Application of ultrasonic waves in retarding senescence state of lens epithelial cells

Abstract

The invention discloses an application of ultrasonic waves in retarding the senescence state of lens epithelial cells. The invention finds that the content expression of the senescence marker P21 gene and beta-galactosidase of the lens epithelial cells irradiated by ultraviolet rays is increased, and the cells are in a senescence state. In addition, the cell also shows a reduced cell nucleus area. The two groups of cells thus produce different nuclear mechanical properties. On the basis, the lens epithelial cells are exposed to ultrasonic waves, the ultrasonic waves can apply short-time pressure waves to the aged lens epithelial cells so as to generate mechanical stress, the P21 gene content and the beta-galactosidase content of the aged cells are reduced, and the aging state of the aged cells is relieved. Compared with other in-vitro methods for slowing down the senescence state of the lens epithelial cells, the method has the advantages that no chemical reagent is added, the senescent LECs are directionally and accurately focused, operation is easy and the like, can be applied to treatment of age-related cataract closely related to senescence of the lens epithelial cells, and has wide development prospects.

Description

Technical Field

[0001] The invention belongs to the field of ophthalmology, relates to a new strategy for treating age-related cataracts, and specifically relates to the application of ultrasound in slowing down the aging state of lens epithelial cells. Background Art

[0002] As life expectancy increases, the aging of the population has attracted widespread attention from all walks of life. As a result, more and more people suffer from age-related diseases, such as arthritis, cardiovascular disease, cataracts, etc. These diseases usually impair the physical functions and quality of life of the elderly. Many problems with age-related diseases can be attributed to the results of cellular aging. Senescent cells cannot perform normal cellular functions and secrete cytokines that promote the aging of neighboring cells. Therefore, it is urgent to develop therapies that can slow down, prevent or even reverse cellular aging.

[0003] Age-related cataract is the main blinding eye disease in the world, which increases the burden of life for many families. Under normal circumstances, lens epithelial cells (LECs) are distributed under the anterior capsule and equatorial part of the lens, playing a role in maintaining the metabolic transport function of the lens and ensuring the transparency of the lens. However, with age, aging LECs will cause lens metabolic disorders and eventually lead to the occurrence of cataracts. However, there is currently no specific drug for this, and the main way to treat cataracts is still surgical intervention. Therefore, this is also accompanied by corresponding side effects, such as damage to the adjacent tissues of the lens, such as the cornea, causing postoperative complications.

[0004] Therefore, targeted treatment of senescent LECs has become a hot topic in current research. At present, the research on cell senescence mainly focuses on: cell cycle arrest, inflammation, changes in proliferation and apoptosis, abnormal energy metabolism, epigenetic modification regulation, etc. However, chemical reagents and drugs for the treatment of the above targets have certain side effects, so trying to use physical means to alleviate or even reverse cell senescence has certain advantages.

[0005] Therapeutic ultrasound is a form of treatment that uses ultrasound waves, which can pass through human tissues to achieve the effect of treating specific tissues. Currently, ultrasound waves have been used to treat various diseases, including ligament sprains, muscle strains, tendinitis, osteoarthritis, etc. However, there are no reports of its use in slowing down the aging of lens epithelial cells or treating age-related cataracts. Summary of the invention

[0006] In view of this, the present invention provides the application of ultrasound in slowing down the aging state of LECs. This method can accurately and contactlessly apply mechanical stress to aging LECs without the use of chemical drugs, thereby achieving the effect of slowing down the aging state of LECs to prevent lens opacity, and provide a reliable research basis for further research on ultrasound treatment of age-related cataracts.

[0007] To achieve the above object, the present invention provides the following technical solution: application of ultrasound in slowing down the aging state of lens epithelial cells.

[0008] Furthermore, the ultrasound waves exert pressure waves on the lens epithelial cells to change the mechanical properties of the cell nucleus, thereby achieving the effect of slowing down the aging of the lens epithelial cells.

[0009] Wherein, the lens epithelial cell senescence is 3-5 mj / cm 2 UV irradiation induced, preferably 5mj / cm 2 , causing decreased cell activity, reduced nuclear area, increased P21 expression, and increased β-galactosidase.

