A stem cell-hydride composition for repairing renal tubular damage

Abstract

The application discloses a kind of stem cell-negative hydrogen ion compositions for repairing renal tubular injury, belong to biological medicine technical field.The stem cell-negative hydrogen ion compositions include stem cell, negative hydrogen ion donor, tianshan viola extract and pharmaceutically acceptable carrier.The stem cell is added concentration 1×10 6 -1×10 8 mg / mL, the negative hydrogen ion donor is added 0.1-0.5mg / mL, and the addition amount of tianshan viola extract is 2-5mg / mL.Animal experiment shows that the stem cell-negative hydrogen ion compositions can significantly restore the body weight growth of acute renal tubular injury model rat, reduce blood creatinine level, reduce renal tubular necrosis, inhibit renal tubular epithelial cell apoptosis, reduce kidney tissue malondialdehyde and active oxygen level, improve ATP content, and correct the imbalance state of serum proinflammatory factor increase and anti-inflammatory factor decrease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a stem cell-negative hydrogen ion composition for repairing renal tubular damage. Background Technology

[0002] Renal tubular injury is a core pathological component of many kidney diseases, especially acute kidney injury, and can lead to renal function loss or even end-stage renal disease in severe cases. Currently, clinical treatment for renal tubular injury mainly involves symptomatic support, anti-inflammatory, and immunosuppressive drugs (such as glucocorticoids and tacrolimus). However, these drugs often fail to reverse existing renal tubular structural damage, and long-term use has significant toxic side effects. Therefore, developing novel treatment strategies that can effectively repair renal tubular epithelial cells and restore renal tubular structure and function is of significant clinical importance.

[0003] In recent years, mesenchymal stem cell (MSC) therapy has shown therapeutic potential in the field of renal regenerative medicine due to its paracrine immune regulation, anti-inflammatory, and tissue repair functions. However, conventional MSCs rely on natural paracrine effects, resulting in problems such as insufficient targeting, low homing efficiency, and decreased survival rate in the injury microenvironment, leading to unstable and poor reproducibility of repair effects. Tonsil-derived MSCs (T-MSCs), as an abundant and easily obtained adult stem cell source, exhibit good proliferation and differentiation capabilities. On the other hand, negative hydrogen ion donors (such as hydrogen-rich water, magnesium hydride, and calcium hydride nanoparticles) can release hydrogen molecules with selective antioxidant effects. Studies have suggested that they can enhance the anti-fibrotic and anti-oxidative stress effects of MSCs, but the duration of action of exogenous small molecules in vivo is short, making it difficult to maintain sustained therapeutic concentrations. In addition, Viola yedoensis extract contains a variety of active ingredients with anti-inflammatory and antioxidant pharmacological effects, but its application in repairing renal tubular damage has not yet been reported.

[0004] Transcription factor NF-YA (Nuclear transcription factor Y subunit alpha) is one of the subunits of the nuclear transcription factor Y complex. It can specifically recognize and bind to the CCAAT box in the promoter region, regulating the expression of various genes related to cell proliferation, differentiation, and stress response. NF-YA generates two main isoforms through alternative splicing, with the short isoform NF-YAs being highly expressed in stem cells. Studies have shown that NF-YAs are mainly involved in cell cycle regulation and transcriptional activation of differentiation-related genes, and may play an important role in regulating stem cell stemness maintenance and directed differentiation. However, research on using NF-YAs to modify mesenchymal stem cells to enhance renal tubular injury repair capacity remains lacking, and the specific mechanism by which it intervenes in kidney injury needs to be elucidated.

[0005] Based on this, this invention is the first to combine NF-YAs modified T-MSCs, negative hydrogen ion donors (such as calcium hydride nanoparticles), and Viola yedoensis extract, aiming to solve the comprehensive technical problems of insufficient targeting, short duration of action of exogenous antioxidant molecules, and lack of synergistic application of natural products with stem cells in existing MSC therapies, thereby more effectively promoting the repair of renal tubular damage. Summary of the Invention

[0006] Therefore, the present invention provides a stem cell-negative hydrogen ion composition for repairing renal tubular damage, in order to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a stem cell-negative hydrogen ion composition for repairing renal tubular damage is provided, comprising stem cells, a negative hydrogen ion donor, Viola yedoensis extract, and a pharmaceutically acceptable carrier.

[0008] Furthermore, the concentration of the added stem cells is 1×10⁻⁶. 6 -1×10 8 The amount of negative hydrogen ion donor added is 0.1-0.5 mg / mL, and the amount of Viola tianshanense extract added is 2-5 mg / mL.

[0009] Furthermore, the concentration of the added stem cells is 1×10⁻⁶. 7 The amount of negative hydrogen ion donor added is 0.2 mg / mL, and the amount of Viola tianshanense extract added is 3 mg / mL.

[0010] Furthermore, the stem cells are tonsillar mesenchymal stem cells; the negative hydrogen ion donor is selected from at least one of hydrogen-rich water, magnesium hydride, calcium hydride, calcium hydride nanoparticles, and negative hydrogen ion powder.

[0011] Furthermore, the tonsillar mesenchymal stem cells are NF-YAs modified cell lines.

[0012] Furthermore, the negative hydrogen ion donor is calcium hydride nanoparticles coated with liposomes.

[0013] Furthermore, the amino acid sequence of the NF-YAs is shown in SEQ ID NO.1, and the nucleotide sequence of the NF-YAs is shown in SEQ ID NO.2.

[0014] According to a second aspect of the present invention, a method for preparing a stem cell-negative hydrogen ion composition for repairing renal tubular injury is provided, comprising the following steps: Constructing NF-YAs-modified amygdala mesenchymal stem cells; The above-mentioned NF-YAs modified tonsillar mesenchymal stem cells, negative hydrogen ion donor, and Viola yedoensis extract were mixed evenly in a certain proportion, and then a pharmaceutically acceptable carrier was added to prepare a stem cell-negative hydrogen ion composition.

[0015] Furthermore, the method for constructing NF-YAs-modified amygdala mesenchymal stem cells is as follows: S1. Construct a recombinant lentiviral expression vector containing the NF-YAs gene; S2. Co-transfect host cells with the recombinant lentiviral expression vector and lentiviral packaging plasmid from step S1, and package them to obtain lentiviral particles. S3. Transfect the lentiviral particles from step S2 into tonsillar mesenchymal stem cells and screen to obtain NF-YAs modified tonsillar mesenchymal stem cells.

[0016] Furthermore, the preparation method of the Viola tianshanense extract is as follows: Take dried whole herb of Viola tianshanense, pulverize and sieve to obtain powder; add purified water at a material-to-liquid ratio of 1g:(10-20)mL, and adjust the pH to 4.0-5.5; add compound enzyme equivalent to 0.5%-2% of the powder mass, and enzymatically hydrolyze at 45-60℃ for 60-120min; after enzymatic hydrolysis, inactivate the enzyme at 80-100℃ for 10-15min; filter, collect the filtrate, concentrate under reduced pressure, and dry to obtain Viola tianshanense extract; The complex enzyme comprises cellulase and pectinase in a mass ratio of (1-2):1.

[0017] According to a third aspect of the present invention, the use of the stem cell-negative hydrogen ion composition prepared by the aforementioned method in the preparation of a drug for repairing renal tubular damage is provided.

[0018] According to a fourth aspect of the invention, the use of Viola tianshanense extract in the preparation of a drug for repairing renal tubular damage is provided.

[0019] The present invention has the following advantages: This invention achieves a synergistic effect of multi-target and multi-level repair of renal tubular damage by combining NF-YAs genetically engineered amygdala mesenchymal stem cells, calcium hydride nanoparticles, and Viola yedoensis extract. The specific mechanism is as follows: (1) NF-YAs modification can significantly enhance the repair activity of tonsillar mesenchymal stem cells. These engineered stem cells not only retain their natural homing ability and can actively integrate into the damaged renal tubular epithelial cell layer to replace necrotic cells, but also maintain the self-renewal and anti-apoptotic properties of stem cells by overexpressing short NF-YAs isoforms. At the same time, they can enhance paracrine function, effectively downregulate endoplasmic reticulum stress markers, and block apoptosis pathways, playing a core role from both structural repair and cell protection levels.

