A photobiomodulation method for improving high glucose-induced injury of human renal tubular epithelial cells and application thereof

Abstract

The application provides a photobiomodulation method for improving high-sugar-induced human renal tubular epithelial cell damage, the photobiomodulation method comprising the step of treating high-sugar-induced human renal tubular epithelial cells by light irradiation; the light irradiation simultaneously satisfies the following conditions: light wavelength 530nm-1050nm, energy density 2-10J / cm 2 And power density 10-50mw / cm 2 The application also comprises the application of the photobiomodulation method in the preparation of non-drug intervention products for improving high-sugar-induced renal tubular epithelial cell damage, and in the preparation of drugs or auxiliary treatment devices for improving diabetic nephropathy; the photobiomodulation method can regulate cell activity, inhibit the level of interstitial fibrosis and related signal pathways, and become a potential treatment method for DKD.

Description

Technical Field

[0001] This application belongs to the field of diabetic nephropathy prevention and treatment technology, specifically involving a photobiological regulation method and its application for improving high glucose-induced damage to human renal tubular epithelial cells. Background Technology

[0002] Diabetic kidney disease (DKD) is a prevalent and serious microvascular complication of diabetes, and a core pathogenic factor in end-stage renal disease (ESRD). Its incidence is increasing annually, posing a serious threat to patients' lives and health. DKD is a secondary chronic kidney disease, with persistent hyperglycemic metabolic disorder being its core inducing factor, continuously damaging nephrons. Among these factors, oxidative stress, inflammation activation, and cell damage and apoptosis induced by the hyperglycemic microenvironment in human renal tubular epithelial cells are key pathological mechanisms inducing renal epithelial-interstitial fibrosis and driving the continuous progression of DKD. Currently, clinical interventions primarily use hypoglycemic and renal-protective drugs for symptomatic treatment, but these can only delay disease progression and cannot target and repair damaged renal tubular cells, nor can they effectively block the process of renal fibrosis. Significant technical bottlenecks remain in the radical cure and control of DKD.

[0003] Photobiomodulation (PBM) is a non-invasive and safe novel biotechnology. It is a non-invasive physical intervention technique with low side effects and represents a relatively new research area in medical technology. Unlike traditional drug treatments, PBM uses specific wavelengths of low-energy visible or near-infrared light to irradiate biological tissues. This light can penetrate cells, activate mitochondrial functions, and regulate key pathways such as oxidative stress, inflammation, and apoptosis. Currently, this technology is being gradually applied in basic research on skin injuries, organ ischemic injuries, and inflammatory diseases, effectively promoting the repair of damaged cells, inhibiting tissue fibrosis, and reducing inflammatory damage.

[0004] Based on the aforementioned effects of PBM, its potential in anti-inflammatory, anti-fibrotic, and tissue repair aspects has made it a research hotspot in kidney disease in recent years. However, current research is limited to basic preliminary exploration. Studies on its protective effect on renal tubular epithelial cells and its anti-fibrotic mechanism in DKD are still relatively limited. Furthermore, a standardized intervention system adapted to high glucose-induced renal tubular epithelial cell damage has not yet been established. The target of action, optimal light parameters, and regulatory mechanisms remain unclear. There are no mature technologies applied to the field of DKD damage repair, and relevant research is urgently needed to fill the technological gaps. Summary of the Invention

[0005] The purpose of this application is to overcome the shortcomings and deficiencies in the prior art and to provide a photobiological regulation method for improving high glucose-induced damage to human renal tubular epithelial cells and its application.

[0006] The technical solution of this invention is achieved through the following means: In a first aspect, a photobiological regulation method for improving high glucose-induced damage to human renal tubular epithelial cells is disclosed, the photobiological regulation method comprising the step of treating high glucose-induced human renal tubular epithelial cells with light; wherein the light irradiation simultaneously meets the following conditions: light wavelength 590 nm to 1050 nm, energy density 4 to 10 J / cm³. 2 and power density of 30-50 mw / cm³ 2 The irradiation time is set according to the matching of power density and energy density.

[0007] Furthermore, the illumination simultaneously satisfies the following conditions: illumination wavelength 780nm, energy density 8J / cm². 2 and power density 30mw / cm 2 .

[0008] Furthermore, the illumination simultaneously satisfies the following conditions: illumination wavelength 880nm, energy density 10J / cm². 2 and power density 30mw / cm 2 .

[0009] Furthermore, the wavelength of the illumination is 780nm.

[0010] Furthermore, the energy density is 8 J / cm³. 2 .

[0011] Furthermore, the power density is 30 mW / cm². 2 .

[0012] Secondly, the application of photobiological regulation methods in the preparation of non-drug intervention products that improve high glucose-induced renal tubular epithelial cell damage; the photobiological regulation methods are used to enhance the proliferation activity, cell migration ability and cell cloning ability of high glucose-induced human renal tubular epithelial cells, thereby repairing the physiological function of renal tubular epithelial cells.

[0013] Furthermore, the photobiological regulation method is used to downregulate the expression of α-SMA, a marker of high glucose-induced interstitial fibrosis in human renal tubular epithelial cells, upregulate the expression of E-cadherin, and / or inhibit the abnormal activation of the PI3K / AKT / Smad2 signaling pathway, thereby inhibiting interstitial fibrosis of renal tubular epithelial cells and blocking high glucose-induced damage to human renal tubular epithelial cells.

[0014] Thirdly, the application of photobiological regulation methods in the preparation of drugs or adjunctive therapeutic devices for improving diabetic nephropathy, which is related to renal tubular damage.

[0015] Furthermore, the photobiological regulation method can intervene in high glucose-mediated renal tubular damage and renal epithelial-interstitial fibrosis, thereby helping to delay or improve the progression of diabetic nephropathy.

[0016] Compared to related technologies, this invention found that culturing HK-2 cells in a medium with a glucose concentration of 30 mmol / L for 48 h significantly inhibited cell proliferation activity, and determined (780 nm + 8 J / cm) 2 +30mw / cm 2 () or (880nm+10J / cm) 2 +30mw / cm 2 Phototherapy combined with other methods can maximally improve the proliferative activity of HK-2 cells. Furthermore, phototherapy (PBM) can significantly promote the migration and clonal capacity of HK-2 cells under high glucose conditions, thereby further protecting basic kidney function. At the molecular level, PBM downregulates the interstitial fibrosis marker α-SMA, upregulates the expression level of E-cadherin, and inhibits the activation of the PI3K / AKT / Smad2 signaling pathway. These results indicate that PBM, by regulating cell activity and inhibiting interstitial fibrosis levels and related signaling pathways, may become a potential treatment for kidney disease (DKD). Attached Figure Description

[0017] Figure 1 The survival rate of HK-2 cells after culturing for different times with different sugar concentrations.

