2.2 Methods
2.2.1 Primary culture of guinea pig orbital fibroblasts
The guinea pigs were sacrificed by intraperitoneal injection of pentobarbital sodium (150mg/kg), and then the treated guinea pigs were immersed in 75% alcohol for 5 minutes. After that, the eyeballs were bluntly separated with scissors, and the intact eyeballs were removed for the required tissue samples. The tissue samples were soaked in Pbs solution to rinse the blood, and then transferred to the ultra-clean table in the cell room. Cut the tissue block into pieces with ophthalmic scissors and ophthalmic tweeds, put it into a 50ml centrifuge tube, add trypsin, and when the tissue block was bloated and flocculent, put it in a centrifuge for centrifugation at 1000rpm for 5 minutes. Discard the digestion liquid, add DMEM slowly along the wall of the bottle, and stop the digestion reaction. After 2–3 times of rinsing, wash. After that, the digested tissue blocks were resuspended in complete medium containing 10% fetal bovine serum and evenly placed in small dishes. The medium did not flow in each dish, and just infiltrated the tissue blocks was better, and put into 37℃, 5%CO2 incubator for primary cell extraction.
2.2.2 Subculture of orbital fibroblasts from guinea pig
After the cells occupied 80 to 90% of the bottom of the T-25 cell culture bottle, passage was started. The medium in the bottle was collected, and the cells were washed 2–3 times with PBS solution. Add trypsin to the T-25 cell culture flask for a 2-min digestion process. After the digestion, complete medium was added to abort the digestion process. This was made into a cell suspension and centrifuged at 1000rpm for 5min in a centrifuge; After centrifugation, 2 ml of complete medium was added to the centrifuge tube, and the centrifuged cells were resuspended. The resuspended cells were transferred to two T-25 cell culture flasks, placed smoothly, shaken and mixed to make the cell density uniform, and the culture flasks were placed in a 37℃, 5%CO2 incubator for static culture.
2.2.3 In vitro pressure culture of guinea pig orbital fibroblasts
As shown in Fig. 1, the pressurization device was constructed, and the OFs of guinea pigs of the third to fifth passages were taken for pressure culture. The guinea pig OFs were divided into blank group (no pressure group), 1KPa group, 2KPa group, and 3KPa group. Air in 37℃, 5%CO2 incubator was injected into T-25 cell culture bottle with 20 ml syringe, and the pressure culture was performed for 36 hours. The blank group (non-pressure group) was directly cultured in 37℃, 5%CO2 incubator, and the 1KPa group, 2KPa group, and 3KPa group were respectively connected with pressure devices and cultured in 37℃, 5%CO2 incubator. After 36 hours of culture, the expression levels of α-SMA, collagenⅠ\Ⅲ\Ⅴ, and piezo1 were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot (WB). CCK8 assay was used to detect the cell proliferation rate, and flow cytometry was used to detect the apoptosis rate of each group.
2.2.4 Co-culture orbital fibroblasts under inhibitor of Pizeo 1 and constant pressure of 3KPa.
The third to fifth passages of guinea pig OFs were taken, and when the cells covered about 80% of the bottom of the bottle, they were cultured under pressure. The primary guinea pig CFs were taken and cultured under a constant pressure of 3KPa to construct TAO cell model of high orbital pressure. 3KPa + 0µM GsMTx-4, 3KPa + 1µM GsMTx-4, 3KPa + 5µM GsMTx-4, and 3KPa + 10µM GsMTx-4 were used to culture for 36 hours. Real-time quantitative polymerase chain reaction (PCR) and Western blot (WB) were used to detect the expression of GsmTX-4. The expression levels of α-SMA, collagenⅠ\Ⅲ\Ⅴ and piezo1 in each group were detected. CCK8 assay was used to detect the cell proliferation rate, and flow cytometry was used to detect the apoptosis rate of each group.
