Application of evaluation model based on Teocytes cells in evaluation of sleep quality improvement of external product

Abstract

The invention discloses an application of an evaluation model based on Teocytes cells in evaluating sleep quality improvement of an external product. According to the method, the exact relation between the TCs and the sleep quality is explored, the application feasibility of the TCs serving as an evaluation index in external sleep nursing product research is achieved, and the TCs can serve as an accurate index for sleep quality improvement evaluation of the external sleep nursing product. Before this, TCs have not been used as a recognized sleep quality evaluation index. By monitoring the change of TCs, the effect of the external sleep nursing product can be evaluated, the risk of sleep disorder related diseases can be predicted, a new potential treatment target for insomnia is provided, and a new thought and strategy are provided for studying the pathological mechanism and diagnosis and treatment of insomnia.

Description

TECHNICAL FIELD

[0001] The application relates to application of a Telocytes cell evaluation model in evaluation of sleep quality improvement of an external use product and belongs to the Telocytes cell application field. BACKGROUND

[0002] Sleep is a physiological process necessary for life. In recent years, the sleep time per capita in China has been continuously reduced, and the sleep quality has been reduced compared with that in the past. Sleep problems have become a public health problem that cannot be ignored. In the theory of traditional Chinese medicine, insomnia is called 'not sleeping' or 'not being able to sleep', which refers to a disease that cannot obtain normal sleep frequently. The earliest record is in Huangdi Neijing. Insomnia is caused by disharmony of yin and yang, blood and qi, and dysfunction of viscera, so that the spirit is disturbed and the spirit is not at ease. Meridians are the channels that run blood and qi, connect viscera and limbs, communicate inside and outside, and run through the upper and lower parts. Dredging the meridians can make the blood circulation, and improve the sleep quality.

[0003] Telocytes (TCs), also known as telocytes, are widely distributed in the interstitium of the body, have very long protrusions (Tps), and have string-like swollen nodes on the Tps. The TCs and the Tps form a cell connection network with the same type of cells or different type of cells, and the TCs and the Tps have rich endoplasmic reticulum, mitochondria and calcium cave. The TCs secrete extracellular vesicles (including exosomes), and realize direct or indirect communication between cells by forming cell connections and secreting extracellular vesicles. Studies have shown that telocytes and the network formed thereby have various characteristics of meridian substantive cells, and the structure and function thereof are highly consistent with the meridian phenomenon, and may become the cytological basis of the meridian substance. The reduction or dysfunction of TCs is related to various diseases, plays a key role in maintaining tissue homeostasis, regulating morphogenesis and promoting tissue repair, and has potential therapeutic target value.

[0004] As a new type of interstitial cell, TCs have made significant progress in recent years, and scientists have carried out extensive exploration on the structure, distribution and function thereof. However, in the current research, TCs are not used as an index for measuring sleep regulation. SUMMARY

[0005] The technical problem to be solved by the application is to provide an application of a Telocytes cell evaluation model in evaluation of sleep quality improvement of an external use product.

[0006] Technical scheme: In order to solve the above technical problem, the application provides an application of a Telocytes cell evaluation model in evaluation of sleep quality improvement of an external use product.

[0007] The evaluation methods for the Telocytes cell evaluation model include immunofluorescence staining, Cd34 / Pdgfra dual immunofluorescence labeling, and transmission electron microscopy observation.

[0008] The external products include one or more of the following: cream, lotion, serum, gel, plaster, and transdermal patch.

[0009] The application includes detecting one or more of the morphology, number, or length of Telocytes.

[0010] The application includes testing the sleep-regulating effects of topical sleep care products.

[0011] The application includes detecting changes in Telocytes at the site where the topical product is applied.

[0012] The topical product in question is a sleep management cream.

