Application of lithium salt in preparation of product with function of promoting activation of ZBP1

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of a lithium salt in preparation of a product with an effect of promoting ZBP1 activation. Researches show that lithium citrate and lithium carbonate have the following effects: (1) up-regulating expression of skin fibroblasts ZBP1; (2) promoting activation of ZBP1; (3) the activity of beta-galactosidase (SA-beta-Gal) is reduced; (4) a relatively large non-toxic concentration range is shown; therefore, the lithium salt disclosed by the invention plays a role in delaying skin cell aging by up-regulating the expression level of ZBP1 in fibroblasts, promoting ZBP1 activation and / or reducing beta-galactosidase activity. Particularly, the effects of lithium citrate on the aspects of improving ZBP1 expression and relieving cell senescence phenotypes are obviously better than those of lithium carbonate. Therefore, the lithium salt serving as an active ingredient has important application value in preparation of cosmetics or medicines with an anti-aging effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a lithium salt in the preparation of products that promote ZBP1 activation. Background Technology

[0002] Skin aging is a complex biological process involving multiple molecular mechanisms, primarily manifested as decreased fibroblast function, cell cycle arrest, reduced matrix components, and the persistent secretion of chronic inflammatory factors. With age, skin fibroblasts gradually accumulate various endogenous damages such as oxidative stress, splicing abnormalities, and activation of reverse transcription elements, leading to the formation of abnormal nucleic acid conformations within the cells, particularly the abnormal accumulation of Z-RNAs. Previous studies have shown that persistent Z-RNAs can activate chronic inflammatory responses and accelerate skin tissue aging. ZBP1 (Z-DNA binding protein 1) is a nucleic acid sensor capable of recognizing Z-RNA structures, normally maintaining cellular homeostasis by recognizing and clearing abnormal nucleic acids. However, during skin aging, ZBP1 expression levels decline, preventing the timely recognition and clearance of Z-RNAs, further triggering inflammatory pathway activation and skin function degeneration. Currently, existing anti-aging products on the market mainly focus on scavenging reactive oxygen species and repairing DNA damage, while interventions targeting the regulation of abnormal cellular nucleic acid homeostasis remain lacking.

[0003] Lithium citrate and lithium carbonate are classic drugs for treating bipolar disorder and have a long history of clinical application. They stabilize mood and reduce the frequency and severity of manic and depressive episodes by regulating neurotransmitter levels in the brain. Although these two drugs are widely used in the field of mental illness, current technology has not reported that lithium citrate and lithium carbonate can significantly upregulate the expression level of ZBP1 in fibroblasts, promote ZBP1 activation, and thereby bind accumulated Z-RNA, thus effectively alleviating cellular senescence.

[0004] Therefore, providing new uses for lithium citrate and lithium carbonate will not only help to further broaden their application scope, but may also provide new treatment options for many diseases for which there are currently no effective treatments. Summary of the Invention

[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides an application of lithium salt in the preparation of products that promote ZBP1 activation.

[0006] The technical solution of the present invention is as follows: This invention first provides the application of lithium salt in the preparation of products that promote ZBP1 activation; the lithium salt is selected from lithium citrate or lithium carbonate.

[0007] The present invention also provides the application of lithium salt in the preparation of products that upregulate the expression level of ZBP1 in cells; wherein the lithium salt is selected from lithium citrate or lithium carbonate.

[0008] Preferably, the cells are skin fibroblasts.

[0009] The present invention also provides the application of lithium salt in the preparation of products that reduce β-galactosidase activity; wherein the lithium salt is selected from lithium citrate or lithium carbonate.

[0010] Preferably, the product is a cosmetic or a pharmaceutical.

[0011] Beneficial Effects: This invention provides a novel use for lithium salts; studies have shown that lithium citrate and lithium carbonate have the following effects: (1) upregulating the expression of ZBP1 in skin fibroblasts; (2) promoting ZBP1 activation; (3) reducing β-galactosidase activity (SA-β-Gal); (4) exhibiting a large non-toxic concentration range; thus, it can be seen that the lithium salts described in this invention delay skin cell aging by upregulating the expression level of ZBP1 in fibroblasts, promoting ZBP1 activation, and / or reducing β-galactosidase activity. In particular, lithium citrate is significantly more effective than lithium carbonate in increasing ZBP1 expression and alleviating cellular senescence phenotypes.

[0012] Therefore, using the lithium salt described in this invention as an active ingredient to prepare cosmetics or drugs with anti-aging effects has important application value. Attached Figure Description

[0013] Figure 1 Figure showing the experimental results of activating the ZBP1 promoter with lithium citrate and lithium carbonate.

[0014] Figure 2 The figure shows the results of the CCK8 assay for the cytotoxicity of lithium citrate and lithium carbonate.

[0015] Figure 3 Figure showing the results of flow cytometry assay for β-galactosidase activity. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.

[0017] I. Materials 293T cells were previously cryopreserved in the laboratory. HFF-1 cells were purchased from Zhejiang Meisen Company; lithium citrate and lithium carbonate were purchased from SELLECK Company; fetal bovine serum, DMEM, and penicillin antibodies were purchased from Gibco Company, USA. β-galactosidase staining probes were purchased from Tongren Chemical Company; ZBP1 promoter plasmid was synthesized in its entirety by Tianyi Huiyuan Company. CCK8 reagent and dual luciferase assay kit were purchased from Yisheng Biotechnology Company.