[0010] In some specific embodiments, the ultrasonic transmitter is mounted on a robotic arm, which can be controlled to position the ultrasonic transmitter to a desired position or orientation to perform ultrasonic treatment on the target lens epithelial cells; and the ultrasonic transmitter is sealed and waterproof; the ultrasonic transmitter can emit repetitive periodic force, wherein parameters such as frequency, power, time, and duty cycle can be adjusted.

[0011] In some embodiments, the ultrasonic transmitter can be placed below the surface of the cell culture medium.

[0012] Furthermore, the ultrasonic treatment frequency is 1 to 5 MHz.

[0013] Furthermore, the ultrasonic treatment power is 0.1-0.5 W / cm 2 .

[0014] Furthermore, the ultrasonic treatment time is 10 seconds to 5 minutes.

[0015] In some specific embodiments, it is also necessary to repeat periodically, 1 to 3 times a day.

[0016] Preferably, the cycle is repeated once a day.

[0017] Furthermore, the ultrasonic wave adopts a duty cycle of 10% to 50%.

[0018] Preferably, the ultrasonic wave adopts a frequency of 1 MHz and a power of 0.3 w / cm 2 , duty cycle is 30%, and processing time is 3min.

[0019] Furthermore, the slowing down of aging of lens epithelial cells includes: the expression of aging-related markers of aging lens epithelial cells is reduced after being treated with ultrasound; the aging-related markers include: P21, β-galactosidase.

[0020] The beneficial effects of the present invention are:

[0021] The present invention achieves the effect of slowing down aging by ultrasonically treating aged LECs. The present invention utilizes UV-induced aged LECs, which can better simulate the environment in which people live in real life, and is also the main pathogenic mechanism of current age-related cataracts. This application is based on the different mechanical properties exhibited by LECs in normal and aged states, and applies specific ultrasonic mechanical stress accordingly, so as to treat senescent cells in a targeted manner without damaging normal LECs. The method and system for using ultrasound to slow down the aging of LECs provided in this application utilize physical means rather than previous chemical and biological means for treatment, which reduces the side effects of chemical drugs, and focuses precisely on aged LECs, reducing damage to surrounding normal cells; and the ultrasonic parameters can be adjusted according to actual needs to achieve the desired ultrasonic effect, providing a new treatment strategy for the treatment of age-related cataracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For those without UV irradiation and after UV5mj / cm 2 Comparison of LECs cell activity after irradiation with different doses;

[0023] Figure 2 For those without UV irradiation and after UV5mj / cm 2 Comparison of β-galactosidase and P21 expression in LECs after irradiation; (A) shows the control group and UV5mj / cm by western blot technology 2 The expression levels of P21 in the two irradiation groups, (B) the difference in β-galactosidase expression levels between the two groups and the quantitative analysis results;

[0024] Figure 3 For those without UV irradiation and after UV5mj / cm 2 Comparison of the nuclear area of ​​senescent LECs after irradiation; (A) is the control group and UV5mj / cm 2 Nuclear immunofluorescence staining images of LECs in the irradiated group, (B) is the statistical graph of quantitative analysis of the nuclear cells in the two groups;

[0025] Figure 4It is a comparison diagram of the aged LECs treated with the control group, UV irradiation group and UV irradiation + ultrasound treatment group; wherein, (A) is the β-galactosidase content and quantitative analysis results of each group, and (B) is the P21 expression content of each group;

[0026] Figure 5 This is a comparison chart of the slowing effect of P21 gene expression in senescent cells under various ultrasound parameters. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] In view of the fact that there is no good method to slow down the aging of LECs and prevent age-related cataracts, the present invention studies aging LECs and finds that the cell activity is significantly decreased, the P21 content and β-galactosidase content are significantly increased, the cell nucleus area is significantly reduced, and normal and aging LECs have certain differences in the mechanical properties of the cells. Since the cell nucleus may change in response to mechanical stimulation, the present invention attempts to apply mechanical stress to LECs in order to slow down or reverse the reduction of the cell nucleus area. Among various stress sources, ultrasound has strong directionality and can pass through human tissue to achieve the effect of treating aging cells in specific tissues without affecting normal cells. It also has high energy transfer efficiency and strong safety, and can accurately apply stress without contact. Therefore, the present invention uses ultrasound to conduct a large number of experiments on LECs and finds that because ultrasound applies short-term pressure waves to LECs to generate mechanical stress, LECs in different states respond differently to this stress, the P21 gene and β-galactosidase content of aging cells are reduced, and their aging state is alleviated. This method can accurately and contactlessly apply mechanical stress to aging LECs without the use of chemical drugs, thereby slowing down the aging of LECs and providing a reliable research basis for further research on ultrasound treatment of age-related cataracts.