[0013] The components of the sleep management cream include:

[0014] Phase A materials: PEG-10 / 15 cross-linked polymer / polydimethyl silicone oil 1.0-4.0%, PEG-10 polydimethylsiloxane 1.0-2.0%, cyclopentamethoxysiloxane / polydimethylsiloxane 5.0-10.0%, glyceryl tris(behenic acid / isostearate / eicosanoic acid) ester 0.1-2.0%, isononyl isononanoate 3.0-6.0%;

[0015] Phase B materials: dextrin palmitate / ethylhexanoate 0.15-0.5%, propylene glycol 1.0-5.0%, magnesium sulfate 0.1-1.0%, siloxane triol alginate 1.0-4.0%, water 1.0-100.0%;

[0016] Phase C materials: Turmeric root extract 0.1-1.0%, Apocynum venetum extract 0.5-3.0%, Poria cocos extract 0.5-1.5%, Lilium brownii extract 0.5-2.0%, Cymbidium goeringii extract 0.5-2.0%, Cocoa seed extract 0.5-2.0%, Longan extract 1.0-2.0%, Amino acids 0.5-2.0%, Hydrolyzed yeast protein 0.1-1.0%, Hops extract 0.1-2.0%, Ginseng root extract 0.5-3.0%, Angelica sinensis extract 0.5-3.0%, Oligopeptide -10.01-2.0%;

[0017] D-phase materials: Valerian oil 0.01-0.05%, Lavender oil 0.05-0.15%, Melaleuca alternifolia leaf oil 0.1-1.0%.

[0018] In this invention, after using the sleep management cream, the 5-HT content in the hippocampus of rats increased; the number of TCs at the cream application site increased significantly, their morphology changed, Tps lengthened, and cell connections between TCs increased, enhancing communication. This indicates that during the use of topical sleep care products, TCs can respond to external stimuli and exert physiological functions, influencing the secretion of 5-HT and other related sleep factors. Furthermore, in human trials, the proportion of deep sleep time increased after using the sleep management cream, indicating that the cream is beneficial for improving sleep quality. Previous studies have also reported the presence of TCs in human skin (WANG J et al. ADVEX PMEDIOL. 2016, 913: 1-21). Therefore, TCs can serve as an indicator for assessing sleep quality, and this approach has a certain degree of scientific validity and feasibility.

[0019] In subcutaneous fascia tissue, counting the number of TCs requires staining with immunofluorescence markers and identifying cell morphology using transmission electron microscopy.

[0020] This invention points out that sleep management creams significantly regulate sleep through changes in the morphology and quantity of total toxic substances (TCs), improving sleep quality. This is of great significance for the development of topical medications for treating insomnia and for the evaluation of insomnia treatment.

[0021] The present invention provides a sleep regulation marker cell, which includes the number and morphology of TCs, as well as the cell connections between TCs and homologous and heterologous cells, and the secretion of extracellular vesicles.

[0022] TCs are a novel type of mesenchymal cell with unique morphology and function discovered in recent years. In human skin, TCs are mainly distributed in the reticular layer of the dermis, surrounding hair follicles, sweat glands, blood vessels, and nerve endings, forming a three-dimensional network. Their most prominent feature is their extremely long telopodes (Tps), which can reach hundreds of micrometers in length but only 50-100 nm in diameter, requiring transmission electron microscopy (TEM) for observation. These telopodes exhibit a beaded structure, composed of alternating swollen podoms (containing mitochondria and endoplasmic reticulum) and narrow podomers.

[0023] Trough cells (TCs) can connect with neighboring cells, blood vessels, nerves, and collagen bundles through their slender processes, constructing complex 3D network structures. TCs are commonly identified using dual immunofluorescence labeling with Cd34 / PDGFR and transmission electron microscopy. Immunohistochemical studies have shown that human skin TCs mainly express CD34 and PDGFR-α, with some subsets expressing c-kit (CD117). Research has found that TCs can form specific connections with hair follicle stem cells and release extracellular vesicles, suggesting their potential involvement in skin regeneration regulation. Furthermore, the close contact between TCs and mast cells suggests a potential role in allergic reactions. Recent research has also found that multiple growth factor receptors expressed by TCs may synergistically interact with PRP (platelet-rich plasma) therapy, promoting skin repair.

[0024] The reduction or dysfunction of TCs is associated with a variety of diseases. They play a key role in maintaining tissue homeostasis, regulating morphogenesis and promoting tissue repair, and have potential therapeutic target value.