[0020] (2) Liposome-coated calcium hydride nanoparticles avoid water reaction during storage and mixing, ensuring formulation stability and cell survival. After injection, the liposomes gradually degrade in vivo, releasing calcium hydride and generating hydrogen, calcium ions, and hydroxide ions upon contact with water: hydrogen scavenges free radicals, calcium ions participate in repair signaling, and hydroxide ions neutralize the acidic environment, creating a favorable microenvironment for stem cells. This sustained-release strategy overcomes the shortcomings of the short-lived effect of ordinary hydrogen-rich water and the violent gas production of naked calcium hydride.

[0021] (3) Viola tianshanense extract, as an anti-inflammatory and antioxidant auxiliary component, can effectively improve the renal tubular microenvironment by inhibiting the NF-κB / NLRP3 pathway, scavenging free radicals, and upregulating the activity of antioxidant enzymes. Based on the binary combination of engineered stem cells and calcium hydride, although the hydroxide ions released by calcium hydride can neutralize acidic metabolites, they may cause local alkalinity fluctuations, posing a potential pressure on stem cell activity. The active ingredients contained in Viola tianshanense extract (such as phenolic acids and flavonoids) can buffer this alkalinity fluctuation, while further reducing residual inflammation and oxidative stress, protecting stem cell vitality, and activating the Nrf2 endogenous defense pathway. This compensates for the shortcomings of the binary combination from three dimensions: environmental repair, cell empowerment, and endogenous activation, achieving a triple synergistic effect and highlighting its indispensable necessity.

[0022] (4) Animal experiments showed that the triple combination of the present invention can significantly restore the body weight gain of model animals, greatly reduce serum creatinine levels, alleviate renal tubular necrosis scores, inhibit renal tubular epithelial cell apoptosis, reduce the content of malondialdehyde and reactive oxygen species in renal tissue, increase ATP levels, and correct the imbalance between elevated pro-inflammatory factors and decreased anti-inflammatory factors in serum. All repair indicators are significantly better than any single component or the combination of two components, and the effect is comparable to or better than that of the positive control drug dexamethasone, fully demonstrating the outstanding technological progress of the three components synergistic effect and comprehensive repair of renal tubular damage. Attached Figure Description

[0023] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0024] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0025] Figure 1 The results show the detection of NF-YAs protein levels in NF-YAs modified T-MSCs according to the present invention. Detailed Implementation

[0026] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] According to a first aspect of the present invention, a stem cell-negative hydrogen ion composition for repairing renal tubular damage is provided, comprising stem cells, a negative hydrogen ion donor, Viola yedoensis extract, and a pharmaceutically acceptable carrier.

[0028] Furthermore, the concentration of added stem cells was 1×10⁻⁶. 6 -1×10 8 The concentration of negative hydrogen ion donors was 0.1-0.5 mg / mL, and the concentration of Viola tianshanense extract was 2-5 mg / mL.

[0029] Furthermore, the concentration of added stem cells was 1×10⁻⁶. 7 The concentration of negative hydrogen ion donors was 0.2 mg / mL, and the concentration of Viola tianshanense extract was 3 mg / mL.

[0030] Furthermore, the stem cells are tonsillar mesenchymal stem cells; the negative hydrogen ion donor is selected from at least one of hydrogen-rich water, magnesium hydride, calcium hydride, calcium hydride nanoparticles, and negative hydrogen ion powder.

[0031] Furthermore, the tonsillar mesenchymal stem cells are NF-YAs modified cell lines.

[0032] Furthermore, the negative hydrogen ion donor is calcium hydride nanoparticles coated with liposomes.

[0033] Furthermore, the amino acid sequence of NF-YAs is shown in SEQ ID NO.1, and the nucleotide sequence of NF-YAs is shown in SEQ ID NO.2.

[0034] According to a second aspect of the present invention, a method for preparing a stem cell-negative hydrogen ion composition for repairing renal tubular injury is provided, comprising the following steps: Constructing NF-YAs-modified amygdala mesenchymal stem cells; The above-mentioned NF-YAs modified tonsillar mesenchymal stem cells, negative hydrogen ion donor, and Viola yedoensis extract were mixed evenly in a certain proportion, and then a pharmaceutically acceptable carrier was added to prepare a stem cell-negative hydrogen ion composition.

[0035] Furthermore, the method for constructing NF-YAs-modified amygdala mesenchymal stem cells is as follows: S1. Construct a recombinant lentiviral expression vector containing the NF-YAs gene; S2. Co-transfect host cells with the recombinant lentiviral expression vector and lentiviral packaging plasmid from step S1, and package them to obtain lentiviral particles. S3. Transfect the lentiviral particles from step S2 into tonsillar mesenchymal stem cells and screen to obtain NF-YAs modified tonsillar mesenchymal stem cells.

[0036] Furthermore, the preparation method of Viola tianshanense extract is as follows: Take dried whole herb of Viola tianshanense, pulverize and sieve to obtain powder; add purified water at a material-to-liquid ratio of 1g:(10-20)mL, and adjust the pH to 4.0-5.5; add compound enzyme equivalent to 0.5%-2% of the powder mass, and enzymatically hydrolyze at 45-60℃ for 60-120min; after enzymatic hydrolysis, inactivate the enzyme at 80-100℃ for 10-15min; filter, collect the filtrate, concentrate under reduced pressure, and dry to obtain Viola tianshanense extract; The complex enzyme contains cellulase and pectinase in a mass ratio of (1-2):1.

[0037] According to a third aspect of the present invention, the use of a stem cell-negative hydrogen ion composition prepared by the method is provided in the preparation of a drug for repairing renal tubular damage.

[0038] According to a fourth aspect of the invention, the use of Viola tianshanense extract in the preparation of a drug for repairing renal tubular damage is provided.

[0039] Preparation Example 1 NF-YAs modified tonsillar mesenchymal stem cells (T-MSCs) high-NF-YAs Construction of ) 1. Construction of lentiviral vector overexpressing NF-YAs 1.1 Construction of recombinant plasmid pCDH-NF-YAs Based on the human NF-YAs gene sequence in UniProt (accession number: P23511-2), which encodes 318 amino acids (as shown in SEQ ID NO.1), the full-length nucleotide sequence (as shown in SEQ ID NO.2) was synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned into the pUC57 vector. After confirmation by sequencing, the plasmid pUC57-NF-YAs was obtained. Using this plasmid as a template, primers for amplifying the NF-YAs coding region were designed, and the specific nucleotide sequences are as follows: Forward primer: 5'-CGC ACCGGT AATGGAACAGTACACGGCCAAC-3' (SEQ ID NO. 3); Reverse primer: 5'-CGC GCTAGC TCAGGACACGCGGATGATCTG-3' (SEQ ID NO.4); Note: In the forward primer, ACCGGT is the recognition site for restriction endonuclease AgeⅠ; in the reverse primer, GCTAGC is the recognition site for restriction endonuclease NheⅠ.

[0040] Using pUC57-NF-YAs as a template, PCR amplification was performed using the above primer pair. The product and the lentiviral vector pCDH-CMV-MCS-EF1-copGFP were digested with AgeⅠ / NheⅠ enzymes, ligated, and transformed into DH5α. Positive clones were screened and confirmed by sequencing to obtain the recombinant plasmid pCDH-NF-YAs.

[0041] 1.2 Preparation of Lentivirals Recombinant plasmid pCDH-NF-YAs was co-transfected into 293T cells with packaging plasmids pLP1, pLP2, and pLP-VSVG at a mass ratio of 4:2:3:3. The medium was changed 6-8 hours post-transfection. Cell supernatants were collected at 48 and 72 hours post-transfection, centrifuged at 3000 rpm for 10 min at 4°C, filtered through a 0.45 μm filter, and then ultracentrifuged at 25000 rpm for 2 h. The supernatant was discarded. The pellet was resuspended in 100 μL of serum-free DMEM, aliquoted, and stored at -80°C.