[0018] Figure 2 The survival rate of HK-2 cells under different light wavelengths.

[0019] Figure 3 The survival rate of HK-2 cells under different energy densities.

[0020] Figure 4 The survival rate of HK-2 cells under different power densities.

[0021] Figure 5 The survival rate of HK-2 cells under different phototherapy combinations.

[0022] Figure 6 The effect of photobiological therapy on the migration ability of HK-2 cells, among which Figure 6 Image (a) in the image is a light microscopic observation of cell migration. Figure 6 (b) in the figure is a data analysis graph of the corresponding cell migration results.

[0023] Figure 7 The effect of photobiological therapy on the cloning ability of HK-2 cells, among which Figure 7 (a) in the figure is a cell clonal observation diagram. Figure 7 (b) in the figure is a statistical chart of the data analysis of the corresponding cell cloning results.

[0024] Figure 8 The effect of photobiological therapy on the fluorescence expression intensity of α-SMA in HK-2 cells, among which Figure 8 (a) in the figure is the fluorescence expression intensity observation diagram of α-SMA. Figure 8 (b) in the figure is a data analysis graph of the corresponding α-SMA fluorescence expression intensity.

[0025] Figure 9 The effect of photobiological therapy on the expression level of α-SMA protein in HK-2 cells, among which Figure 9 (a) in the figure is a graph showing the expression level of α-SMA protein. Figure 9 (b) in the figure is a data analysis graph corresponding to the expression level of α-SMA protein.

[0026] Figure 10 The effect of photobiological therapy on the fluorescence expression intensity of E-cadherin in HK-2 cells, among which Figure 10 (a) in the figure is the fluorescence expression intensity observation diagram of E-cadherin. Figure 10 (b) in the figure is a data analysis graph of the corresponding E-cadherin fluorescence expression intensity.

[0027] Figure 11 The effect of photobiological therapy on the expression level of E-cadherin protein in HK-2 cells, among which Figure 11 (a) in the figure shows the observation of E-cadherin protein expression level. Figure 11 (b) in the figure is a data analysis graph of the corresponding E-cadherin protein expression level.

[0028] Figure 12 This is an immunofluorescence image showing the effect of photobiological therapy on the fluorescence expression intensity of the PI3K / AKT / Smad2 pathway in HK-2 cells.

[0029] Figure 13 The effect of photobiological therapy on the fluorescence expression intensity of the PI3K / AKT / Smad2 pathway in HK-2 cells. Figure 13 (a) in the figure is a data analysis graph of PI3K immunofluorescence expression. Figure 13 (b) in the figure is the AKT immunofluorescence expression intensity analysis diagram.

[0030] Figure 14 The effect of photobiological therapy on the fluorescence expression intensity of the PI3K / AKT / Smad2 pathway in HK-2 cells. Figure 14 (a) in the figure is the p-PI3K immunofluorescence expression intensity analysis diagram. Figure 14 (b) in the figure is the p-AKT immunofluorescence expression intensity analysis diagram. Figure 14(c) in the figure is an immunofluorescence expression intensity analysis of the Smad2 signaling pathway.

[0031] Figure 15 The effect of photobiological therapy on the expression levels of PI3K / AKT pathway proteins in HK-2 cells, among which Figure 15 (a) in the figure shows the observation of PI3K / p-PI3K / AKT / p-AKT protein expression levels. Figure 15 (b) in the figure is a data analysis graph corresponding to the expression levels of p-PI3K / PI3K proteins. Figure 15 (c) in the figure is a data analysis graph of the corresponding p-AKT / AKT protein expression levels.

[0032] Figure 16 The effect of photobiological therapy on the expression level of Smad2 pathway proteins in HK-2 cells, among which Figure 16 (a) in the figure shows the observation of Smad2 protein expression level. Figure 16 (b) in the figure is a data analysis graph corresponding to the expression level of Smad2 protein. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0034] All experimental data in the following examples, including results from immunofluorescence staining, cell scratch assays, and Western blot analysis, were statistically analyzed using GraphPad Prism 8.0.2 software. Data are expressed as mean ± standard deviation (Mean ± SD), and comparisons between groups were performed using independent samples t-tests or one-way ANOVA. All experiments were independently repeated at least three times to ensure data reliability and reproducibility. The statistical significance level was set at *. p <0.05 (*) p <0.05,** p <0.01, *** p <0.001, **** p <0.0001), where p A value <0.05 is considered statistically significant.

[0035] The human renal tubular epithelial cells used in the following examples were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (catalog number: SCSP-502) and identified by STR. All experimental reagents used were commercially available products. Unless otherwise specified, the preparation methods for the experimental reagents were based on publicly available literature.

[0036] Example 1: Experiment on detection of different sugar concentrations and different incubation times 1) Construction of HK-2 cell injury models with different high glucose concentrations: Complete culture medium for HK-2 cells was prepared in advance with glucose concentrations of 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, and 50 mmol / L. Human proximal renal tubular epithelial HK-2 cells with good growth and uniform morphology were selected. After trypsin digestion, the cells were resuspended in complete culture medium containing 5.6 mmol / L glucose. The cells were then evenly seeded into two 96-well cell culture plates, with approximately 1 × 10⁶ cells seeded in each well. 4 Six groups with varying glucose concentrations were set up for the experiment, with four replicates per group to eliminate experimental errors and ensure the reliability of the results. The seeded cell plates were placed in a constant temperature cell culture incubator at 37℃ and 5% CO2 for 24 hours. After complete cell adhesion, the medium was changed. Group 1 was replaced with complete medium at a glucose concentration of 5.6 mmol / L as the control group, while groups 2–6 were replaced with complete medium at glucose concentrations of 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, and 50 mmol / L respectively as the experimental groups, and all were continuously cultured in an incubator at 37℃ and 5% CO2.

[0037] 2) Cell proliferation assay: CCK8 cell proliferation assays were performed at two time points: 24 h and 48 h after medium change. 10 μL of CCK8 assay reagent was added to each well and the blank well, gently mixed, and the cell plate was incubated at 37℃ in a 5% CO2 incubator for 1.5 h in the dark. After incubation, the culture plates were removed, and the absorbance of each well was measured at 450 nm using a microplate reader. The data from both culture plates were recorded.

[0038] 3) Statistics: Measure the average absorbance of the blank wells, the average absorbance of each experimental group minus the absorbance of the blank wells, and the average absorbance of the control group minus the absorbance of the blank wells. Cell proliferation rate (%) = (average absorbance of each experimental group minus the absorbance of the blank wells) / (average absorbance of the control group minus the absorbance of the blank wells) × 100%.