[0025] In this invention, a sleep management cream was applied to the skin of healthy and insomniac rats. The number, length, and cell connections of TCs at the application site all increased. The results indicate that changes in the number and length of TCs have a significant and reliable indicative effect on the improvement of sleep quality.

[0026] This invention uses Telocytes as a novel indicator for assessing sleep quality. Through multiple sets of double immunofluorescence labeling and transmission electron microscopy, and by analyzing cell morphology, quantity, and cell connectivity, the potential relationship between TCs and sleep quality was revealed. This addresses the feasibility of its application in clinical practice and provides clinicians with an accurate indicator for evaluating the sleep quality of insomnia patients. Currently, the universal cell markers for TCs are Cd34 and Pdgfra double-positive markers, Cx43 is a gap junction marker, and Cdh1 is a calcium-dependent cell adhesion molecule. The TCs studied in this invention include Cd34+ / Pdgfra+ TCs.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0028] 1. Observations showed that, compared with the healthy group, the number of total cellular structures (TCs) in the subcutaneous fascia of rats in the insomnia group was significantly reduced (P<0.01), and the length of TCs was significantly shortened (P<0.05). After treatment with the sleep management cream, compared with the insomnia group, the number of TCs in the subcutaneous fascia of rats in the insomnia treatment group was significantly increased (P<0.05), and the length of TCs was significantly increased (P<0.01). Parallel Tps were clearly observed, cell connections between TCs were increased, and extracellular vesicles were present around TCs, indicating enhanced cell communication. This suggests that TCs have a significant and reliable indicative role in improving sleep quality.

[0029] 2. In human trials, the sleep management cream increased the percentage of deep sleep in the test subjects, which is beneficial to improving sleep quality. Furthermore, TCs are also present in human skin tissue, indirectly proving the feasibility of applying TCs in the research of topical sleep care products.

[0030] 3. This invention explores the precise relationship between total sleep tract infections (TCs) and sleep quality, providing an accurate indicator for evaluating the sleep quality improvement effects of topical sleep care products. Previously, TCs were not recognized as a standard indicator for sleep quality assessment. Furthermore, monitoring changes in TCs may not only help assess the effectiveness of sleep disorder treatments but also predict the risk of sleep disorder-related diseases, providing a new potential therapeutic target for insomnia and offering new ideas and strategies for studying the pathological mechanisms and diagnosis and treatment of insomnia. Attached Figure Description

[0031] Figure 1 The regulatory effect of sleep management cream on sleep in rats: Differences in 5-HT content in the hippocampus of rats in different experimental groups; *P<0.05; **P<0.01;

[0032] Figure 2 Effects of sleep management cream on the number and morphology of TCs in the skin and subcutaneous fascia: A. Arrows represent TCs that are positive for both Cd34 (green) and Pdgfra (red) immunofluorescence staining; the bar chart shows the comparison results of the differences in the number of Cd34 and Pdgfra positive cells in each group; B. Arrows represent TCs; the bar chart shows the comparison results of the differences in the length of TCs in each group;

[0033] Figure 3 A shows the fluorescence double-labeled images of Cd34 (green) and Cx43 (red) in each group. White arrows represent TCs, and the bar chart shows the comparison results of the differences in the number of Cd34 and Cx43 positive cells in each group. Figure 3 B shows the fluorescence double-labeled images of Cd34 (green) and Cdh1 (red) in each group. The white arrows represent TCs, and the bar chart shows the comparison results of the differences in the number of Cd34 and Cdh1 positive cells in each group. Detailed Implementation

[0034] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings.