[0018] II. Methods 1. Activate ZBP1 bootloader detection The effects of lithium citrate and lithium carbonate on ZBP1 promoter activity were detected using a dual-luciferase reporter assay system. 293T cells were co-transfected with the pGL3-luc reporter plasmid containing the human ZBP1 promoter region, and pRL-TK was used as an internal control. Twenty-four hours after transfection, cells were treated with 5 μM lithium citrate and lithium carbonate for 48 hours, respectively. Luciferase activity was detected using the Dual-Luciferase Reporter Assay System, and ZBP1 promoter activity was expressed as the Firefly / Renilla ratio.

[0019] 2. Cell culture, grouping, modeling, and intervention. HFF-1 cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics at 37°C, 5% CO2, and saturated humidity, with medium changes every 2–3 days. HFF-1 cells in the logarithmic growth phase were divided into Blank group, Model group, lithium citrate intervention group, and lithium carbonate intervention group. The Blank group was cultured in 5% medium throughout the entire process. Other groups were treated with 40 mg / mL D-galactose for 72 hours to induce a senescence model. The Model group received no lithium citrate or lithium carbonate intervention, while the intervention groups received 5 μM and 20 μM lithium citrate and lithium carbonate, respectively, for 48 hours.

[0020] 3. CCK8 assay was used to determine the toxicity of lithium citrate and lithium carbonate to HFF-1 cells after 48 hours of treatment. Human fibroblasts were resuspended in complete culture medium at a concentration of 200 μL per well (3 × 10⁶ cells per well). 3 Cells were seeded into 96-well plates, and the plates were then transferred to an incubator and cultured at 37°C with 5% CO2. After 24 h, the cell culture medium was aspirated, and 100 μL of complete culture medium containing different concentrations of lithium citrate and lithium carbonate (2.5-80 μM) was added for 48 h. A control group without samples and a blank group without cells and samples were also set up. After treatment according to the CCK8 kit instructions, the absorbance (OD) at 450 nm was measured using a microplate reader, and the cell viability was calculated using the formula.

[0021] 4. Flow cytometry analysis of the effects of lithium citrate and lithium carbonate on β-galactosidase activity β-galactosidase activity was detected using the SPiDER-βGal fluorescent probe. After staining according to the manufacturer's instructions, FITC channel signals were detected by flow cytometry to obtain the percentage of positive cells and the average fluorescence intensity. Three biological replicates were performed for each group, with at least 5000 positive cells detected per replicate.

[0022] 5. Statistical Analysis Data are expressed as mean ± SD. T-tests were used for comparisons between groups. P <0.05 indicates a significant difference.

[0023] III. Results 1. ZBP1 promoter activation effect The results of the dual-luciferase reporter assay are shown in Table 1. Figure 1 The results showed that lithium citrate dose-dependently enhanced ZBP1 promoter activity. At a concentration of 5 μM, ZBP1 promoter activity was significantly increased, approximately 1.5-fold compared to the control group. P The value <0.01 indicates that lithium citrate and lithium carbonate can activate ZBP1 expression by upregulating its transcription. Among them, lithium citrate has a better activation effect.

[0024] 2. Non-toxic concentrations of lithium citrate and lithium carbonate After incubation with 0-80 μM lithium citrate and lithium carbonate for 48 hours, the cell viability was above 85%, indicating that these concentrations had no significant cytotoxic effect on human skin fibroblasts (Table 2). Figure 2 ).

[0025] 3. β-galactosidase activity Flow cytometry results (Table 3, Figure 3 In the D-Gal-induced group, the positive rate of β-galactosidase was significantly increased, while the positive rate and average fluorescence intensity decreased significantly after the intervention of lithium citrate and lithium carbonate, indicating that lithium citrate can alleviate the aging phenotype and reduce intracellular β-galactosidase activity. P <0.05). In comparison, lithium carbonate only needs a concentration of 20 μM to inhibit β-galactosidase activity.

[0026] Table 1. ZBP1 promoter activation multiple

[0027] ** indicates a significant difference compared to transfected empty PLG3, P < 0.01; ** indicates a significant difference compared to transfected empty PLG3, P < 0.001;

[0028] Table 2. Effects of different concentrations of lithium carbonate and lithium citrate on HFF-1 cell viability

[0029] Cell viability above 85% is considered a non-toxic concentration.

[0030] Table 3. Effects of different concentrations of lithium carbonate and lithium citrate on β-galactosidase.

[0031] ### indicates a significant difference compared to the blank group, P < 0.01; ** indicates a significant difference compared to the aging model group, P < 0.01; ** indicates a significant difference compared to the transfected empty PLG3 group, P < 0.001.

Claims

1. The application of a lithium salt in the preparation of a product that promotes ZBP1 activation; wherein the lithium salt is selected from lithium citrate or lithium carbonate.

2. The use of a lithium salt in the preparation of a product that upregulates the expression level of ZBP1 in cells; wherein the lithium salt is selected from lithium citrate or lithium carbonate.

3. The application according to claim 2, characterized in that, The cells mentioned are skin fibroblasts.

4. The use of a lithium salt in the preparation of a product having the effect of reducing β-galactosidase activity; wherein the lithium salt is selected from lithium citrate or lithium carbonate.

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