[0039] Experimental results are as follows Figure 1As shown, the proliferation activity of HK-2 cells after culturing in complete culture medium with different glucose concentrations at different time points was as follows: ① Compared with the control group, the average proliferation rate of HK-2 cells in the 10 mmol / L glucose concentration culture group was approximately 95.33% after 24 hours of culture, with no significant difference, and the result was not statistically significant; after 48 hours of culture, the average proliferation rate of HK-2 cells was 93.25%, which was lower than that of the control group, and the result was statistically significant. ② Compared with the control group, the average proliferation rate of HK-2 cells in the 20 mmol / L glucose concentration culture group was approximately 99.75% after 24 hours of culture, with no significant difference, and the result was not statistically significant; after 48 hours of culture, the average proliferation rate of HK-2 cells was 76.25%, which was lower than that of the control group, and the result was statistically significant. ③ Compared with the control group, the average proliferation rate of HK-2 cells in the 30 mmol / L glucose concentration culture group was approximately 97.00% after 24 hours of culture, with no significant difference, and the result was not statistically significant; after 48 hours of culture, the average proliferation rate of HK-2 cells was 55.75%, which was significantly lower than that of the control group, and the result was statistically significant. ④ Compared with the control group, the average proliferation rate of HK-2 cells in the 40 mmol / L glucose concentration culture group was approximately 87.70% after 24 hours of culture, with no significant difference, and the result was not statistically significant. After 48 hours of culture, the average proliferation rate of HK-2 cells was 45.00%, significantly lower than that of the control group, and the result was statistically significant. ⑤ Compared with the control group, the average proliferation rate of HK-2 cells in the 50 mmol / L glucose concentration culture group was approximately 85.25% after 24 hours of culture, with no significant difference, and the result was not statistically significant. After 48 hours of culture, the average proliferation rate of HK-2 cells was 42.25%, significantly lower than that of the control group, and the result was statistically significant. ⑥ Based on this, the IC50 (the concentration of drug required to inhibit 50% of biological activity) was further calculated: culture in complete medium with a glucose concentration of 256 mmol / L for 24 hours inhibited 50% of HK-2 cell activity, and culture in complete medium with a glucose concentration of 37.94 mmol / L for 48 hours inhibited 50% of HK-2 cell activity. The experimental results suggest that different glucose concentrations and different culture times have different degrees of damage to the proliferation activity of HK-2 cells. Based on the fact that HK-2 cells can be significantly inhibited from cell proliferation by culturing in a medium with a glucose concentration of 30 mmol / L for 48 h, subsequent experiments with different power, energy densities, and power densities all used a medium with a glucose concentration of 30 mmol / L (the concentration was changed after 24 h) to construct cell damage models.

[0040] Example 2: Detection experiments with different power, different energy densities, and different power densities 1) Setting of light wavelength, energy density, and power density: The experiment was conducted using the controlled variable method. After installing the optical instruments, the light wavelengths were set to 530nm, 590nm, 680nm, 780nm, 880nm, 970nm, and 1050nm, with light energy densities of 2 J / cm². 2 4 J / cm 2 6 J / cm 2 8 J / cm 2 10J / cm 2 The optical power density is 10 mW / cm². 2 30mw / cm 2 50mw / cm 2 .

[0041] 2) Detection of different light wavelengths: The preliminary steps are the same as in Example 1. After culturing for 24 hours, groups 2-8 are replaced with culture medium containing 30 mmol / L glucose. After 48 hours, groups 1-8 are replaced with complete culture medium containing 5.6 mmol / L glucose and subjected to light irradiation at a fixed power density (30 mw / cm²). 2 Energy density (8J / cm³) 2 Groups 2-8 were irradiated with light at wavelengths of 530nm, 590nm, 680nm, 780nm, 880nm, 970nm, and 1050nm, respectively, and then cultured in a constant temperature incubator at 37℃ and 5% CO2 for 24 hours. Cell proliferation was detected according to the method in Example 1.

[0042] 3) Detection of different energy densities: The preliminary steps were the same as in Example 1. After culturing for 24 hours, groups 2-6 were replaced with complete culture medium containing 30 mmol / L glucose. After 48 hours, groups 1-6 were replaced with complete culture medium containing 5.6 mmol / L glucose. Irradiation was then performed, with a fixed wavelength (780 nm) and power density (30 mW / cm²). 2 Groups 2-6 received 2 J / cm 2 4 J / cm 2 6 J / cm 2 8 J / cm 2 10J / cm 2 The cells were irradiated with energy density and then incubated in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. Cell proliferation was then detected according to the method in Example 1.

[0043] 4) Detection of different power densities: The preliminary steps were the same as in Example 1. After culturing for 24 hours, groups 2-4 were replaced with complete culture medium containing 30 mmol / L glucose, and group 1 was replaced with complete culture medium containing 5.6 mmol / L glucose. The media were incubated at 37°C and 5% CO2 for 48 hours. After 48 hours, groups 1-4 were replaced with complete culture medium containing 5.6 mmol / L glucose and subjected to light irradiation, with a fixed wavelength (780 nm) and energy density (8 J / cm²). 2 Groups 2-4 received 10 mw / cm 2 30 mw / cm 2 50mw / cm 2 The cells were irradiated with high power density and then incubated in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. Cell proliferation was detected according to the method in Example 1.

[0044] The experimental results are as follows: (1) Effects of different wavelengths on the proliferation of HK-2 cells under high glucose conditions, such as Figure 2 As shown: The cell viability of different light wavelength groups (530 nm, 590 nm, 680 nm, 780 nm, 880 nm, 970 nm, and 1050 nm) after culturing in high-glucose medium for 48 h was detected by CCK8 assay. It was found that the viability of HK-2 cells in the 590 nm, 680 nm, 780 nm, 880 nm, 970 nm, and 1050 nm groups was higher than that in the control group. Among them, the viability of HK-2 cells in the 780 nm group was significantly higher, while the viability of HK-2 cells in the 530 nm group was not significantly different from that in the control group.

[0045] In addition, this experiment also found that within a certain range, increasing the wavelength of light helps promote cell proliferation. However, beyond a certain wavelength (corresponding to 780nm in this experiment), the increased wavelength of light has a limited effect on promoting cell proliferation. This may be related to the different penetrating power and heat generated by different wavelengths. Theoretically, the longer the wavelength, the stronger the penetrating power, and the better it can reach the inside of the cell to produce an effect. However, in the same amount of time, the longer the wavelength, the more heat is generated. Excessive heat accumulation can damage the cells and lead to cell death.