[0029] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0030] The present invention provides an application of ultrasound in reversing lens epithelial cell aging, comprising the following steps:

[0031] (1) Use 3~5mj / cm 2UV irradiation of LECs induces cell senescence, preferably 5 mj / cm 2 , cell activity, nuclear area, P21 and β-galactosidase expression levels were detected.

[0032] (2) Extend the ultrasonic transmitter below the surface of the cell culture medium and adjust the parameters within the following ranges: frequency is 1, 3, 5 MHz or higher, preferably 1 MHz; power is 0.1, 0.2, 0.3, 0.4, 0.5 W / cm 2 or higher, preferably 0.3 W / cm 2 ; The time is 10 seconds to 5 minutes, preferably 30 seconds to 3 minutes; some need to be repeated periodically, including 1 time, 2 times, 3 times a day, preferably 1 time a day; the duty cycle is 10% to 50%, preferably 30%.

[0033] (3) After ultrasonic treatment, the expression levels of P21 and β-galactosidase in aged LECs were detected.

[0034] Example 1

[0035] This example studies aging LECs, and by comparing normal LECs with aging LECs, it is found that there are significant differences in the mechanical properties of their nuclei, and the area of ​​the nuclei of aging LECs is significantly reduced. The details are as follows:

[0036] 1. Induction of Senescence in LECs

[0037] On the first day, normal LECs were planted in the well plate and cultured overnight in an incubator at 37°C with 5% CO2. On the second day, when the cell density occupied about 60%-70% of the bottom area of ​​the well plate, the original culture medium was aspirated and half the volume of PBS buffer was added. Then, the UV crosslinker was turned on and the irradiation dose was adjusted to 5 mj / cm 2 After the irradiation is completed, the PBS is aspirated and an appropriate amount of culture medium is added to continue culturing for 24 hours.

[0038] 2. Detection of Cell Activity

[0039] Cell activity was detected using CCK8. The cells were seeded in a 96-well plate and LECs were treated with the above method. On the third day after seeding the cells, 10 μl of CCK8 was added to each well and incubated at 37°C for 1 hour. Finally, the absorbance (A) value at 450nm was measured using an ELISA reader. The calculation formula is cell activity = (absorbance value of the experimental group - absorbance value of the blank control) / (absorbance value of the control group - absorbance value of the blank control) × 100%. The results showed that at 5 mj / cm 2 After irradiation with energy of Figure 1 (The sample size of each group is 6, *P<0.05).

[0040] 3. Detection of P21 expression in LECs by Western blotting

[0041] Protease inhibitors, phosphatase inhibitors, and phenylmethylsulfonyl fluoride were added to the lysate at a ratio of 1:100 to extract the total protein of LECs in the control group and the aging group; then the protein concentration was determined using the BCA method, and 5X loading buffer was added to the quantitative protein solution to dilute it to 1X loading buffer, and then placed in a 100°C metal bath for 5 minutes. The cooked protein was then added to the SDS-PAGE gel to separate the protein, and the separated protein was transferred to the PVDF membrane. Then it was blocked with 10% skim milk powder at room temperature for 1 hour. After the blocking was completed, it was washed with TBST solution for 2×5 minutes, and after the primary antibody was diluted with TBST, it was placed in a 4°C refrigerator overnight. On the second day, the primary antibody liquid was aspirated, the membrane was washed with TBST for 3×10 minutes, and then the corresponding diluted secondary antibody was added, incubated at room temperature for 1 hour, and the membrane was washed with TBST for 3×10 minutes. Finally, the ECL luminescent developer was prepared according to the ratio in the instruction manual, added to the membrane and placed in the exposure instrument to observe the expression of P21 protein, refer to Figure 2 A, it can be seen that the P21 content in LECs increased significantly after being irradiated with UV 5mj energy.