[0042] Viral titer was determined by qPCR: 293T cells were infected with serially diluted viral solution, and genomic DNA was extracted after 72 hours. Real-time quantitative PCR was performed using WPRE gene-specific primers. Simultaneously, a standard curve was established using serially diluted WPRE plasmid standards. Based on the Ct values ​​of the test samples, the integrated viral copy number was calculated from the standard curve, and thus the viral titer was calculated.

[0043] The viral titer was determined to be between 1×10⁻⁶. 8 -1×10 9 (TU) / mL is between 1.6 × 10 8 (TU) / mL.

[0044] 2. Establishment and identification of T-MSCs cell lines stably expressing NF-YAs 2.1 Cell Preparation Tonsil mesenchymal stem cells (T-MSCs) (purchased from Wuhan Yuansheng Primary Biotechnology Co., Ltd.) were harvested and revived strictly according to the instructions. The cells were then seeded into a special culture medium for human tonsil mesenchymal cells (purchased from Shanghai Jingkang Biotechnology Co., Ltd.) and cultured routinely in a 37℃, 5% CO2 incubator, passaged every 2-3 days.

[0045] 2.2 Infection and Screening Take P3 generation T-MSCs with good growth status, digest them, and then feed them with 1×10⁻⁶ TMSCs. 5 Seeds were placed in 6-well plates at a density of cells / well and cultured overnight to achieve 60%-70% confluence. The old medium was discarded, and complete medium containing lentiviral particles (MOI=10) was added, along with polybrene to a final concentration of 8 μg / mL, and gently mixed. The plates were incubated overnight at 37°C, and the medium was replaced with fresh complete medium the following day. 48 hours post-transfection, the medium was replaced with fresh medium, and the plates were cultured for another 72 hours. 72 hours post-infection, GFP-positive cells were sorted by flow cytometry. Cells were digested and collected, resuspended in sterile PBS, filtered through a 70 μm filter, and sorted. GFP-positive cells (>90%) were collected and seeded into culture dishes for further culture, designated as T-MSCs. high-NF-YAs (Overexpression group). GFP-negative cells were also collected and cultured extensively, designated as T-MSCs. Empty (Empty vector control).

[0046] 2.3 Overexpression Validation Western blot results showed that T-MSCs high-NF-YAs A specific band appeared at 32 kDa, while no band was observed in the empty vector control. After grayscale scanning and normalization with the internal control GAPDH, T-MSCs... high-NF-YAs The relative expression level was 5.98 times higher than that of the empty vector control (e.g., Figure 1 (As shown).

[0047] 2.4 Cellular characteristics Flow cytometry analysis revealed high expression of stem cell surface markers CD73, CD90, and CD105, while CD14, CD34, CD45, and HLA-DR were not expressed, meeting the identification criteria for mesenchymal stem cells of the International Society for Cell Therapy (ISCT).

[0048] The above results indicate that an NF-γAs-modified T-MSCs cell line was successfully constructed, and this cell line is designated as T-MSCs. high-NF-YAs .

[0049] Preparation Example 2 Preparation of liposome-coated calcium hydride nanoparticles (1) Take commercially available calcium hydride powder (purchased from Jiangsu Aikon Biomedical R&D Co., Ltd.) and add it to dried N-methylpyrrolidone at a ratio of 1g:10mL, and mix well. Under ice-water bath protection and nitrogen atmosphere, use an ultrasonic cell disruptor (power 400W, working 2s / interval 2s) to perform liquid phase exfoliation for 20min. Centrifuge the resulting suspension at 2000r / min for 5min, take the supernatant and centrifuge at 12000r / min for 15min, collect the precipitate, and dry it in a vacuum drying oven at room temperature for 24h to obtain calcium hydride nanoparticle dry powder, which is sealed and stored for later use.

[0050] (2) Weigh 120mg of soybean lecithin, 30mg of cholesterol and 15mg of DSPE-PEG 2000, dissolve them in 10mL of chloroform-methanol mixed solvent (chloroform and methanol volume ratio of 2:1), transfer to a round bottom flask, and remove the organic solvent by rotary evaporation in a 40℃ water bath to form a uniform lipid film on the flask wall. Continue vacuum drying for 2h to completely remove the residual solvent.

[0051] (3) Take another 20 mg of the calcium hydride nanoparticle powder obtained in step (1) and disperse it in 10 mL of anhydrous cyclohexane (containing 1% anhydrous ethanol as a solubilizer). Disperse it ultrasonically for 5 min under nitrogen protection. Add the dispersion to the flask containing the lipid film mentioned above and mix it by rotation at 40 °C for 30 min to ensure that the calcium hydride nanoparticles are in full contact with the lipids. Then, remove the cyclohexane and ethanol by rotary evaporation to obtain lipid-coated calcium hydride nanoparticle solids.

[0052] (4) Add 5 mL of sterile water for injection (containing 5% trehalose as a lyophilization protectant) to the lipid-coated calcium hydride nanoparticle solid obtained in step (3), and gently shake to hydrate the liposomes, forming a liposome suspension. Dispense the suspension into vials, pre-freeze at -80℃ for 2 h, and then transfer it to a freeze dryer and freeze-dry at -50℃ and 0.1 mbar for 48 h to obtain the lyophilized powder of liposome-coated calcium hydride nanoparticles. Store in a sealed container under nitrogen protection at -20℃.

[0053] Preparation Example 3 Preparation of Viola tianshanense extract Take 1g of whole Viola tianshanense herb, pulverize it through a 40-mesh sieve to obtain powder; add 10mL of purified water, adjust the pH of the system to (5.0±0.2) with citric acid, add 1% of the powder mass of compound enzyme (prepared from cellulase and pectinase in a mass ratio of 1:1), and enzymatically hydrolyze at 50℃ for 90min; after enzymatic hydrolysis, inactivate the enzyme at 95℃ for 10min, filter, collect the filtrate, concentrate under reduced pressure at 60℃ and -0.09MPa to 1 / 5 of the original volume, and freeze-dry at -50℃ and 0.1mbar for 48h to obtain Viola tianshanense extract.

[0054] Example 1 (1) Prepare the T-MSCs from Example 1 high-NF-YAs Cells were resuspended in sterile physiological saline containing 5% HSA and the cell concentration was adjusted to 1×10⁻⁶. 7 T-MSCs were obtained per mL. high-NF-YAs Cell suspension; (2) Take the liposome-coated calcium hydride nanoparticle dispersion from Preparation Example 2 and prepare an aqueous dispersion with a concentration of 0.2 mg / mL using sterile physiological saline to obtain a calcium hydride nanoparticle liposome suspension. (3) Take the Viola tianshanensis extract from Preparation Example 3, dissolve it in sterile physiological saline to a concentration of 3 mg / mL, and filter it through a 0.22 μm filter membrane for sterilization; (4) The above T-MSCs high-NF-YAs Cell suspension, calcium hydride nanoparticle liposome suspension and Viola tianshanensis extract were mixed evenly at a volume ratio of 1:1:1, and then diluted to 1 mL with sterile physiological saline containing 5% HSA to obtain the stem cell-negative hydrogen ion composition. This preparation should be used immediately, and the cell survival rate should be ensured to be ≥90% before administration.

[0055] All of the above steps were performed under aseptic conditions.

[0056] Example 2 The difference between Example 2 and Example 1 is that: T-MSCs high-NF-YAs The concentration of the cell suspension was 1×10⁻⁶. 6The concentration of calcium hydride nanoparticle liposome suspension was 0.5 mg / mL; the concentration of Viola tianshanense extract was 5 mg / mL.

[0057] Example 3 The difference between Example 3 and Example 1 is that: T-MSCs high-NF-YAs The concentration of the cell suspension was 1×10⁻⁶. 8 The concentration of calcium hydride nanoparticle liposome suspension was 0.1 mg / mL; the concentration of Viola tianshanense extract was 2 mg / mL.