[0046] (2) Effects of different energy densities on the proliferation of HK-2 cells under high glucose conditions, such as Figure 3 As shown: Different light wavelength groups (2 J / cm) were detected using the CCK8 experiment. 2 4 J / cm 2 6 J / cm 2 8 J / cm 2 , 10J / cm2 The cell viability after culturing in high glucose medium for 48 hours was found to be 2 J / cm. 2 Compared with the control group, 2J / cm 2 The proliferation activity of HK-2 cells in group 4J / cm showed no significant increase, while... 2 Group, 6J / cm 2 Group, 8J / cm 2 Group, 10J / cm 2 Compared with the control group, the survival rate of HK-2 cells increased, with 8J / cm² cells showing improved survival. 2 The group showed a significant increase.

[0047] Different energy densities have different effects on the survival rate of HK-2 cells. As the energy density increases, the cell survival rate gradually increases, but after a certain limit, the increase in cell survival rate is not significant. This may be related to the cellular inflammatory response and free radical formation caused by excessive energy density.

[0048] (3) Effects of different power densities on the proliferation of HK-2 cells under high glucose conditions, such as Figure 4 As shown: Different light power density groups (10 mW / cm²) were tested using the CCK8 experiment. 2 30 mw / cm 2 50mw / cm 2 Cell viability after 48 hours of culture in high glucose medium, of which 10 mw / cm 2 Compared to the control group, the survival rate of HK-2 cells in the group was decreased, while the survival rate of cells at 30 mw / cm² was lower. 2 50mw / cm 2 The survival rate of HK-2 cells increased in group 30mw / cm 2 The group showed a significant increase, and the experimental results indicate that different power densities can reduce the damage to the proliferative activity of HK-2 cells under high glucose stimulation to varying degrees.

[0049] Example 3: Detection experiments with different power, different energy densities, and combinations of different power densities 1) Setting the wavelength, energy density, and power density of the light wave: After installing the optical instrument, set the light wave... The combination of wavelength, energy density, and power density, wherein the light wavelength is selected from 530nm, 590nm, 680nm, 780nm, 880nm, 970nm, and 1050nm, and the light energy density is selected from 2 J / cm². 2 4 J / cm 2 6 J / cm 2 8 J / cm 2 10J / cm 2 The optical power density is 30 mW / cm². 2 The specific combinations are as follows: (530nm+2 J / cm 2 +30mw / cm 2) 、(530nm+4J / cm 2 +30mw / cm 2) 、(530nm+6 J / cm 2 +30mw / cm 2) 、(530nm+8 J / cm 2 +30mw / cm 2) 、(530nm+10 J / cm 2 +30mw / cm 2);( 590nm+2 J / cm 2 +30mw / cm 2) 、(590nm+4 J / cm 2 +30mw / cm 2) 、(590nm+6J / cm 2 +30mw / cm 2) 、(590nm+8 J / cm 2 +30mw / cm 2) 、(590nm+10 J / cm 2 +30mw / cm 2);( 680nm+2 J / cm 2 +30mw / cm 2) 、(680nm+4 J / cm 2 +30mw / cm 2) 、(680nm+6 J / cm 2 +30mw / cm 2) 、(680nm+8J / cm 2 +30mw / cm 2) 、(680nm+10 J / cm 2 +30mw / cm 2);( 780nm+2 J / cm 2 +30mw / cm 2) 、(780nm+4 J / cm 2 +30mw / cm 2) 、(780nm+6 J / cm 2 +30mw / cm 2) 、(780nm+8 J / cm 2 +30mw / cm 2) 、(780nm+10 J / cm 2 +30mw / cm 2);( 880nm+2 J / cm 2 +30mw / cm2) (880nm+4 J / cm) 2 +30mw / cm 2) (880nm+6 J / cm) 2 +30mw / cm 2) (880nm+8 J / cm) 2 +30mw / cm 2) (880nm+10 J / cm) 2 +30mw / cm 2); ( 970nm+2 J / cm 2 +30mw / cm 2) (970nm+4 J / cm) 2 +30mw / cm 2) (970nm+6 J / cm) 2 +30mw / cm 2) (970nm+8 J / cm) 2 +30mw / cm 2) (970nm+10J / cm) 2 +30mw / cm 2);( 1050nm+2 J / cm 2 +30mw / cm 2) (1050nm+4 J / cm) 2 +30mw / cm 2) (1050nm+6J / cm) 2 +30mw / cm 2) (1050nm+8 J / cm) 2 +30mw / cm 2) (1050nm+10J / cm) 2 +30mw / cm 2). 2) The preliminary steps were the same as in Example 1. After culturing for 24 hours, groups 2-36 were replaced with complete culture medium containing 30 mmol / L glucose as the experimental group, and group 1 was replaced with complete culture medium containing 5.6 mmol / L glucose as the control group. They were cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 hours. After 48 hours, groups 1-36 were replaced with complete culture medium containing 5.6 mmol / L glucose and subjected to light irradiation. Based on the above light irradiation combinations, groups 2-36 were irradiated under different light irradiation conditions and then cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. Cell proliferation was detected and statistically analyzed according to the method in Example 1.

[0050] The effects of different combinations of light wavelengths, power, and energy densities on cell viability after 48 hours of culture in high-glucose medium were detected using the CCK8 assay. The results are as follows: Figure 5 As shown: the cell viability rates of the 530nm, 590nm, and 680nm groups were not significantly different from the control group, indicating that the above light conditions did not significantly improve the proliferation damage of HK-2 cells; however, the cell viability rates of the 780nm, 880nm, and 970nm groups were improved to varying degrees, which further verifies the beneficial effect of the above higher wavelengths on cell proliferation damage, and among them (780nm + 8J / cm 2 +30mw / cm 2 ) group and (880nm+10J / cm 2 +30mw / cm 2 Compared with the control group, the survival rate of HK-2 was significantly higher in the control group.

[0051] In this study, the system for photobiological intervention of high glucose-induced renal tubular epithelial cell injury showed discrepancies between the optimal wavelength, power density, and energy density obtained through single-variable screening and the optimal combination of illumination parameters obtained through multi-parameter coupling orthogonal screening. This is a typical biological-optical coupling effect in the field of photobiology, rather than an experimental error. The underlying mechanism can be explained from three aspects: optical physical characteristics, cellular energy reception response, and biological regulatory pathways. First, single-parameter gradient screening only fixes the remaining variables and independently examines the biological effects of wavelength, power density, and energy density, without considering the coupling synergy and mutual constraints between the optical parameters. In contrast, under the multi-parameter combined intervention mode, wavelength, power density, and energy density are not simply linearly superimposed. This leads to an overall change in the light wave propagation mode, irradiance uniformity, photon flux density, and photon interaction efficiency, resulting in significant differences between the actual effective light dose received by the cells and the photon interaction energy level under the single-parameter optimal condition. Secondly, from the perspective of cellular response, renal tubular epithelial cells in a high-glucose microenvironment are already in a pathological state characterized by oxidative stress imbalance, abnormal activation of inflammatory pathways, and impaired mitochondrial function. Their photobiological response is parameter-dependent and microenvironment-sensitive. A single optimal parameter can only target and regulate a specific damaged pathway. However, changes in the combination of parameters shift the penetration depth of light, the efficiency of cellular light absorption, and the degree of photosensitivity activation of mitochondrial cytochrome c oxidase. This, in turn, induces differentiated responses in the intracellular regulation of ATP synthesis, reactive oxygen species levels, inflammatory factors, and the expression of apoptosis-related genes. Ultimately, this results in a mismatch between the protective effects of a single optimal parameter and the combined optimal parameter.