[0042] 4. Detection of β-galactosidase expression in LECs

[0043] Similarly, LECs were irradiated with UV, and the subsequent steps were completed according to the instructions of the cell senescence β-galactosidase staining kit. Pictures were taken with an inverted biological microscope. 2 The β-galactosidase content in LECs after energy irradiation was significantly increased. Figure 2 B (sample size n for each group is 4, ****P<0.0001)

[0044] 5. Measurement of nuclear area

[0045] Similarly, cells were seeded on a 24-well plate cell slide. After UV irradiation of LECs on the second day, the original culture medium was aspirated on the third day after inoculation, and the cells were washed with PBS for 3×10 minutes. They were fixed with 4% paraformaldehyde fixative at room temperature for 15 minutes, and the 4% paraformaldehyde fixative was aspirated again, and washed with PBS for 3×10 minutes. The cells were then treated with 0.5% PBS-Triton for 15 minutes to permeabilize the membrane, and then blocked with 10% goat serum at room temperature for 1 hour. The serum was then aspirated, incubated in the dark with 5μg / ml DAPI staining solution for 5 minutes, and then washed with PBS for 3×10 minutes. Finally, the slides were sealed and photographed with a confocal microscope. The images obtained were used to measure the nuclear area using Image J software. Reference Figure 3 , it was found that the nuclear area of ​​​​the cells in the aging state was significantly reduced (the sample size of each group was 20-28, **P<0.01), and the mechanical properties changed.

[0046] Example 2

[0047] Because the cell nucleus responds to mechanical stimulation, its morphology and structure may change dynamically. This example attempts to apply specific mechanical stress to LECs, selects ultrasound as a stress source, treats aged LECs, and uses different ultrasound conditions to conduct multiple experiments to obtain the best ultrasound conditions suitable for reversing the aging state of lens epithelial cells, which can be used to treat age-related cataracts. The details are as follows:

[0048] (1) On the first day, LECs were divided into a control group (ctl), a UV irradiation group (uv5), and a UV irradiation + ultrasound treatment group (uv5+us1). They were seeded in a six-well plate at a rate of 12 w / well, and 2 ml of cell culture medium was added. The plates were cultured overnight at 37°C in an incubator containing 5% CO2. On the second day, the original culture medium of the two groups was aspirated, 1 ml of PBS was added, and then the cells were treated with a 5 mj / cm 2 The LECs in the UV irradiation group were treated with a UV crosslinker with high energy. After irradiation, the PBS was aspirated and 2 ml of cell culture medium was added to continue incubation in the incubator for 24 h. On the third day, ultrasound was set to 1 MHz and 0.5 W / cm 2The ultrasonic power and duty cycle of 30% were continued for 1 minute, which was the uv5+ultrasound1 (uv5+us1) group (ultrasound parameters refer to the ultrasonic treatment condition parameter setting table). The ultrasonic transmitter was placed below the liquid level of the LECs culture medium in the UV irradiation group. After the ultrasound was completed, the original culture medium of the three groups was aspirated, and 2 ml of culture medium was added again and placed in the incubator for 24 hours. On the fourth day, the β-galactosidase content was detected according to the method for measuring the expression content of β-galactosidase in LECs as described above. It was found that the β-galactosidase content of the aged LECs treated with ultrasound was significantly lower than that of the aged LECs not treated with ultrasound. Figure 4 A(the sample size n for each group is 3, ****P<0.0001).

[0049] Subsequently, the cells were treated with the same culture medium as above. On the fourth day, the culture medium was aspirated and each well was washed with PBS for 3 × 10 minutes. Then, 1 ml of Trizol was added to each well and lysed on ice for 15 minutes. Total RNA was then extracted according to the instructions. TM RT Master Mix (Takara) was used for reverse transcription to obtain cDNA for each group, and SYBR Premix Ex Tag was used. TM The reagents were used to prepare the reaction system, and the ABI Fast 7500 RT-PCR system was used to measure the P21 gene expression content of each component. Figure 4 B, it can be seen that the P21 gene expression of senescent LECs treated with ultrasound is significantly lower than that of senescent LECs not treated with ultrasound. The results show that the ultrasound condition can effectively reverse the senescence of LECs.