[0058] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that: T-MSCs Empty Cell suspension replacement for T-MSCs high-NF-YAs The cell suspension was prepared using the same method as step (1) in Example 1.

[0059] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that T-MSCs are absent. high-NF-YAs The cell suspension was replenished with an equal volume of sterile saline containing 5% HSA.

[0060] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 lacks the calcium hydride nanoparticle liposome suspension and is supplemented with an equal volume of sterile saline containing 5% HSA.

[0061] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 lacks Viola tianshanense extract and is supplemented with an equal amount of sterile saline containing 5% HSA.

[0062] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that Comparative Example 5 contains only T-MSCs. high-NF-YAs The cell suspension was replenished with an equal volume of sterile saline containing 5% HSA.

[0063] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that Comparative Example 6 contains only calcium hydride nanoparticle liposome suspension, supplemented with an equal volume of sterile physiological saline containing 5% HSA.

[0064] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that Comparative Example 7 contains only Viola yedoensis extract, supplemented with an equal amount of sterile saline containing 5% HSA.

[0065] Test Example 1 Cytotoxicity assay (CCK-8 assay) Logarithmic growth phase HK-2 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were digested with 0.25% trypsin-EDTA, resuspended in DMEM / F12 medium containing 10% FBS, and the cell density was adjusted to 5×10⁶ cells / year. 4 Cells / mL. Add 100 μL of cell suspension (i.e., 5 × 10⁶ cells / mL) to each well of a 96-well plate. 3 Cells / well). Place the prepared 96-well plates in a 37°C, 5% CO2 incubator for 24 hours (CCK-8 assay was performed 30 minutes after the positive control group to avoid excessive cell killing), allowing cells to adhere and recover. Discard the old culture medium and add 100 μL of the corresponding treatment solution to each well according to the following groups, with 10 replicates per group: Positive control group: treated with 1% Triton X-100; Negative control group: treated with DMEM / F12 medium containing 10% FBS; Example 1 group: using the stem cell-negative hydrogen ion composition from Example 1; Example 2 group: using the stem cell-negative hydrogen ion composition from Example 2; Example 3 group: using the stem cell-negative hydrogen ion composition from Example 3; After each of the above treatments was completed, 10 μL of CCK-8 solution was immediately added, and the mixture was gently tapped to mix. The mixture was then incubated at 37°C in the dark for 2 hours. The absorbance (OD) at 450 nm was measured using a microplate reader. 450 Cell viability (%) is calculated using the following formula: ; Results are expressed as mean ± standard deviation.

[0066] The experimental results are shown in Table 1 below: Table 1. Cell viability test results for each experimental group (n=10)

[0067] As shown in Table 1 above, the cell survival rate of groups 1-3 in Examples 1-3 reached over 90%, indicating that the stem cell-negative hydrogen ion composition of the present invention has no obvious direct cytotoxicity to HK-2 cells.

[0068] Test Example 2 Acute tubular injury (AKI) rat model 1. Modeling and Grouping SPF-grade SD rats, weighing (180-220) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After 7 days of acclimatization, they were randomly divided into sham-operated group and model group.

[0069] Model group: 200 μL of physiological saline containing gentamicin sulfate was injected intraperitoneally at a dose of 100 mg / kg for 7 consecutive days.

[0070] Sham surgery group: Intraperitoneal injection of an equal volume (200 μL) of normal saline.

[0071] 2. Grouping and Intervention The rats in the model group were randomly divided into 12 groups, with 8 rats in each group. A blank control group (n=8, sham-operated rats) was also included, for a total of 13 groups. The cell concentration in all groups containing cell suspension was uniformly set at 1×10⁻⁶. 7 The specific grouping and intervention protocols are as follows: (Units / mL) Blank control group: sham-operated rats, treated with an equal volume of sterile saline containing 5% HSA; Model group: Model rats, treated with an equal volume of sterile saline containing 5% HSA; Positive control group: model rats, treated with 5 mg / kg dexamethasone; Example 1 group: model rats, using the stem cell-negative hydrogen ion composition in Example 1; Example 2 group: model rats, using the stem cell-negative hydrogen ion composition of Example 2; Example 3 group: model rats, using the stem cell-negative hydrogen ion composition of Example 3; Comparative Example 1: Model rats, treated with the stem cell-negative hydrogen ion composition (in T-MSCs) from Comparative Example 1. Empty Cell suspension replacement for T-MSCs high-NF-YAs (cell suspension) Comparative Example 2: Model rats, using the stem cell-negative hydrogen ion composition from Comparative Example 2 (lacking T-MSCs) high -NF-YAs (cell suspension) Comparative Example 3: Model rats, using the stem cell-negative hydrogen ion composition of Comparative Example 3 (lacking calcium hydride nanoparticle liposome suspension); Comparative Example 4: Model rats, using the stem cell-negative hydrogen ion composition of Comparative Example 4 (lacking Viola yedoensis extract). Comparative Example 5: Model rats, using the stem cell-negative hydrogen ion composition (containing only T-MSCs) from Comparative Example 5. high -NF-YAs (cell suspension) Comparative Example 6: Model rats, using the stem cell-negative hydrogen ion composition of Comparative Example 6 (containing only calcium hydride nanoparticle liposome suspension). Comparative Example 7: Model rats, using the stem cell-negative hydrogen ion composition (containing only Viola yedoensis extract) from Comparative Example 7. Except for the positive control group, all groups received a tail vein injection of the therapeutic agent starting on the 4th day of modeling, with an administration volume of 0.2 mL / animal, once daily for 21 consecutive days.

[0072] 3. Sample Collection Blood was collected from the supraorbital venous sinus of rats in each experimental group, allowed to stand at room temperature for 1 hour, centrifuged at 3500 r / min for 10 min, and the serum was collected and stored at -20℃ for serum biochemistry testing.

[0073] After blood collection, the left kidney of each experimental group of rats was taken, and pre-cooled tissue lysis buffer was added at a ratio of 1 mg: 9 μL. The mixture was then homogenized thoroughly in an ice bath. The homogenate was centrifuged at 8000 r / min for 8 min at 4℃, and the supernatant was collected and stored at -80℃ for biochemical analysis. The right kidney was taken, half of which was fixed with 4% paraformaldehyde for pathological experiments, and the other half was frozen at -80℃ for later use.

[0074] 4. Renal function and histopathological examination 4.1 General condition observation and weight changes The general condition of rats in each experimental group was observed daily, including their mental state, activity, diet, water intake, coat condition, and mortality. Rats were weighed one day before modeling and on the first day after treatment. The rate of change in body weight was calculated using the following formula: ; 4.2 Determination of serum creatinine (Scr) levels The serum creatinine (Scr, μmol / L) and blood urea nitrogen (BUN, mmol / L) levels in rats of each experimental group were determined using a fully automated biochemical analyzer (Hitachi 7100).

[0075] 4.3 Measurement of serum TNF-α and IL-10 levels The levels of tumor necrosis factor-α (TNF-α, pg / mL) and interleukin-10 (IL-10, pg / mL) in the serum of rats in each experimental group were determined using an ELISA kit (purchased from Wuhan Elite Biotechnology Co., Ltd.).

[0076] 4.4 Renal tubular necrosis score Kidney tissue from rats in each experimental group was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (3±0.2μm), and stained with PAS. Under an optical microscope (×400), 10 non-overlapping fields of view were randomly selected from each section, and semi-quantitatively scored according to the following criteria: 0 points: Normal, no damage; 1 point: Renal tubular damage area <10%; 2 points: The area of ​​renal tubular damage is 10%-25%; 3 points: Renal tubular damage area is 26%-50%; 4 points: Renal tubular damage area is 51%-75%; 5 points: Renal tubular damage area > 75%; Note: The above-mentioned damaged renal tubules are characterized by tubular dilation, vacuolar degeneration of epithelial cells, brush border shedding, cast formation, and cell necrosis.