[0052] Finally, from the perspective of photobiophysical mechanisms, different wavelengths paired with specific power and energy densities will alter the irradiation sequence and energy deposition rate: at the same energy density, differences in power density will lead to different photon input rates, thereby affecting the instantaneous energy load of cells and subsequent metabolic cascade reactions; and the coupling of wavelength and power will further regulate the targeted enrichment effect of light in the cell membrane, cytoplasm, and mitochondria, causing the antioxidant, anti-inflammatory, and anti-damage regulatory networks initiated by cells under combined parameters to deviate from the biological response pathways under single parameter screening conditions, thus resulting in inconsistencies between the results of a single optimal parameter and the overall optimal parameter combination.

[0053] Example 4: Cell Scratch Assay To simulate the pathological microenvironment of diabetic nephropathy, a high-glucose medium was prepared by adding 1000 mmol / L glucose solution to ME medium to establish an in vitro cell model of diabetic nephropathy. Cells were seeded in culture plates, and after cell attachment, experimental groupings were performed: A.NC group: HK-2 cells were placed in complete culture medium with a glucose concentration of 5.6 mmol / L and cultured for 96 h.

[0054] B.HG group: HK-2 cells were placed in complete medium with a glucose concentration of 5.6 mmol / L and cultured for 24 h, then replaced with complete medium with a glucose concentration of 30 mmol / L and cultured for 72 h.

[0055] C.HG+780 group: HK-2 cells were placed in complete medium with a glucose concentration of 5.6 mmol / L and cultured for 24 h. The medium was then replaced with complete medium with a glucose concentration of 30 mmol / L and cultured for 48 h. The cells were then exposed to light (780 nm + 8 J / cm²). 2 +30mw / cm 2） After exposure to light, continue culturing for 24 hours.

[0056] D.HG+880 group: HK-2 cells were placed in complete medium with a glucose concentration of 5.6 mmol / L and cultured for 24 h. The medium was then replaced with complete medium with a glucose concentration of 30 mmol / L and cultured for 48 h. Cells were then exposed to light (880 nm + 10 J / cm²). 2 +30mw / cm 2） After exposure to light, continue culturing for 24 hours.

[0057] The steps of the cell scratch assay are as follows: 1) Cell culture and treatment: After trypsin digestion and resuspending, cells were seeded into six-well plates pre-marked with cross-shaped scratches on the bottom, approximately 3 × 10⁶ cells per well. 4Add culture medium and incubate at a constant temperature for 24 hours. Then, change the culture medium to a different glucose concentration for each group and incubate for 48 hours. Next, change the culture medium to a 5.6 mmol / L glucose concentration for each group. Then, provide light to the light-treated groups and incubate again in a cell culture incubator for 24 hours. When the cell density reaches 95% or higher, subsequent experimental procedures can be performed.

[0058] 2) Cell Scratching: Using a 200 μL sterile yellow pipette tip, gently and evenly scratch a straight line perpendicular to the scratch reference line at the bottom of the six-well plate, ensuring the pipette tip remains perpendicular to the bottom of the plate at all times. After scratching, remove the original culture medium and detached cell debris. Then add serum-free HK-2 complete cell culture medium and incubate at 37°C, 5% CO2 to continue culturing to observe cell migration.

[0059] 3) Photography and Statistical Analysis: Images of the scratched area were observed and acquired using a conventional optical microscope at 0h, 12h, 24h, and 36h. ImageJ software was used to analyze the scratch images and quantitatively calculate the area of ​​the scratched region. Using the scratch area at 0h as a baseline control, the cell migration rate at each time point was calculated using the following formula: Cell migration rate (%) = [(0h scratch area - current time point scratch area) / 0h scratch area] × 100%.

[0060] Results of HK-2 cell migration level detection by cell scratch assay are as follows: Figure 6 As shown: Compared with the NC group, the HG group showed a significant decrease in HK-2 cell migration at 36 h, which was statistically significant. Compared with the HG group, the HG+780 group showed a significant increase in cell migration at 36 h, which was statistically significant. Compared with the HG group, the HG+880 group showed a significant increase in cell migration at 36 h, which was statistically significant.

[0061] Experimental results suggest that high glucose stimulation significantly inhibits the migration ability of HK-2 cells, while photobiological therapy can effectively reverse this damage. In this experiment, by comparing the migration area of ​​HK-2 cells in different groups, it was found that the migration ability of HK-2 cells under simulated diabetic conditions was significantly weakened compared with that under normal conditions. This further corroborates the pathogenesis related to renal tubular epithelial cell atrophy and absence in patients with diabetic nephropathy. At the same time, under improved light conditions, the migration ability of HK-2 cells improved compared with those before, but was still lower than the normal level. The improvement effect of the 880nm group was weaker than that of the 780nm group, which further indicates that the light conditions of the 780nm group were more conducive to improving the migration ability of HK-2 cells.

[0062] Example 5: Cell Cloning Experiment 1) Cell culture and fixation: After trypsin digestion and resuspending of cells, 10 μl of the resuspended solution was placed in a cell counting chamber and counted under a biological microscope. The cells were then seeded into a six-well plate, and the cell density was adjusted to 100-300 cells / well. The twenty-four-well plate was placed in a 37°C, 5% CO2 cell culture incubator for 24 h.

[0063] 2) Cell treatment and fixation: Change the complete culture medium with different sugar concentrations (same as in Example 4) according to the group. After changing the culture medium, continue to culture the cells for 48 hours and then discard the culture medium. Wash the cells with PBS 2-3 times, add complete culture medium with a glucose concentration of 5.6 mmol / L, and treat the light group with light. After treatment, place the cells in a 37°C, 5% CO2 cell culture incubator and continue to culture. Observe the cells under a microscope for 2-3 days and change the complete culture medium until the cells form clusters under the microscope, with more than 50 cells per cluster, or the formation of cell clusters can be seen with the naked eye. Discard the culture medium and add 1-1.5 ml of 4% paraformaldehyde solution per well. Fix at room temperature for 15-20 min.

[0064] 3) Stain the cells with 0.1% crystal violet solution. After staining, rinse the six-well plate with water and invert the plate to air dry.

