumatoid arthritis or muscular senescence.

[0012] Preferably, the irradiation time of the 630nm LED light source is 30min, and the power density is 8.2mW / cm 2 , carried out in dark conditions, with an energy density of 14 J / cm 2 , twice a day, with each illumination time of 30 minutes, for 20 days.

[0013] Among them, preferably, irradiation with a 630nm LED light source can significantly restore the richness and diversity of intestinal bacterial communities in patients with rheumatoid arthritis, activate the photosensitive genes of microorganisms in vivo and in vitro, induce and promote the secretion and synthesis of alanine, and regulate the metabolic homeostasis of alanine in the intestinal flora, ultimately achieving the purpose of inhibiting inflammatory damage in patients and improving muscle strength.

[0014] Among them, preferably, the device is an abdominal irradiation device.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a new method for regulating the body's flora and its metabolic homeostasis, by which the purpose of treating diseases related to intestinal flora and its metabolic disorders can be achieved. Through research, the present invention found that irradiation with a 630nm LED light source can significantly restore the community richness and diversity of intestinal bacteria in patients with rheumatoid arthritis, can activate the photosensitive genes of microorganisms in vivo and in vitro, induce and promote the secretion and synthesis of alanine, regulate the metabolic homeostasis of alanine in the intestinal flora, and ultimately achieve the purpose of inhibiting inflammatory damage in patients and improving muscle strength. The present invention provides a new method for regulating intestinal flora and its metabolic disorders, which can replace probiotics, prebiotics, and postbiotics, does not require oral administration, only requires in vitro irradiation, is easy to use, has no side effects, and will have broad application prospects in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flow chart of the experiment using 630nm LED to irradiate TNF-Tg mice;

[0018] Figure 2 The effect of 630nm LED irradiation on the RA score of mice in each group;

[0019] (A) RA scores of mice in each group before 630nm LED treatment; (B) RA scores of mice in each group after 630nm LED treatment. One-way ANOVA was used to analyze the differences among the control group, TNF-α transgenic group, and LED treatment group.

[0020] Figure 3 The effect of 630nm LED irradiation on the muscle tension level of mice in each group;

[0021] Figure 4 The effect of 630nm LED irradiation on the α-diversity index of intestinal flora in TNF-T mice;

[0022] Figure 5 The effect of 630nm LED irradiation on the β-diversity index of intestinal flora in TNF-Tg mice;

[0023] Figure 6 Effects of 630nm LED irradiation on the intestinal microbiota of TNF-Tg mice at the genus level;

[0024] Figure 7 This is a network diagram showing the interaction between rheumatoid arthritis scores in TNF-Tg mice after 630nm LED irradiation and mouse muscle strength and intestinal microbial abundance;

[0025] Figure 8 Changes in the content of alanine and unsaturated fatty acid biosynthesis pathways in the intestine of TNF-Tg mice after 630nm LED irradiation;

[0026] Figure 9 The rheumatoid arthritis score of TNF-Tg mice after 630nm LED irradiation and the interaction network diagram between mouse muscle strength and intestinal microbial metabolites;

[0027] Figure 10 Changes in the expression levels of photosensitized genes of Enterobacter and Lactobacillus in the feces of TNF-Tg mice after 630nm LED irradiation;

[0028] Figure 11 The changes in the expression levels of photosensitivity genes in Escherichia coli and Lactobacillus acidophilus after irradiation with 630nm LED;

[0029] Figure 12 The changes in alanine content in the supernatant of Lactobacillus acidophilus and Escherichia coli after 630 nm LED irradiation. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0031] Example 1630nm LED improves rheumatoid arthritis and muscle aging in TNF-Tg mice

[0032] (1) Experimental animals and grouping: A total of 30 8-week-old male TNF-Tg and WT mice (purchased from Suzhou Saiye Biological Co., Ltd.) weighing 18-22 g were used. After 2 weeks of adaptive feeding, the mice were divided into three groups (10 mice each): WT mouse group (control group), TNF-Tg mouse group (TNF-α transgenic group), and TNF-Tg mouse group irradiated with 630 nm LED (LED treatment group).

[0033] 630nm LED light panel irradiation method and irradiation process Figure 1As shown, the 630nm LED irradiation time is 30min and the power density is 8.2mW / cm 2 , carried out under dark conditions, with an energy density of 14 J / cm 2, twice a day, with a light exposure time of 30 minutes each time, for 20 days.

[0034] (2) Animal observation indicators and analysis

[0035] (2.1) Rheumatoid arthritis score in mice: The rheumatoid arthritis score (RA score) is an important indicator for assessing the severity of rheumatoid arthritis affecting the limbs of mice. Scoring is based on the degree of swelling of the mouse paws (0, no change; 1, redness or swelling of the toes; 2, redness and swelling of less than two toes or swelling of the entire paw without ankle involvement; 3, redness and swelling of more than two toes or swelling of the entire paw without ankle involvement; 4, lesions involving the ankle joint). The sum of the scores for all four limbs is the RA score for TNF-induced rheumatoid arthritis in mice.