[0077] 4.5 Apoptosis rate assay (TUNEL staining method) Paraffin sections of right kidney tissue from rats in each experimental group were dewaxed to water, repaired with proteinase K, and then endogenous peroxidase was blocked with 3% H2O2. TUNEL reaction solution (purchased from Wuhan Yilairuit Biotechnology Co., Ltd.) was added, and the mixture was incubated at 37℃ in the dark for 60 min. DAB staining was performed, followed by hematoxylin counterstaining. The tissues were observed and photographed under a light microscope; apoptotic cell nuclei appeared brownish-yellow, while normal cell nuclei appeared blue.

[0078] Five non-overlapping high-power fields (×400) were randomly selected from each slice, and the apoptosis rate was calculated using the following formula: ; Note: a This indicates the number of brownish-yellow apoptotic cell nuclei (number) within the count field. b This indicates the total number of cell nuclei counterstained with hematoxylin.

[0079] 4.6 Determination of malondialdehyde (MDA) content in kidney tissue The supernatant of kidney tissue homogenate from rats in each experimental group was used to determine the malondialdehyde (MDA) content using an ELISA kit (purchased from Wuhan Elite Biotechnology Co., Ltd.), expressed as nmol / mg prot. The reactive oxygen species (ROS) level was determined using a reactive oxygen species (ROS) detection kit (purchased from Wuhan Elite Biotechnology Co., Ltd.), expressed as RFU / mg prot.

[0080] 4.7 Determination of ATP content in kidney tissue The supernatant of kidney tissue homogenate from rats in each experimental group was collected, and the ATP content was determined using an ATP assay kit (chemiluminescence method, purchased from Wuhan Yilairuit Biotechnology Co., Ltd.), expressed as nmol / mg prot.

[0081] 5. Data Analysis and Processing All experimental data are expressed as mean ± standard deviation and were analyzed using GraphPad Prism 9.3.0 software. p A value <0.05 is considered statistically significant.

[0082] 6 Results and Analysis 6.1 Observation results of general condition of rats The changes in body weight of rats in each experimental group throughout the experiment are shown in Table 2 below: Table 2. Results of body weight change rate of rats in each experimental group during the entire experiment (n=8)

[0083] Note: Compared with the blank control group #### p <0.0001; compared with the model group, **** p <0.0001, ** p <0.01, * p <0.05, ns p >0.05; compared with the positive control group, there was no significant difference in the rate of weight change in groups 1-3 of Examples ( p >0.05); compared with Examples 1-3, the weight change rate of Comparative Examples 1-7 was significantly different ( p <0.05).

[0084] It can be seen from Table 2 above: Compared with the blank control group, the rate of change in body weight of rats in the model group was significantly reduced ( p <0.0001), indicating that the gentamicin sulfate-induced acute tubular injury model resulted in severely impaired weight gain in rats.

[0085] Compared with the model group, the rate of weight change in Examples 1-3 was significantly higher. p <0.0001), ranging from 40.6% to 51.8%. Among them, the weight change rate of groups 1-3 in Examples 1-3 was not significantly different from that of the positive control group ( p >0.05), notably, the values ​​in Example 1 and Example 3 were slightly higher than those in the positive control group. This indicates that the stem cell-negative hydrogen ion composition of the present invention is comparable in effect to the positive control drug (dexamethasone) in improving weight gain impairment in rats with acute renal tubular injury.

[0086] Compared with the model control group, T-MSCs Empty Cell suspension replacement for T-MSCs high-NF-YAs Comparative Example 1, containing cell suspension; and Comparative Example 3, containing only T-MSCs, lacking calcium hydride nanoparticle liposome suspension. high-NF-YAs The weight change rates of control group 5 (cell suspension) and control group 6 (liposome suspension containing only calcium hydride nanoparticles) ranged from 24.2% to 33.4%, both significantly higher than those of the model control group. p<0.05), but still significantly different compared to groups 1-3 of Examples ( p <0.05). Among them, the weight recovery rate of control group 3, which was further supplemented with Viola tianshanense extract, was higher than that of control group 5, which contained only stem cells, confirming that Viola tianshanense extract can enhance the repair effect of stem cells.

[0087] Compared with the model control group, the weight change rate of the control group lacking Viola tianshanensis extract was (22.5±4.4)%, which was slightly lower than that of the control group (containing only calcium hydride nanoparticle liposome suspension). This indicates that the engineered stem cells and calcium hydride failed to produce a synergistic effect in the absence of Viola tianshanensis extract, suggesting that Viola tianshanensis extract plays an indispensable key role in the synergistic repair system of this invention.

[0088] Compared with the model control group, the comparative group 2 (lacking T-MSCs) high-NF-YAs The weight change rates of the cell suspension and the 7 control groups (containing only Viola yedoensis extract) were between 20.3% and 22.5%, which were higher than those of the model control group, but there was no significant difference. p >0.05). This indicates that the absence of engineered stem cells as the main repair agent cannot effectively reverse the weight loss induced by gentamicin; and the binary combination of engineered stem cells and calcium hydride is significantly weaker than the triple combination with Viola yedoensis extract (Examples 1-3, p<0.05).

[0089] During the experiment, the rats in the blank control group were in good spirits, active, with smooth fur, and ate normally. From the third day after modeling, the rats in the model group gradually showed signs of lethargy, piloerection, arched back, reduced activity, rapid breathing, and a significant decrease in food and water intake. Compared with the model group, the general condition of the rats in the positive control group and each treatment group (Examples 1-3 and Comparative Examples 1-5) improved to varying degrees, with the most significant improvement observed in the positive control group and Examples 1-3, where rats exhibited good spirits and activity levels, smooth or nearly smooth fur, and a return to normal appetite. No rats died during the entire experiment.

[0090] 6.2 Results of serum Scr content determination The results of serum Scr content determination in rats of each experimental group are shown in Table 3 below: Table 3. Results of serum Scr content measurement in rats of each experimental group (n=8)

[0091] Note: Compared with the blank control group #### p <0.0001; compared with the model group, **** p <0.0001,** p <0.01; Compared with the positive control group, there was no significant difference in serum Scr levels in rats in groups 1-3 of Examples ( p >0.05); compared with groups 1-3 of Examples, the serum Scr content of rats in groups 1-7 of Comparative Examples was significantly different ( p <0.05).

[0092] It can be seen from Table 3 above: Compared with the blank control group, the serum Scr content in the model group rats was significantly increased ( p <0.0001), indicating that the gentamicin sulfate-induced acute tubular injury model was successfully established and that functional damage to the kidney tissue was observed.

[0093] Compared with the model group, the serum Scr content in rats in Examples 1-3 was significantly reduced ( p <0.0001), ranging from 40.7 to 49.6 μmol / L. Among them, the Scr content in groups 1-3 was not significantly different from that in the positive control group ( p >0.05), it is worth noting that the values ​​in Example 1 and Example 3 were slightly lower than those in the positive control group. This indicates that the stem cell-negative hydrogen ion composition of the present invention has an effect on improving renal function comparable to that of the positive control drug (dexamethasone).

[0094] Compared with the model group, the serum Scr levels in rats in comparative groups 1-7 were also significantly reduced. p <0.01), indicating that each control preparation also has a certain effect in improving renal function.

[0095] Compared with Examples 1-3, T-MSCs Empty Cell suspension replacement for T-MSCs high-NF-YAs Comparative Example 1, containing cell suspension; and Comparative Example 3, containing only T-MSCs, lacking calcium hydride nanoparticle liposome suspension. high-NF-YAs The serum Scr levels in rats in comparative group 5 (cell suspension) and comparative group 6 (liposome suspension containing only calcium hydride nanoparticles) ranged from 58.1 to 72.6 μmol / L, with significant differences. p <0.05). Among them, the Scr content of the control group 3, which further added Viola tianshanense extract, was lower than that of the control group 5, which contained only stem cells, confirming that Viola tianshanense extract can enhance the repair effect of stem cells and further improve glomerular filtration function.