[0065] 4) Cell count: Count by eye or ImageJ software. The clone formation rate (%) = number of clones / number of inoculated cells × 100%.

[0066] The results of the cell cloning experiment to detect the cloning ability of HK-2 cells are as follows: Figure 7 As shown: Compared with the NC group, the clonal capacity of HK-2 cells in the HG group was significantly decreased, and the results were statistically significant. Compared with the HG group, the clonal capacity of cells in the HG+780 group was significantly increased, and the results were statistically significant. Compared with the HG group, the clonal capacity of cells in the HG+880 group was significantly increased, and the results were statistically significant. The experimental results suggest that photobiological therapy can improve the damage to the clonal capacity of HK-2 cells under high glucose stimulation.

[0067] This experiment compared the number of HK-2 cell clones in different groups and found that the clonal capacity of HK-2 cells under simulated diabetic conditions was significantly reduced compared to normal conditions. This further corroborates the important role of renal tubular epithelial cell damage in the pathogenesis of diabetic nephropathy. Simultaneously, under different light wavelengths, the clonal capacity of HK-2 cells improved compared to the control group, with the 780nm group showing a significantly enhanced clonal capacity. This strongly demonstrates the ability of photobiological therapy to reverse damage to HK-2 cells, restore their proliferative capacity, delay the transformation of renal tubular epithelial cells to mesenchymal cells (EMT) in the DKD process (an important pathological feature of renal fibrosis), improve renal function deterioration, and provide strong experimental support for the clinical application of photobiological therapy in patients with diabetic nephropathy.

[0068] Example 6: Detection of related protein expression levels 6.1 Immunofluorescence staining method for detection: 1) Cell culture: After trypsin digestion and resuspending, cells are seeded into 24-well plates pre-placed with climbing slides, and the cell density is adjusted to 4 × 10⁶ cells / well. 4 Cells per well were used to incubate the 24-well plate in a 37°C, 5% CO2 cell culture incubator for 24 hours.

[0069] 2) Cell treatment and fixation: The complete culture medium with different sugar concentrations (same as in Example 4) was changed according to the group. After changing the culture medium, the cells were cultured for 48 hours and then the culture medium was discarded. The cells were rinsed with PBS 2-3 times and then a complete culture medium with a glucose concentration of 5.6 mmol / L was added. The light group was treated with light and then placed in a 37°C, 5% CO2 cell culture incubator for 24 hours. Finally, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes.

[0070] 3) Cell membrane permeation and blocking: Discard the 4% paraformaldehyde solution and wash the cells twice with pre-chilled PBA buffer at 4°C for 5 min each time. Add 0.5% Triton X-100 solution and permeate the cells at room temperature for 15 min to enhance cell membrane permeability. After permeation, wash twice with pre-chilled PBA buffer for 5 min each time. Then, add 2% BSA solution and block at room temperature for 30 min to block non-specific binding sites.

[0071] 4) Primary antibody incubation: Discard the blocking solution and dilute the primary antibody according to the ratio recommended in the antibody instructions (PI3K 1:500, p-PI3K 1:500, AKT 1:500, p-AKT 1:400, Smad2 1:200). Add 200 μL of the diluted primary antibody solution to each well, and add a small amount of PBS between wells to prevent liquid evaporation. Seal the 24-well plate with sealing film and incubate overnight at 4°C. The next day, remove the 24-well plate and continue incubating at room temperature for 1 hour to ensure sufficient binding of the antibody to the antigen.

[0072] 5) Secondary antibody incubation: After recovering the primary antibody, gently wash each well three times with 1 mL of PBA buffer for 5 min each time to remove unbound primary antibody. Then, add 200 μL of diluted fluorescently labeled secondary antibody (1:500 for 488 goat anti-rabbit secondary antibody and 1:500 for 488 goat anti-mouse secondary antibody) to each well and incubate in a 37°C incubator in the dark for 1 h to ensure that the secondary antibody fully binds to the primary antibody.

[0073] 6) Nuclear staining: After discarding the secondary antibody, rinse three times with 1 mL of PBA buffer for 5 min each time to remove unbound secondary antibody. Then, add 300 μL of DAPI staining solution (1 μg / mL) to each well and stain for 10 min at room temperature in the dark to label cell nuclei.

[0074] 7) Mounting and Imaging: After discarding the DAPI staining solution, rinse three times with 1 mL of PBA buffer for 5 min each time to remove unbound DAPI. Add a small amount of PBA buffer to each well to keep the cell slide moist. Take a clean glass slide and label it with the grouping information, then add an appropriate amount of anti-fluorescence quencher to the slide. After mounting the slide on the mounting medium, immediately acquire images under a confocal microscope, ensuring that the fluorescence signal is clear and unquenched.

[0075] 8) Statistics: The average immunofluorescence intensity of each group of images was analyzed using ImageJ, and then Graphpad Pism was used for statistical analysis.

[0076] 6.2 Western blotting of proteins 1) Cell culture: After trying and resuspending cells, they were seeded into six-well plates, and the cell density was adjusted to 4 × 10⁶ cells / well. 5 Cells per well were used to incubate the six-well plate in a 37°C, 5% CO2 cell culture incubator for 24 hours.

[0077] 2) Cell treatment: The complete culture medium with different sugar concentrations (same as in Example 4) was replaced according to the group. After culturing in a 37°C, 5% CO2 cell culture incubator for 48 h, the complete culture medium of each group was replaced with 5.6 mmol / L. The light group was irradiated. Then, the six-well plate was placed in a 37°C, 5% CO2 cell culture incubator for 24 h. Finally, the culture medium was discarded, and the cells were rinsed 3 times with pre-cooled PBS and the PBS was thoroughly aspirated.

[0078] 3) Cell protein extraction: Place a six-well plate on ice, add 100 μL of pre-chilled cell lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor) to each well, gently shake to evenly cover the bottom of the well, and lyse on ice for 15 min. Then, collect the lysis buffer using a sterile cell scraper and transfer it to a pre-chilled 0.2 mL EP tube, and continue incubating on ice for 15 min. Next, place the EP tube on an ice box for sonication lysis. The sonication parameters are: power 60 W, working time 10 seconds, interval 20 seconds, 10 cycles. After sonication, transfer the sample to a centrifuge pre-chilled to 4°C and centrifuge at 15000 × g for 15 min. After centrifugation, transfer the supernatant to a new 1.5 mL EP tube, label it, and store it on ice for later use.