[0035] A method for evaluating sleep quality through TCs, comprising the following steps:

[0036] S1: Select healthy SD female rats (purchased from Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing City) as the research objects, and divide them into a healthy group, an insomnia group, and an insomnia treatment group. The method of modeling rats in the insomnia group is as follows: Inject PCPA suspension (Sigma-Aldrich, C6506, 350 mg / kg) intraperitoneally for short-term insomnia modeling, once a day for 2 consecutive days. The rats in the insomnia treatment group are smeared with a cream for sleep management on the basis of the insomnia group: The cream for sleep management (product number: GD Makeup Preparation No. 2024030644, company: Shenzhen Rongda Biotechnology Co., Ltd.) is smeared on the back of the rats in the insomnia treatment group between about 1 cm on the left and right of the midline, from the first lumbar vertebra to the sacral region, once a day for 5 consecutive days. Samples are taken in batches after 5 days of treatment. The tissues are cut into 6-μm thin slices by paraffin embedding technology, dried and reserved;

[0037] S2: Use a rat 5-HT enzyme-linked immunosorbent assay kit to strictly measure the 5-HT level according to the instructions. As Figure 1 shown, the content of 5-HT in the hippocampus of rats is detected by ELISA method. The results show that there are extremely significant differences in the content of 5-HT in the hippocampus of rats after 5 days (P < 0.01), and the content of 5-HT increases with the extension of the smearing days. Compared with the healthy group, the content of hippocampal 5-HT in the insomnia group rats decreased significantly (from 2616.04 ng / g to 1678.96 ng / g) (P < 0.01); compared with the insomnia group, the content of hippocampal 5-HT in the treatment group rats increased significantly (from 1678.96 ng / g to 2434.85 ng / g) (P < 0.01).

[0038] S3: Take the skin in the middle of the rat's back, fix the tissue in 4% paraformaldehyde (Shanghai Macklin Biochemical Co., Ltd., P804536-500g) for 48 h, and then perform trimming, dehydration, clearing, wax impregnation and embedding; Cut the tissue into 6-μm thin slices, dry and store for later use;

[0039] S4: Immunofluorescence staining using Cd34 (highly glycosylated type I transmembrane glycoprotein) / Pdgfra (platelet-derived growth factor receptor) markers: Tissue sections were dewaxed to water and placed in 0.01 mol / L sodium citrate solution at 95°C to repair the antigen. After cooling to room temperature, they were washed with phosphate-buffered saline (PBS). The sections were blocked with 5% bovine serum albumin (BSA) solution at 37°C for 1 h. Then, 500 μL of rabbit Cd34 polyclonal antibody (A13929, Wuhan Aibotek Biotechnology Co., Ltd., diluted 1:200 with 1% BSA) and mouse Pdgfra monoclonal antibody (sc-398206, Santa Cruz Biotechnology (Shanghai) Co., Ltd., diluted 1:200 with 1% BSA) were added to the tissue sections and incubated overnight at 4°C. The tissue was washed with PBS and incubated at 37°C for 1 h with 500 μL of fluorescent secondary antibody (CoraLite488-conjugated goat anti-rabbit IgG (H+L), SA00013-2 and 500 μL of CoraLite594-conjugated goat anti-mouse IgG (H+L), SA00013-3 (Wuhan Sanying Biotechnology Co., Ltd.)) (diluted to PBS at a ratio of 1:100). Nuclear staining was performed with DAPI. After incubation with autofluorescence quencher at room temperature for 10 min, the tissue was mounted with antifluorescence quencher. Images were collected using a confocal microscope, and statistical analysis was performed. Figure 2 As shown in Figure A, compared to the healthy group, the number of TCs in the subcutaneous fascia of rats in the insomnia group was significantly reduced (from 657.77 / mm²). 2 Reduced to 386.71 per mm 2 (P < 0.01). After treatment with sleep management cream, compared with the insomnia group, the number of TCs in the subcutaneous fascia of rats in the treatment group was significantly increased (from 386.71 / mm²). 2 Increased to 714.00 pieces / mm 2 (P < 0.01).

[0040] S5: TCs cells were identified by dual immunofluorescence labeling with Cd34 / Pdgfra and observation by transmission electron microscopy.