[0096] Compared with groups 1-3 of Examples, the serum Scr content of rats in Comparative Example 4, which lacked Viola tianshanense extract, was (76.3±5.4) μmol / L, slightly higher than that in Comparative Example 6 (which only contained calcium hydride nanoparticle liposome suspension). This result indicates that in the absence of Viola tianshanense extract, engineered stem cells and calcium hydride failed to synergistically enhance each other, suggesting that Viola tianshanense extract plays an indispensable key role in the synergistic repair system of this invention.

[0097] Compared with Examples 1-3, T-MSCs were missing. high-NF-YAs In comparative group 2 (cell suspension) and comparative group 7 (containing only *Viola yezoensis* extract), the serum Scr levels in rats ranged from 82.0 to 85.3 μmol / L, showing the weakest reduction effect. This indicates that the absence of engineered stem cells, the main repair agent, cannot effectively reverse gentamicin-induced glomerular filtration function damage. Furthermore, the binary combination of engineered stem cells and calcium hydride was significantly weaker than the triple combination with *Viola yezoensis* extract (Examples 1-3). p <0.05).

[0098] 6.3 Results of serum TNF-α and IL-10 level measurement The serum TNF-α and IL-10 levels in rats from each experimental group are shown in Table 4 below: Table 4. Results of serum TNF-α and IL-10 levels in rats of each experimental group (n=8)

[0099] Note: Compared with the blank control group #### p <0.0001; compared with the model group, **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, ns p >0.05.

[0100] Compared with the positive control group, the serum TNF-α levels in rats in Example 1 and Example 2 groups were significantly different from those in the positive control group. p <0.05), the serum TNF-α level in the rat group of Example 3 was not significantly different from that in the positive control group ( p >0.05); compared with groups 1-3 of Examples, the serum TNF-α levels in rats in groups 1-7 of Comparative Examples were significantly different ( p <0.05).

[0101] Compared with the positive control group, there was no significant difference in serum IL-10 levels in rats in Examples 1-3. p >0.05); compared with the groups of Examples 1-3, the serum IL-10 levels in rats of Comparative Examples 1, 2 and 4-7 were significantly different ( p <0.05), there was no significant difference in serum IL-10 levels among the three groups of rats in the comparative example. p >0.05).

[0102] It can be seen from Table 4 above: Compared with the blank control group, the serum TNF-α level in the model group rats was significantly increased ( p <0.0001), IL-10 levels were significantly reduced ( p <0.0001), indicating that gentamicin sulfate-induced acute tubular injury leads to a severe systemic inflammatory response and significantly inhibits anti-inflammatory capacity.

[0103] Compared with the model group, the serum TNF-α level in rats in Examples 1-3 was significantly reduced ( p <0.0001), ranging from 26.3 to 44.8 pg / mL. Among them, the TNF-α level in the Example 1 group was significantly lower than that in the positive control group ( p <0.05), the value of the Example 2 group was significantly higher than that of the positive control group ( p <0.05), the 3rd example group was comparable to the positive control group ( p >0.05, but still significantly lower than the model group ( p <0.0001). This indicates that the stem cell-negative hydrogen ion composition of the present invention is highly effective in reducing pro-inflammatory factors, even superior to the positive control drug dexamethasone. Compared with the model group, the serum IL-10 level in rats in Examples 1-3 was significantly increased ( p <0.0001), ranging from 31.0 to 39.8 pg / mL. Among them, the IL-10 levels in groups 1-3 were not significantly different from those in the positive control group ( p >0.05), but the levels in Example 1 and Example 3 were slightly higher than those in the positive control group, indicating that the stem cell-negative hydrogen ion composition of the present invention has an effect on increasing the level of anti-inflammatory factors comparable to that of the positive drug (dexamethasone).

[0104] Compared with the model group, the serum TNF-α levels in rats in comparative groups 1-7 were significantly reduced ( p <0.0001), and the IL-10 levels in comparative group 1 and comparative groups 3-6 were also significantly increased ( p <0.05), indicating that each control preparation also has a certain anti-inflammatory effect.

[0105] Compared with Examples 1-3, T-MSCs Empty Cell suspension replacement for T-MSCs high-NF-YAs Comparative Example 1, containing cell suspension; and Comparative Example 3, containing only T-MSCs, lacking calcium hydride nanoparticle liposome suspension. high-NF-YAs In Comparative Example 5 (cell suspension) and Comparative Example 6 (calcium hydride nanoparticle liposome suspension only), the serum TNF-α levels in rats ranged from 53.1 to 70.3 pg / mL, and the IL-10 levels ranged from 21.6 to 28.7 pg / mL, respectively, both inferior to those in Examples 1-3. p <0.05). Among them, the TNF-α level of the group with further addition of Viola tianshanense extract (comparative group 3) was lower than that of the group with modified stem cells alone (comparative group 5), and the IL-10 level was higher than that of the group with modified stem cells alone (comparative group 5), which confirms that Viola tianshanense extract can enhance the anti-inflammatory effect of stem cells.

[0106] Compared with groups 1-3, the serum TNF-α level in group 4 rats, which lacked Viola tianshanense extract, was (70.3±5.8) pg / mL, and the IL-10 level was (19.2±5.9) pg / mL, slightly worse than that in group 6 (containing only calcium hydride nanoparticle liposome suspension). This indicates that in the absence of Viola tianshanense extract, engineered stem cells and calcium hydride failed to produce a synergistic anti-inflammatory effect, confirming that Viola tianshanense extract plays an indispensable key role in the synergistic anti-inflammatory system of this invention.

[0107] Compared with Examples 1-3, T-MSCs were missing. high-NF-YAs The serum TNF-α levels in rats in Comparative Example 2 (cell suspension) and Comparative Example 7 (containing only Viola yedoensis extract) were between 92.3 and 94.3 pg / mL, significantly higher than those in Examples 1-3. p <0.05), IL-10 levels were between 18.1-20.1 pg / mL, significantly lower than in groups 1-3 of Examples ( p <0.05), indicating the weakest improvement effect, suggesting that the absence of engineered stem cells as the main repair agent cannot effectively reverse the inflammatory response induced by gentamicin; and the binary combination of engineered stem cells and calcium hydride is significantly weaker than the triple combination with Viola yedoensis extract (Examples 1-3). p <0.05).

[0108] 6.4 Renal tubular necrosis scoring results The renal tubular necrosis scores of rats in each experimental group are shown in Table 5 below: Table 5. Results of renal tubular necrosis scores in rats of each experimental group (n=8)

[0109] Note: Compared with the blank control group #### p <0.0001; compared with the model group, **** p <0.0001. Compared with the positive control group, there was no significant difference in the renal tubular necrosis score of rats in Examples 1-3. p >0.05); compared with groups 1-3 of Examples, the renal tubular necrosis scores of rats in groups 1-7 of Comparative Examples were significantly different ( p <0.05).

[0110] It can be seen from Table 5 above: Compared with the blank control group, the renal tubular necrosis score of rats in the model group was significantly increased ( p <0.0001), indicating that the gentamicin sulfate-induced acute tubular injury model was successfully established and that significant damage was observed in the kidney tissue.

[0111] Compared with the model group, the renal tubular necrosis scores of rats in Examples 1-3 were significantly reduced ( p <0.0001), ranging from 0.9 to 1.4 points. Among them, the renal tubular necrosis scores of groups 1-3 in Examples showed no significant difference from the positive control group ( p >0.05), it is worth noting that the values ​​in Example 1 and Example 3 groups were slightly lower than those in the positive control group. This indicates that the stem cell-negative hydrogen ion composition of the present invention is comparable to the positive control drug (dexamethasone) in its effect of repairing renal tubular damage.

[0112] Compared with the model group, the renal tubular necrosis scores of rats in comparative groups 1-7 were also significantly reduced. p <0.01), indicating that each control preparation also has a certain effect in repairing renal tubular damage.