[0079] 4) BCA Protein Concentration Assay: Following the instructions of the BCA protein concentration assay kit, prepare a series of standard protein solutions (concentration gradients of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL). Add 25 μL of the standard protein solution to each 96-well plate, with 3 replicates per group. Simultaneously, add 25 μL of the sample to be tested, diluted 10-fold, with 3 replicates per group, to each 96-well plate. Add 200 μL of freshly prepared BCA working solution (reagent A and reagent B mixed at a 50:1 ratio) to each well, gently mix, and incubate at 37°C for 30 min. Measure the OD value of each well at 562 nm using a microplate reader. Establish a standard curve with the standard protein solution concentration on the x-axis and the corresponding OD value on the y-axis, ensuring the R² value of the fitted curve is close to 1. Calculate the protein concentration of the sample to be tested using the standard curve, and dilute all samples to the same concentration using 1× sample loading buffer.

[0080] 5) Protein denaturation: Transfer the diluted protein sample to an EP tube and heat in a 100°C metal bath for 10 minutes to fully denature the protein. The denatured sample can be stored in a -20°C freezer for a long period of time, not exceeding 6 months.

[0081] 6) Western Blot a. Gel Preparation: SDS-PAGE electrophoresis gels were prepared using the NewSemi 10% ExpressCast PAGE Color Gel Rapid Kit, following the standard preparation procedure for single gels with a thickness of 1.5 mm. Separating Gel Preparation: 4.0 mL of the lower gel solution, 4.0 mL of the lower gel buffer, and 80 μL of modified coagulant were mixed thoroughly and poured into the gel mold. 1 mL of isopropanol or anhydrous ethanol was added on top for liquid sealing and coagulation. After the separating gel had completely solidified, the top sealing layer was discarded, and residual organic solvent was removed from the plate surface using clean filter paper. Stacking Gel Preparation: 1.0 mL of the upper gel solution, 1.0 mL of the upper gel buffer, and 20 μL of modified coagulant were mixed thoroughly and poured evenly onto the top layer of the separating gel. The electrophoresis comb was inserted smoothly, and the gel was allowed to stand until completely solidified before removing the comb.

[0082] b. Sample loading: Assemble the complete electrophoresis apparatus and add 1×SDS electrophoresis buffer to the electrophoresis tank; add 10μL of the protein sample to be tested to each gel sample well in sequence, and add 3μL of pre-stained protein molecular weight standard marker to the blank sample well for protein molecular weight control.

[0083] c. Electrophoresis: Electrophoresis experiments were conducted in constant voltage mode. The initial electrophoresis voltage was set to 80 V. After the sample band was completely in the separating gel area, the voltage was increased to 110 V. Electrophoresis was continued until the indicator dye migrated to the bottom of the gel and then the electrophoresis operation was terminated.

[0084] d. Wet Transfer: Activate the PVDF membrane in anhydrous methanol for 30 seconds. After activation, transfer it to transfer buffer for thorough wetting and equilibration. Simultaneously, soak the transfer-specific filter paper and sponge pad in the transfer buffer for later use. Cut the separating gel according to the experimental objectives. Assemble the transfer fixture strictly in the following layer order: negative electrode – filter paper – gel – PVDF membrane – filter paper – positive electrode. Thoroughly remove air bubbles at the gel-PVDF membrane interface to avoid uneven transfer. Place the assembled transfer fixture into the transfer tank, add pre-cooled rapid transfer buffer, and confirm the electrode polarity is correct. Under ice bath conditions, set a constant current of 400 mA for 0 minutes to complete the protein sample transfer.

[0085] 7) Membrane sealing treatment: Immerse the PVDF membrane after transfer in 5% skim milk sealing solution and seal it on a low-speed shaker at room temperature for 1 h to effectively seal non-specific protein binding sites on the membrane surface and reduce non-specific stray band interference in subsequent experiments. 8) Primary antibody incubation: Dilute the specific primary antibodies according to the antibody product instructions. Specific dilution ratios are: PI3K antibody 1:1000, phosphorylated PI3K (p-PI3K) antibody 1:1000, AKT antibody 1:25000, phosphorylated AKT (p-AKT) antibody 1:5000, and Smad2 antibody 1:1000. After blocking, wash the PVDF membrane three times with 1×TBST washing buffer, each wash lasting 10 min. After washing, place the membranes in the corresponding primary antibody dilution buffers and incubate overnight at 4°C on a shaker.

[0086] 9) Secondary antibody incubation: Prepare horseradish peroxidase (HRP) labeled secondary antibody dilution solution according to the instructions. The dilution ratio of goat anti-mouse HRP secondary antibody and goat anti-rabbit HRP secondary antibody is 1:10000. Take out the PVDF membrane after incubation with the primary antibody, wash it 3 times with 1×TBST washing solution for 10 min each time, and then immerse it in the secondary antibody dilution solution of the matching species and incubate it at room temperature on a shaker at a constant speed for 1 h.

[0087] 10) Chemiluminescence imaging detection: After the secondary antibody incubation, the PVDF membrane was washed three times with 1×TBST, each wash lasting 10 min. The working chromogenic solution was prepared by mixing equal volumes of chemiluminescence chromogenic solution A and solution B. The chromogenic solution was then evenly applied to the surface of the PVDF membrane. The membrane was placed in a fully automated chemiluminescence imager under light-protected conditions for exposure imaging. Protein-specific band images were collected for result analysis.

[0088] 6.3 Experimental Results (1) The results of immunofluorescence staining for detecting the fluorescence intensity of α-SMA protein expressed in HK-2 cells are as follows: Figure 8 As shown, compared with the NC group, the fluorescence intensity of α-SMA protein in the HG group was significantly increased. Compared with the HG group, the fluorescence intensity of α-SMA protein in the HG+780 group was significantly decreased, and the results were statistically significant. Compared with the HG group, the fluorescence intensity of α-SMA protein in the HG+880 group was significantly decreased, and the results were statistically significant.

[0089] To further clarify the effect of photobiological therapy on interstitial fibrosis in HK-2 cells under high glucose stimulation, we extracted proteins from HK-2 cells in each group and performed Western blot analysis. The results are as follows: Figure 9As shown, compared with the NC group, the expression level of α-SMA protein in the HG group was significantly increased. Compared with the HG group, the expression level of α-SMA protein in the HG+780 group was significantly decreased, and the results were statistically significant. Compared with the HG group, the expression level of α-SMA protein in the HG+880 group was significantly decreased, and the results were statistically significant. These results collectively suggest that photobiological therapy can inhibit the expression level of α-SMA and reduce the degree of interstitial fibrosis in HK-2 cells.

[0090] This experiment used immunofluorescence intensity and Western blotting analysis to detect the expression of α-SMA factor in different groups under photobiological therapy. It was found that compared with the control group, photobiological therapy could significantly reduce the expression of α-SMA in HK-2 cells. This further confirms the ability of phototherapy to improve the EMT transition of HK-2 cells and suggests the important role of phototherapy in the process of renal fibrosis. However, this mechanism of action still needs to be verified through further animal experiments and clinical studies.