[0036] Depend on Figure 2 It can be seen that before treatment (day 0), the RA scores of the TNF-α transgenic group and the LED treatment group were significantly increased compared with the control group. The significant increase in RA scores indicates that the severity of rheumatoid arthritis in TNF mice has worsened. After 630nm irradiation, the RA scores of mice were significantly reduced, indicating that 630nm LED irradiation has a positive effect on the recovery of TNF-α inflammatory state. The significant decrease in RA scores after 630nm LED treatment reflects that red light irradiation can inhibit TNF-induced inflammatory damage in mice.

[0037] (2.2) Mouse tensile strength test: Mouse tensile strength test is an important indicator for evaluating its muscle strength, motor ability or neuromuscular function. Through a special device (such as a metal grid, lever or crossbar), the mouse is allowed to grasp with its forelimbs or hindlimbs, and then the device is pulled horizontally or vertically. The sensor records the maximum tensile force value. According to the mouse's forelimb grasping the crossbar, gently pull the tail until it is released, and record the peak tensile force. Repeat 3-5 times and take the average value to avoid fatigue. The obtained peak tensile force of the mouse is divided by the mouse body weight, and the resulting value is the relative muscle strength of the TNF mouse (referred to as "muscle strength").

[0038] Depend on Figure 3It can be seen that before treatment (day 0), compared with the control group, the muscle strength of the TNF-α transgenic group and the LED treatment group decreased significantly. The significant decrease in muscle strength indicates that the severity of muscle aging in TNF mice has worsened. After 630nm irradiation, the muscle strength of mice increased significantly, indicating that 630nm LED irradiation has a positive effect on the recovery of TNF-α muscle aging. The significant increase in muscle strength of mice after 630nm LED treatment reflects that red light irradiation can inhibit TNF-induced muscle aging in mice.

[0039] Example 2630nm LED irradiation can cause changes in intestinal flora and metabolism in mice and is related to the therapeutic effect

[0040] (1) Experimental animals and groups: same as in Example 1

[0041] (2) Detection of Alpha and Beta Diversity of Mouse Gut Microbiota: The alpha diversity of the intestinal microbiota of the three groups of mice was detected by 16s rRNA sequencing. The alpha diversity of intestinal bacteria, including the Shannon and Simpson indices, was calculated using Mothur software. Alpha diversity refers to the diversity within a specific region or ecosystem and is a comprehensive indicator reflecting richness and diversity.

[0042] Figure 4 The results showed that after 630nm LED irradiation, the Shannon index of the control group and the LED treatment group increased significantly, and the Simpson index decreased significantly compared with the TNF-α transgenic mice, indicating that 630nm LED irradiation can significantly restore the richness and diversity of the intestinal bacterial community in TNF-Tg mice (P<0.05). The results of principal component analysis (PCoA) based on weighted UniFrac distance showed that the intestinal microbiota between different groups showed significant separation in similarity, suggesting that there was a significant difference in β diversity between the TNF-Tg and WT and 630nm LED treatment groups (PERMANOVAR, p=0.001, Figure 5 ).

[0043] (3) Detection of the composition of the mouse intestinal flora: The species OTU table obtained by 16s rRNA sequencing was split, and the relative abundance of the species composition of the three groups of bacteria at the phylum level and genus level was counted respectively. The difference analysis at the phylum level and genus level was performed using Metastats. At the phylum level, the overall intestinal microbiome (accounting for approximately 98.17%) was mainly composed of Firmicutes (62.60%), followed by Bacteroidetes (27.28%), Verrucomicrobia (7.13%) and Proteobacteria (1.34%). Compared with the control group, the relative abundance of Firmicutes, Actinobacteria, Proteobacteria, and Tenericutes in the TNF-α transgenic group mice increased (p < 0.05), while the relative abundance of Verrucomicrobia decreased (p < 0.05) (Table 1). Compared with the TNF-α transgenic mice, the relative abundance of Verrucomicrobia increased, while the relative abundance of Actinobacteria decreased after 630nm LED treatment (p<0.05). In addition, after 630nm LED treatment, the contents of Verrucomicrobia, Proteobacteria, Actinobacteria, Candidatus_Saccharibacteria, and Tenericutes approached the levels of WT. In the TNF-Tg group, the abundance of Lactonifactor, Escherichia, Turicibacter, and Alistipes increased. The abundance of Blautia, Lactobacillus, Allobaculum, and Odoribacter decreased (p<0.05). Compared with the TNF-Tg group, the abundance of Lactobacillus, Allobaculum, Clostridium, and Staphylococcus increased after 630nm LED irradiation, while the abundance of Alistipes, Bacteroides, Lactonifactor, and Echerichia decreased ( Figure 6 ).