[0113] Compared with Examples 1-3, T-MSCs Empty Cell suspension replacement for T-MSCs high-NF-YAs Comparative Example 1, containing cell suspension; and Comparative Example 3, containing only T-MSCs, lacking calcium hydride nanoparticle liposome suspension. high-NF-YAs The renal tubular necrosis scores of rats in Comparative Example 5 (cell suspension) and Comparative Example 6 (calcium hydride nanoparticle liposome suspension) were between 2.3 and 2.7, both significantly higher than those in Examples 1-3. p <0.05). Among them, the renal tubular necrosis score of the control group 3, which was further supplemented with Viola tianshanense extract, was lower than that of the control group 5, which contained only stem cells, confirming that Viola tianshanense extract can enhance the repair effect of stem cells and further improve renal tubular damage.

[0114] Compared with groups 1-3 of Examples, the renal tubular necrosis score of rats in Group 4 of Comparative Example, which lacked Viola tianshanense extract, was (3.1±0.6) points, slightly higher than that of Group 6 of Comparative Example (containing only calcium hydride nanoparticle liposome suspension). This result indicates that in the absence of Viola tianshanense extract, engineered stem cells and calcium hydride failed to produce a synergistic repair effect, confirming that Viola tianshanense extract plays an indispensable key role in the synergistic repair system of the present invention.

[0115] Compared with Examples 1-3, T-MSCs were missing. high-NF-YAs In the control group 2 (cell suspension) and the control group 7 (containing only *Viola yezoensis* extract), the renal tubular necrosis scores of rats were between 3.3 and 3.5, showing the weakest reduction effect. This indicates that the absence of engineered stem cells, the main repair agent, cannot effectively reverse gentamicin-induced renal tubular necrosis. Furthermore, the binary combination of engineered stem cells and calcium hydride was significantly weaker than the triple combination with *Viola yezoensis* extract (Examples 1-3). p <0.05).

[0116] 6.5 Results of apoptosis rate assay The apoptosis status of rats in each experimental group is shown in Table 6 below: Table 6. Results of apoptosis rate measurement in rats of each experimental group (n=8)

[0117] Note: Compared with the blank control group #### p <0.0001; compared with the model group, **** p <0.0001, * p <0.05. Compared with the positive control group, there was no significant difference in the apoptosis rate of rats in groups 1-3 of Examples ( p >0.05); compared with groups 1-3 of Examples, the apoptosis rate of rats in groups 1-7 of Comparative Examples was significantly different ( p <0.05).

[0118] It can be seen from Table 6 above: Compared with the blank control group, the apoptosis rate of rats in the model group was significantly increased. p <0.0001), indicating that the gentamicin sulfate-induced acute tubular injury model was successfully established, and a large number of apoptosis occurred in the renal tubular epithelial cells.

[0119] Compared with the model group, the apoptosis rate of rats in Examples 1-3 was significantly reduced. p <0.0001), ranging from 8.2% to 15.6%. Among them, the apoptosis rate of rats in Examples 1-3 groups was not significantly different from that of the positive control group (p >0.05), it is worth noting that the values ​​in Example 1 and Example 3 were slightly lower than those in the positive control group. This indicates that the effect of the stem cell-negative hydrogen ion composition of the present invention is comparable to that of the positive control drug (dexamethasone).

[0120] Compared with Examples 1-3, T-MSCs Empty Cell suspension replacement for T-MSCs high-NF-YAs Comparative Example 1, containing cell suspension; and Comparative Example 3, containing only T-MSCs, lacking calcium hydride nanoparticle liposome suspension. high-NF-YAs The apoptosis rates of rats in control group 5 (cell suspension) and control group 6 (calcium hydride nanoparticle liposome suspension) ranged from 25.0% to 30.7%, with significant differences. p <0.05). Among them, the apoptosis rate of control group 3, which was further supplemented with Viola tianshanense extract, was lower than that of control group 5, which contained only stem cells, confirming that Viola tianshanense extract can enhance the anti-apoptotic effect of stem cells.

[0121] Compared with groups 1-3, the apoptosis rate of rats in control group 4, which lacked Viola tianshanense extract, was (31.6±4.2)%, slightly higher than that in control group 6 (containing only calcium hydride nanoparticle liposome suspension). This result indicates that in the absence of Viola tianshanense extract, engineered stem cells and calcium hydride failed to produce a synergistic anti-apoptotic effect, confirming that Viola tianshanense extract plays an indispensable key role in the synergistic system of this invention.

[0122] Compared with Examples 1-3, T-MSCs were missing. high-NF-YAs The apoptosis rates of rats in control group 2 (cell suspension) and control group 7 (containing only *Viola yezoensis* extract) ranged from 34.9% to 37.6%, showing the weakest reduction effect. This indicates that the absence of engineered stem cells, the main repair agent, cannot effectively reverse gentamicin-induced apoptosis. Furthermore, the binary combination of engineered stem cells and calcium hydride was significantly weaker than the triple combination containing *Viola yezoensis* extract (Examples 1-3). p <0.05).

[0123] 6.6 Results of MDA content and ROS level measurement in kidney tissue The results of MDA content and ROS level measurements in the kidney tissue of rats in each experimental group are shown in Table 7 below: Table 7. Results of MDA content and ROS level measurements in kidney tissue of rats in each experimental group (n=8)

[0124] Note: Compared with the blank control group #### p <0.0001; compared with the model group, ****p <0.0001. Compared with the positive control group, there was no significant difference in MDA content and ROS level in the kidney tissue of rats in Examples 1-3 ( p >0.05); compared with groups 1-3 of Examples, the MDA content and ROS level in the kidney tissue of rats in groups 1-7 of Comparative Examples were significantly different ( p <0.05).

[0125] It can be seen from Table 7 above: Compared with the blank control group, the MDA content and ROS level in the kidney tissue of rats in the model group were significantly increased. p <0.0001), indicating that the gentamicin sulfate-induced acute tubular injury model was successfully established and that oxidative stress damage occurred in the kidney tissue.

[0126] Compared with the model group, the MDA content in the kidney tissue of rats in Examples 1-3 was between 16.8-29.4 nmol / mg prot, and the ROS level was between 18.9-27.0 RFU / mg prot, both significantly reduced. p <0.0001). Among them, the MDA content and ROS level in groups 1-3 were not significantly different from those in the positive control group ( p >0.05), it is worth noting that the values ​​in Example 1 and Example 3 were slightly lower than those in the positive control group. This indicates that the stem cell-negative hydrogen ion composition of the present invention is comparable to the positive control drug (dexamethasone) in reducing oxidative stress damage.

[0127] Compared with the model group, the MDA content and ROS level in the kidney tissue of rats in comparative groups 1-7 were also significantly reduced. p <0.01), indicating that each control formulation also has a certain effect in improving oxidative stress damage.

[0128] Compared with Examples 1-3, T-MSCs Empty Cell suspension replacement for T-MSCs high-NF-YAs Comparative Example 1, containing cell suspension; and Comparative Example 3, containing only T-MSCs, lacking calcium hydride nanoparticle liposome suspension. high-NF-YAs In comparative group 5 (cell suspension) and comparative group 6 (liposome suspension containing only calcium hydride nanoparticles), the MDA content in rat kidney tissue was between 40.8 and 48.2 nmol / mg prot, and the ROS level was between 38.6 and 56.6 RFU / mg prot, both significantly higher than those in groups 1-3 of Examples. p <0.05). Among them, the MDA content and ROS level of the comparative group 3, which further added Viola tianshanense extract, were lower than those of the comparative group 5, which contained only stem cells, confirming that Viola tianshanense extract can enhance the antioxidant stress effect of stem cells and further improve oxidative stress damage.

[0129] Compared with groups 1-3, the MDA content in the kidney tissue of rats in control group 4 (lacking Viola tianshanense extract) was (50.6±5.7) nmol / mg prot, and the ROS level was (59.4±4.6) RFU / mg prot, both slightly higher than control group 6 (containing only calcium hydride nanoparticle liposome suspension). This result indicates that in the absence of Viola tianshanense extract, engineered stem cells and calcium hydride failed to produce a synergistic repair effect, confirming that Viola tianshanense extract plays an indispensable key role in the synergistic antioxidant stress system of this invention.