[0091] (2) The results of immunofluorescence staining for detecting the fluorescence intensity of E-cadherin protein expressed in HK-2 cells are as follows: Figure 10 As shown, compared with the NC group, the fluorescence intensity of E-cadherin protein in the HG group was significantly decreased. Compared with the HG group, the fluorescence intensity of E-cadherin protein in the HG+780 group was significantly increased, and the results were statistically significant. Compared with the HG group, the fluorescence intensity of E-cadherin protein in the HG+880 group was significantly increased, and the results were statistically significant.

[0092] To further clarify the effect of photobiological therapy on interstitial fibrosis in HK-2 cells under high glucose stimulation, we extracted proteins from HK-2 cells in each group and performed Western blot analysis. The results are as follows: Figure 11 As shown, compared with the NC group, the E-cadherin protein expression level in the HG group was significantly decreased. Compared with the HG group, the E-cadherin protein expression level in the HG+780 group was significantly increased, and the results were statistically significant. Compared with the HG group, the E-cadherin protein expression level in the HG+880 group was significantly increased, and the results were statistically significant. These results collectively suggest that photobiological therapy can promote the expression level of E-cadherin and reduce the degree of interstitial fibrosis in HK-2 cells.

[0093] This experiment used immunofluorescence intensity and Western blotting analysis to detect the expression of E-cadherin in different groups under photobiological therapy. It was found that compared with the control group, photobiological therapy could significantly upregulate E-cadherin expression, which further confirms the ability of phototherapy to improve the EMT transition of HK-2 cells and suggests the important role of phototherapy in the process of renal fibrosis. However, this mechanism of action still needs to be verified through further animal experiments and clinical studies.

[0094] (3) Immunofluorescence staining was used to detect the fluorescence intensity of PI3K, p-PI3K, AKT, p-AKT, and Smad2 proteins expressed in HK-2 cells. The results are as follows: Figure 12 As shown, compared with the NC group, the fluorescence intensity of PI3K, p-PI3K, AKT, p-AKT, and Smad2 proteins in HK-2 cells was significantly increased in the HG group. Compared with the HG group, the fluorescence intensity of PI3K, p-PI3K, AKT, p-AKT, and Smad2 proteins in HK-2 cells was significantly decreased in the HG+780 group. Compared with the HG group, the fluorescence intensity of PI3K, p-PI3K, AKT, p-AKT, and Smad2 proteins in HK-2 cells was significantly decreased in the HG+880 group.

[0095] To further clarify the effects of photobiological therapy on the expression of PI3K, p-PI3K, AKT, p-AKT, and Smad2 proteins in HK-2 cells under high glucose stimulation, we extracted proteins from HK-2 cells in each group and performed Western blot analysis. The results are as follows: Figures 13-16 As shown, compared with the NC group, the levels of p-PI3K / PI3K, p-AKT / AKT, and Smad2 in HK-2 cells were significantly increased in the HG group. Compared with the HG group, the levels of p-PI3K / PI3K, p-AKT / AKT, and Smad2 in the HG+780 group were significantly decreased, and the results were statistically significant. Compared with the HG group, the levels of p-PI3K / PI3K, p-AKT / AKT, and Smad2 in the HG+880 group were also significantly decreased. These results collectively suggest that photobiological therapy can inhibit the expression level of the PI3K / AKT / Smad2 pathway in HK-2 cells.

[0096] In this experiment, immunofluorescence staining and Western blot analysis were used to detect the fluorescence intensity of PI3K, p-PI3K, AKT, and p-AKT proteins expressed in HK-2 cells. The results showed that the fluorescence intensity and Western blot analysis of PI3K, p-PI3K, AKT, and p-AKT proteins in HK-2 cells under simulated diabetic conditions were significantly increased, while the expression of these factors in HK-2 cells under light treatment was significantly decreased. The results of this experiment further demonstrate that photobiological therapy can reduce the level of interstitial fibrosis in HK-2 cells by inhibiting the PI3K / AKT signaling pathway, thereby improving kidney damage.

[0097] In this experiment, the expression level of Smad2 protein in HK-2 cells was detected by immunofluorescence staining and Western blot analysis. It was found that the fluorescence intensity and Western blot expression of Smad2 protein in HK-2 cells under simulated diabetic environment were significantly increased, while the expression of Smad2 in HK-2 cells under light treatment was significantly decreased.

[0098] In summary, photobiological therapy can significantly enhance the anti-interstitial fibrosis ability of renal tubular epithelial cells in the DKD state by specifically activating the PI3K / AKT / Smad2 signaling pathway in HK-2 cells. Building upon previous studies confirming the crucial role of this pathway in renal protection, we further discovered that photobiological therapy can effectively improve the basic functions of renal tubular epithelial cells, such as proliferation, migration, and cloning, by targeting renal tubular epithelial cells and inhibiting the interstitial fibrosis mechanism of the PI3K / AKT / Smad2 signaling pathway. This further curbs the progression of renal interstitial fibrosis and provides a novel, non-invasive, and convenient treatment strategy for DKD.

[0099] The above are merely embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A photobiological modulation method for improving high glucose-induced human renal tubular epithelial cell damage in vitro for non-therapeutic purposes, characterized in that, The photobiological regulation method includes the step of treating human renal tubular epithelial cells induced by high glucose in vitro with light; the light wavelength is selected from 780nm, 880nm, and 970nm, and the energy density is 4–10 J / cm³. 2 The power density is 30 mW / cm³. 2 The irradiation time is set according to the power density and energy density.

2. The photobiological regulation method according to claim 1, characterized in that, The illumination simultaneously meets the following conditions: illumination wavelength 780nm, energy density 8J / cm². 2 and power density 30mw / cm 2 .

3. The photobiological regulation method according to claim 1, characterized in that, The illumination simultaneously meets the following conditions: illumination wavelength 880nm, energy density 10J / cm². 2 and power density 30mw / cm 2 .

4. The photobiological regulation method according to claim 1, characterized in that, The energy density is 8 J / cm³. 2 .

5. The photobiological regulation method according to any one of claims 1-4, characterized in that, The photobiological regulation method is used to enhance the proliferation activity, cell migration ability, and cell cloning ability of high glucose-induced human renal tubular epithelial cells, thereby repairing the physiological function of renal tubular epithelial cells.

6. The photobiological regulation method according to any one of claims 1-4, characterized in that, The photobiological regulation method is used to downregulate the expression of α-SMA, a marker of high glucose-induced interstitial fibrosis in human renal tubular epithelial cells, upregulate the expression of E-cadherin, and / or inhibit the abnormal activation of the PI3K / AKT / Smad2 signaling pathway, thereby inhibiting interstitial fibrosis in renal tubular epithelial cells and blocking high glucose-induced damage to human renal tubular epithelial cells.

Images