[0050] (2) On the first day, LECs were divided into a control group (ctl), a UV irradiation group (uv5), and a UV irradiation + ultrasound treatment group (uv5+us1). They were seeded in a six-well plate at a number of 12 w / well, and 2 ml of cell culture medium was added. The plates were cultured overnight in an incubator at 37°C with a volume fraction of 5% CO2. On the second day, the original culture medium of the two groups was aspirated, 1 ml of PBS was added, and then the cells were treated with a 5 mj / cm 2 The LECs in the UV irradiation group were treated with a UV crosslinker with a high energy level. After irradiation, the PBS was aspirated and 2 ml of cell culture medium was added to continue incubation in the incubator for 24 h. On the third day, the ultrasound energy was set to 1 MHz ultrasound frequency and 0.3 W / cm 2The ultrasound power and duty cycle of 30% were continued for 3 minutes, which was the uv5+ultrasound2 (uv5+us2) group (ultrasound parameters refer to the ultrasonic treatment condition parameter setting table). The ultrasonic transmitter was placed below the liquid level of the LECs culture medium in the UV irradiation group. After the ultrasound was completed, the original culture medium of the three groups was aspirated, and 2 ml of culture medium was added again and placed in the incubator for 24 hours. On the fourth day, the β-galactosidase content and P21 gene expression in LECs were measured according to the above method, and the reference Figure 4 A, B. The results show that the ultrasound condition can effectively reverse the aging of LECs. In addition, as shown in Table 1, the present invention tried multiple sets of ultrasound condition parameter settings, and the results are as follows Figure 5 As shown, the display parameters are set to frequency 1Mhz and power 0.3w / cm 2 When the duty cycle was 30% and the ultrasound time was 3 minutes, ultrasound had the best effect in slowing down the aging state of lens epithelial cells, and could significantly slow down the β-galactosidase content and P21 gene expression of lens epithelial cells.

[0051] Table 1 Ultrasonic treatment condition parameter setting table

[0052] Frequency (MHz) <![CDATA[Power (w / cm 2 )]]> Duty Cycle Time (min) Ctl / / / / us1 1 0.5 30％ 1 us2 1 0.3 30％ 3 us3 1 0.1 30％ 1 us4 1 0.3 50％ 1 us5 1 0.1 40％ 3

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The application of ultrasound in slowing down the aging of lens epithelial cells.

2. The use according to claim 1, characterized in that: The ultrasound waves exert pressure waves on the lens epithelial cells to change the mechanical properties of the cell nucleus, thereby achieving the effect of slowing down the aging of the lens epithelial cells.

3. The use according to claim 1, characterized in that: The lens epithelial cell senescence is 3-5 mj / cm 2 Ultraviolet radiation induces decreased cell activity, reduced nuclear area, increased P21 expression, and increased β-galactosidase.

4. The use according to claim 1, characterized in that: The application is specifically as follows: an ultrasonic transmitter is installed on a mechanical arm, and the mechanical arm is controlled to position the ultrasonic transmitter to a desired position or orientation, so as to perform ultrasonic treatment on target lens epithelial cells; the ultrasonic transmitter is sealed and waterproof.

5. The use according to claim 4, characterized in that: The ultrasonic treatment frequency is 1 to 5 MHz.

6. The use according to claim 4, characterized in that: The ultrasonic treatment power is 0.1-0.5 W / cm 2 .

7. The use according to claim 4, characterized in that: The ultrasonic treatment time is 10 seconds to 5 minutes, and is repeated periodically 1 to 3 times per day.

8. The use according to claim 4, characterized in that: The ultrasonic treatment has a duty cycle of 10% to 50%.

9. The use according to any one of claims 5 to 8, characterized in that: The ultrasonic treatment frequency was 1 MHz and the power was 0.3 w / cm 2 , duty cycle is 30%, and processing time is 3min.

10. The use according to claim 1, characterized in that: The performance of slowing down the aging of lens epithelial cells includes: the expression of aging-related markers of the aging lens epithelial cells is reduced after being treated with ultrasound; the aging-related markers include: P21 and β-galactosidase.

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