[0130] Compared with Examples 1-3, T-MSCs were missing. high-NF-YAs In comparative group 2 (cell suspension) and comparative group 7 (containing only *Viola yezoensis* extract), the MDA content and ROS levels in rat kidney tissue were between 54.5-56.5 nmol / mg prot and 68.2-71.7 RFU / mg prot, respectively, showing the weakest reduction effect. This indicates that the absence of engineered stem cells, the main repair agent, cannot effectively reverse gentamicin-induced oxidative stress damage. Furthermore, the binary combination of engineered stem cells and calcium hydride was significantly weaker than the triple combination with *Viola yezoensis* extract (Examples 1-3). p <0.05).

[0131] 6.7 Results of ATP content measurement in kidney tissue The results of ATP content determination in the kidney tissue of rats in each experimental group are shown in Table 8 below: Table 8. Results of ATP content measurement in kidney tissue of rats in each experimental group (n=8)

[0132] Note: Compared with the blank control group #### p <0.0001; compared with the model group, **** p <0.0001. Compared with the positive control group, the ATP content in the kidney tissue of rats in Example 1 group and Example 2 group was significantly different ( p <0.05), there was no significant difference in ATP content in the kidney tissue of rats in Example 3 group ( p >0.05); compared with groups 1-3 of Examples, the ATP content in the kidney tissue of rats in groups 1-7 of Comparative Examples was significantly different ( p <0.05).

[0133] It can be seen from Table 8 above: Compared with the blank control group, the ATP content in the kidney tissue of rats in the model group was significantly reduced ( p<0.0001), indicating that the gentamicin sulfate-induced acute tubular injury model was successfully established and that energy metabolism disorders occurred in the kidney tissue.

[0134] Compared with the model group, the ATP content in the kidney tissue of rats in Examples 1-3 was significantly increased ( p <0.0001), ranging from 12.3 to 16.7 nmol / mg prot. Among them, the ATP content in the Example 1 group was significantly higher than that in the positive control group ( p <0.05), the level in Example 2 group was significantly lower than that in the positive control group ( p <0.05), the 3rd example group was comparable to the positive control group ( p The concentration was >0.05, but still slightly higher than that of the model group. This indicates that the stem cell-negative hydrogen ion composition of the present invention has an effect on improving renal tissue energy metabolism that is comparable to or better than that of the positive control drug (dexamethasone).

[0135] Compared with the model group, the ATP content in the kidney tissue of rats in comparative groups 1-7 was significantly increased. p <0.01), indicating that each control preparation also has a certain effect on improving energy metabolism.

[0136] Compared with Examples 1-3, T-MSCs Empty Cell suspension replacement for T-MSCs high-NF-YAs Comparative Example 1, containing cell suspension; and Comparative Example 3, containing only T-MSCs, lacking calcium hydride nanoparticle liposome suspension. high-NF-YAs The ATP content in rat kidney tissue of comparative group 5 (cell suspension) and comparative group 6 (liposome suspension containing only calcium hydride nanoparticles) was between 9.3 and 9.9 nmol / mg prot, with a significant difference. p <0.05). Among them, the ATP content of the control group 3, which was further supplemented with Viola tianshanense extract, was higher than that of the control group 5, which contained only stem cells, confirming that Viola tianshanense extract can enhance the energy metabolism support of stem cells in renal tissue.

[0137] Compared with groups 1-3, the ATP content in the kidney tissue of rats in Comparative Example 4, which lacked Viola tianshanense extract, was (9.0±0.5) nmol / mg prot, slightly lower than that in Comparative Example 6 (which only contained calcium hydride nanoparticle liposome suspension). This result indicates that in the absence of Viola tianshanense extract, engineered stem cells and calcium hydride failed to produce a synergistic repair effect, confirming that Viola tianshanense extract plays an indispensable key role in the synergistic repair system of this invention.

[0138] Compared with Examples 1-3, T-MSCs were missing. high-NF-YAsIn comparative group 2 (cell suspension) and comparative group 7 (containing only *Viola yezoensis* extract), the ATP content in the kidney tissue of rats was between 8.4 and 8.6 nmol / mg prot, showing the weakest effect. This indicates that the absence of engineered stem cells, the main repair agent, cannot effectively reverse gentamicin-induced energy metabolism disorders. Furthermore, the binary combination of engineered stem cells and calcium hydride was significantly weaker than the triple combination with *Viola yezoensis* extract (Examples 1-3). p <0.05).

[0139] 7. Conclusion Based on the above experimental results, it is evident that the combination of NF-YAs genetically engineered tonsillar mesenchymal stem cells, calcium hydride nanoparticles, and Viola yedoensis extract in this invention significantly improves renal function, reduces renal tubular necrosis, inhibits renal tubular epithelial cell apoptosis, lowers renal tissue oxidative stress levels, increases renal tissue ATP content, and regulates inflammatory factor balance in model rats. Furthermore, the overall efficacy is comparable to or better than that of the positive control drug dexamethasone. The synergistic effect of the triple combination is significantly superior to any single component or any combination of two components, confirming the clinical application potential of the stem cell-negative hydrogen ion composition of this invention in repairing renal tubular damage.

[0140] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A stem cell-negative hydrogen ion composition for repairing renal tubular damage, characterized in that, This includes stem cells, negative hydrogen ion donors, Viola tianshanense extract, and pharmaceutically acceptable carriers.

2. The stem cell-negative hydrogen ion composition as described in claim 1, characterized in that, The concentration of the added stem cells is 1×10⁻⁶. 6 -1×10 8 The amount of negative hydrogen ion donor added is 0.1-0.5 mg / mL, and the amount of Viola tianshanense extract added is 2-5 mg / mL.

3. The stem cell-negative hydrogen ion composition as described in claim 2, characterized in that, The concentration of the added stem cells is 1×10⁻⁶. 7 The amount of negative hydrogen ion donor added is 0.2 mg / mL, and the amount of Viola tianshanense extract added is 3 mg / mL.

4. The stem cell-negative hydrogen ion composition as described in claim 3, characterized in that, The stem cells are tonsillar mesenchymal stem cells; the negative hydrogen ion donor is selected from at least one of hydrogen-rich water, magnesium hydride, calcium hydride, calcium hydride nanoparticles, and negative hydrogen ion powder.

5. The stem cell-negative hydrogen ion composition as described in claim 4, characterized in that, The tonsillar mesenchymal stem cells are NF-YAs modified cell lines.

6. The stem cell-negative hydrogen ion composition as described in claim 5, characterized in that, The amino acid sequence of the NF-YAs is shown in SEQ ID NO.1, and the nucleotide sequence of the NF-YAs is shown in SEQ ID NO.

2.

7. A method for preparing a stem cell-negative hydrogen ion composition for repairing renal tubular damage, characterized in that, Includes the following steps: Constructing NF-YAs-modified amygdala mesenchymal stem cells; The above-mentioned NF-YAs modified tonsillar mesenchymal stem cells, negative hydrogen ion donor, and Viola yedoensis extract were mixed evenly in a certain proportion, and then a pharmaceutically acceptable carrier was added to prepare a stem cell-negative hydrogen ion composition.

8. The preparation method according to claim 7, characterized in that, The method for constructing NF-YAs-modified amygdala mesenchymal stem cells is as follows: S1. Construct a recombinant lentiviral expression vector containing the NF-YAs gene; S2. Co-transfect host cells with the recombinant lentiviral expression vector and lentiviral packaging plasmid from step S1, and package them to obtain lentiviral particles. S3. Transfect the lentiviral particles from step S2 into tonsillar mesenchymal stem cells and screen to obtain NF-YAs modified tonsillar mesenchymal stem cells.

9. The use of the stem cell-negative hydrogen ion composition prepared by the method of claim 7 or 8 in the preparation of drugs for repairing renal tubular damage.

10. Application of Viola tianshanense extract in the preparation of drugs for repairing renal tubular damage.

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