[0044] Table 1. Effects of 630 nm LED irradiation on the phylum-level composition of the intestinal microbiota in TNF-Tg mice

[0045]

[0046] (4) Correlation analysis between mouse intestinal flora and RA scores and mouse muscle strength: To evaluate the changes in bacterial genera related to mouse muscle strength, the correlation analysis between intestinal microorganisms (based on Euclidean distance) and mouse muscle strength (based on Manhattan distance) showed that intestinal microbiota plays an important role in affecting mouse muscle strength. The correlation results showed that mouse muscle strength was positively correlated with the abundance of Allobaculum, Clostridium, Lactobacillus, Odoribacter and Staphylococcus (p < 0.05); and negatively correlated with the abundance of Bacteroides, Blautia, Escherichia, Shigella, Lachnospiracea and Turicibacter (p < 0.05) ( Figure 7 ).

[0047] (5) Functional prediction analysis of mouse muscle strength-related intestinal flora: To evaluate the changes in the functions of mouse muscle strength-related bacterial genera, the functions of mouse muscle strength-related bacterial genera were predicted using PICRUSt2, and the regulation of 630nm LED on the metabolic homeostasis of TNF-Tg intestinal flora was reflected. Bioinformatics results showed that the biosynthesis of alanine and unsaturated fatty acids in the intestinal flora of TNF-Tg mice was inhibited. After treatment with 630nm LED, the biosynthesis of alanine and unsaturated fatty acids in the intestinal flora was significantly restored, indicating that 630nm LED can regulate the metabolic homeostasis of the intestinal flora ( Figure 8 ).

[0048] (6) Correlation analysis between the abundance of gut microbiota related to muscle strength in mice and metabolites of gut microbiota: To evaluate the changes in metabolites of bacterial genera related to muscle strength in TNF-Tg mice, the correlation analysis between metabolites of gut microbes (based on Euclidean distance) and metabolites related to muscle strength and rheumatoid arthritis scores in mice (based on Manhattan distance) showed that gut microbiota metabolites such as L-alanine, taurine, indolepyruvic acid, indoleacrylic acid, tyrosine, phenylalanine, and stearic acid played an important role in affecting the muscle strength and rheumatoid arthritis inflammation scores of mice ( Figure 9 ).

[0049] Example 3630nm LED can induce the synthesis of microbial metabolites by activating microbial photoreceptors

[0050] (1) Experimental animals and groups: same as in Example 1

[0051] (2) 630nm LED irradiation leads to activation of intestinal microbial photoreceptors in mice: Figure 9 and 10As shown in the results, compared with TNF-Tg transgenic mice, the content of photoreceptors increased significantly after 630nm LED irradiation. In vitro experiments showed that 630nm LED irradiation could lead to increased expression levels of photosensitivity genes in Escherichia coli and Lactobacillus acidophilus ( Figure 11 ), indicating that the intervention of 630nm LED can activate the light-sensitive genes of microorganisms in vivo and in vitro, respectively.

[0052] (3) 630nm LED irradiation leads to increased alanine biosynthesis in the intestinal microorganisms of mice: Figure 12 As shown in the figure, compared with the control group, the microbial alanine biosynthesis increased after 630nm LED irradiation, indicating that the intervention of 630nm LED can induce the increase of microbial metabolite alanine biosynthesis by activating the microbial photosensitive genes, thereby promoting the secretion and synthesis of microbial alanine.

Claims

Application of 1.630nm LED light source in the preparation of devices for regulating the body's microbial flora and its metabolic homeostasis.

2. The use according to claim 1, characterized in that The irradiation time of 630nm LED light source is 30min, and the power density is 8.2mW / cm 2 , carried out in dark conditions, with an energy density of 14 J / cm 2 , twice a day, with each illumination time of 30 minutes, for 20 days.

3. The use according to claim 1, characterized in that The device is an abdominal irradiation device. Application of 4.630nm LED light source in the preparation of devices for treating diseases related to intestinal flora and its metabolic disorders.

5. The use according to claim 4, characterized in that The disease is rheumatoid arthritis or muscular senescence.

6. The use according to claim 4, characterized in that The irradiation time of 630nm LED light source is 30min, and the power density is 8.2mW / cm 2 , carried out in dark conditions, with an energy density of 14 J / cm 2 , twice a day, with each illumination time of 30 minutes, for 20 days.

7. The use according to claim 4, characterized in that Irradiation with a 630nm LED light source can significantly restore the richness and diversity of intestinal bacterial communities in patients with rheumatoid arthritis. It can activate the photosensitive genes of microorganisms both in vivo and in vitro, induce and promote the secretion and synthesis of alanine, and regulate the metabolic homeostasis of alanine in the intestinal flora, ultimately achieving the goal of inhibiting inflammatory damage in patients and improving muscle strength.

8. The use according to claim 4, characterized in that The device is an abdominal irradiation device.

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