2.2.5 Western blot assay (WB)
The cells in each group were precooled, washed 3 times with PBS solution, added with 1 mL RIPA cell lysate, lysed on ice for 30 min, collected into a 1.5 mL centrifuge tube, centrifuged at 4 ℃ at 5 000 r·min1 for 5 min (centrifugation radius: 3 cm), then the supernatant was taken, boiled at 95 ℃, and the protein was collected for later use. 10 mL concentration glue and 20 mL separation glue were prepared. After loading (20 µL per well), the membrane was separated by electrophoresis, and blocked with BSA for 30 min. After blocking, the membrane was washed with PBST solution, repeated 3 times, and the primary antibody was added (diluted 1ะ10 000 and then added to the sample ion membrane (incubation temperature was 4 ℃, time was > 12 h). On the second day, the membranes were washed 3 times with PBST diluent, diluted with secondary antibody (1ะ1 000) and incubated for 1.5 h (incubation temperature at 37 ℃). The membranes were washed 3 times with PBST diluent and the samples were treated with DAB developer. Image J software was used to analyze the protein gray level and calculate the relative expression amount.
2.2.6.Real-time quantitative polymerase chain reaction (PCR) detection
The cells were collected by trypsin, washed 3 times with PBS solution, centrifuged at 1 500 r·min1 at room temperature for 5 min, then the precipitate was removed, and 1 mL Trizol solvent was added to extract total cell RNA. The purity of RNA was ensured to be 1.8 to 2.1, and then the TAKARA reverse transcription kit was used. A 20 µL reaction system was used for reverse transcription to cDNA, which was stored at -80 ℃. The sample DNA was analyzed by FTC 2000 RTqPCR system and 50 µg reaction system according to the requirements of TAKARA real-time PCR kit (SYBR Premix Ex TaqⅡ kit). The Tm value of the samples was determined to be 53.7 ℃ by the lysis curve. The CT values of the samples in each group were counted and recorded. The expression levels of α-SMA, collagenⅠ\Ⅲ\Ⅴ, and piezo1 in each group were detected by 2△△CT method. The expression levels of α-SMA, collagenⅠ\Ⅲ\Ⅴ and Piezo1 were analyzed. Primer design for Piezo1, and the reference gene glyceraldehyde phosphate dehydrogenase (GAPDH) (Table 1).
Table 1: Primer design
2.2.7.CCK8 experiment
The digested cells were blown and counted, and the cell concentration was adjusted to 1×1045 cells /ml. The cell suspension was divided into a 96-well plate, and 100ul was added to each well, that is, 1×10 cells per well. Cell samples were collected at the cells at the specified time points (0h, 24h, 48h, 72h), and CCK-8 solution was added to each sample, 100ul cell culture medium was added to 10ul detection solution; After 3–4 hours of incubation, plates were read by microplate reader and OD450 data were read by CCK-8 assay.
2.2.8. Flow cytometry was used to detect cell apoptosis
The cell culture medium of each group was collected and placed in a centrifuge tube. The cells were washed with pbs, and the pbs solution was discarded. Trypsin was added until the cell wall was removed, and the cell wall was completely removed by continuous beating. The cells were resuspended in the medium of step 2.9.1, so that the cell density was greater than 1×106 cells /ml. 0.5ml of the cell suspension was transferred to a 15ml centrifuge tube, and 1.25 µl of Annexin V-FITC was added. The reaction was kept in the dark for 15min. The cells were detected and analyzed by flow cytometry.
2.2.9.Statistical Methods
Experimental data are presented as mean ± standard deviation (X ± SD). SPSS 29.0 statistical software was used to test the normality of the data, which was consistent with the normal distribution. One-way analysis of variance was used to analyze the data. One-way analysis of variance was used to compare the data between the two groups after homogeneity of variance test. P < 0.05 was considered to indicate statistical significance. Statistical plots were drawn using Graphpad Prism 9.0.