[0041] S6: Trim the skin tissue at the application site to 1mm. 3Small tissue fragments were fixed in pre-cooled 2.5% glutaraldehyde solution at 4°C for 48 h, washed with PBS, and incubated in 1% osmium tetroxide solution at 4°C for 2 h. After washing with PBS again, the fragments were dehydrated using a gradient of alcohol solutions (75%, 85%, 95%, and 100%, 10 min each), and then embedded in epoxy resin. Ultrathin sections (50 nm) were prepared using a microtome. The ultrathin sections were stained with 1% uranium acetate and Reynolds lead (a commonly used staining agent in transmission electron microscopy sample preparation) for 20 min. Finally, the morphology of TCs was observed using a transmission electron microscope on the ultrathin tissue sections. This meticulous process allows for detailed examination of the tissue structure at the ultrastructural level and enables statistical analysis of the data. The results are as follows: Figure 2 As shown in Figure B, compared to the healthy group, the length of transcranial poles (TCs) in the subcutaneous fascia of the insomnia group rats was significantly shortened (from 19.59 μm to 6.60 μm) (P < 0.05). After treatment with the sleep management cream, compared to the insomnia group rats, the length of TCs in the subcutaneous fascia of the treatment group rats was significantly increased (from 6.60 μm to 33.44 μm) (P < 0.01), and parallel Tps could be clearly observed. The results are as follows... Figure 3 As shown in Figure A, compared to the healthy group, the number of TCs expressing Cx43 in the subcutaneous fascia of rats in the insomnia group was significantly reduced (from 527.75 / mm²). 2 Reduced to 180.54 per mm 2 (P < 0.01), compared with the insomnia group, the number of TCs expressing Cx43 in the subcutaneous fascia of rats in the treatment group was significantly increased (from 180.54 / mm²). 2 Increased to 472.19 per mm 2 (P < 0.01), the results are as follows Figure 3 As shown in Figure B, compared to the healthy group, the number of TCs expressing Cdh1 in the subcutaneous fascia of rats in the insomnia group was significantly reduced (from 694.40 / mm²). 2 Reduced to 569.41 per mm 2 (P < 0.05), compared with the insomnia group, the number of TCs expressing Cdh1 in the subcutaneous fascia of rats in the treatment group was significantly increased (from 569.41 cells / mm²). 2 Increased to 708.29 per mm 2 (P < 0.01). The increased number of Cx43 and Cdh1 indicates that their cell connections are more extensive, and extracellular vesicles are present around TCs, which enhances cell communication and enables them to respond to external stimuli and perform physiological functions.

[0042] In a sleep study of 11 participants, changes in sleep were assessed before (D0) and after (D1, D2) application of the cream. The results showed that 54.5% of the participants experienced an increase in total sleep time, with an average increase of 0.55 hours; 81% of the participants experienced a decrease in the proportion of light sleep time, a decrease of 8.85%; and 72% of the participants experienced an increase in the proportion of deep sleep time, an increase of 14.9%. The overall sleep patterns of the participants are shown in Table 1.

[0043] Table 1

[0044]

[0045] The results showed that sleep management cream has a beneficial effect on improving sleep quality.

Claims

1. An application of a Telocytes-based cell evaluation model in assessing the improvement of sleep quality by topical products.

2. The application according to claim 1, characterized in that, The evaluation methods for the Telocytes cell evaluation model include immunofluorescence staining, Cd34 / Pdgfra dual immunofluorescence labeling, and transmission electron microscopy.

3. The application according to claim 1, characterized in that, The evaluation method of the Telocytes cell evaluation model includes detecting one or more of the following: morphology, number, or length of Telocytes.

4. The application according to claim 1, characterized in that, The topical products include one or more of the following: creams, lotions, serums, gels, plasters, and transdermal patches.

5. The application according to claim 1, characterized in that, The application includes detecting whether relevant indicators of sleep quality have improved; these relevant indicators include 5-HT.

6. The application according to claim 1, characterized in that, The application includes detecting changes in Telocytes at the site where topical sleep care products are applied.

7. The application according to claim 1, characterized in that, The application also includes testing the effects of topical products on sleep duration.

8. The application according to claim 1, characterized in that, When the number of Telocytes increases significantly, their morphology changes, Telocytes become longer, and cell connections between Telocytes increase, the secretion of 5-HT in the hippocampus, a key indicator of sleep quality, increases.

Images