الملخص
Objective:To investigate the effect of L-carnitine on calcium oxalate-induced ferroptosis in renal tubular epithelial cells (HK-2).Methods:The effects of calcium oxalate(0, 2, 4 and 8 mmol/L) on the expression of ferroptosis-related protein long chain fatty acyl-CoA synthetase 4 (ACSL4), cystine/glutamate transporter(XCT) and glutathione peroxidase 4 (GPX4) in HK-2 cells were detected by Western blotting. The experiment was then divided into four groups: ①control group, cells were cultured in normal medium for 12 hours, then continued to use normal medium; ②L-carnitine group, cells were pretreated with medium containing 5mmol/L L-carnitine for 12 hours, then changed to medium containing 5mmol/L L-carnitine; ③calcium oxalate group, cells were cultured in normal medium for 12 hours, and then replaced with medium containing 4 mmol/L calcium oxalate; ④calcium oxalate+ L-carnitine group, the cells were pretreated with medium containing 5mmol/L L-carnitine for 12 h, and then replaced with 5mmol/L L-carnitine and 4mmol/L calcium oxalate medium. After changing the culture medium for 24 hours, the cells or supernatants were collected, and the expression levels of ferroptosis-related protein quinone oxidoreductase (NQO1), ACSL4, XCT and GPX4 were detected by Western blotting. The levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde were detected by corresponding kit, and the level of reactive oxygen species in cells was detected by reactive oxygen species kit.Results:The results of Western blotting showed that the expression of ACSL4 protein in 0, 2, 4, 8 mmol/L calcium oxalate was 0.37±0.16, 0.68±0.16, 0.73±0.09, 0.89±0.03 respectively. The expression of XCT protein was 1.11±0.10, 0.91±0.14, 0.83±0.09, 0.80±0.07, respectively. The expression of GPX4 protein was 1.23±0.13, 0.99±0.17, 0.81±0.05, 0.72±0.06, respectively. Compared with 0mmol/L group, the expression of ACSL4 protein increased and the expression of XCT and GPX4 decreased in 2, 4 and 8 mmol/L groups, and the difference was more significant between 4 mmol/L group and 0 mmol/L group. So 4 mmol/L was taken as the optimal concentration for follow-up experiment. The levels of NQO1 in control group, L-carnitine group, calcium oxalate group and calcium oxalate+ L-carnitine group were (0.36±0.06, 0.54±0.05, 0.76±0.07, 0.90±0.03) respectively. There was significant difference between L-carnitine group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of ACSL4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.66±0.10, 0.58±0.08, 0.99±0.03, 0.77±0.09) respectively. There was no significant difference between L-carnitine group and control group(P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of XCT in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.93±0.08, 0.85±0.07, 0.76±0.06, 0.99±0.05). There was no significant difference between L-carnitine group and control group (P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GPX4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (1.10±0.09, 1.09±0.09, 0.85±0.03, 0.99±0.02) respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of LDH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine were (100.00±5.37)%, (99.50±6.38)%, (153.77±6.06)% and (132.50±5.58)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The SOD levels in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±5.79)%, (105.80±3.26)%, (44.74±7.60)% and (85.01±5.15)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GSH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±4.73)%, (107.10±5.48)%, (53.49±3.98)% and (85.18±5.48)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.01). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The levels of MDA in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±2.36)%, (98.00±11.10)%, (129.11±2.59)% and (113.35±5.79)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The fluorescence intensity of ferrous ion in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (39.77±0.68) AU, (68.40±3.14) AU and (48.60±4.30) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The fluorescence intensity of reactive oxygen species in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (63.98±9.41) AU, (145.41±8.39) AU and (85.37±4.51) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). Transmission electron microscopy results showed that mitochondria were wrinkled, cristae were broken or disappeared in the calcium oxalate group compared to the control group, and a double-layer membrane structure was evident. DAPI staining showed that compared with the control group, some of the nuclei in the calcium oxalate group were significantly more damaged, while compared with the calcium oxalate group, the nuclei in the calcium oxalate + L-carnitine were significantly less damaged. The results of crystal adhesion test showed that compared with the control group, calcium oxalate crystals in the calcium oxalate group adhered to the cells in black-like particles and formed clusters. Compared with the calcium oxalate group, the calcium oxalate + L-carnitine showed less black particles adhering to the cells. Conclusions:L-carnitine may reduce the effects of oxidative stress and ferroptosis induced by calcium oxalate, thus reducing cell damage and crystal adhesion.
الملخص
Objective:To investigate the role of ferroptosis in calcium oxalate (Calcium Oxalate, CaOx) crystal-induced injury of human renal tubular epithelial cells (HK-2 cells).Methods:From March 2021 to September 2021, I used calcium oxalate crystal suspension to intervene HK-2 cells to build a HK-2-CaOx reaction model. Set the concentration gradient group and time gradient of calcium oxalate crystal intervention in HK-2 cells: 7 groups of calcium oxalate crystals with different concentrations (0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L) were used to intervene HK-2 cells24 hours, the HK-2 cell protein was extracted after the intervention; HK-2 cells were intervened with calcium oxalate crystals at optimum concentration, and extract proteins at different time points (0, 3, 6, 9, 12, 24, 48 h) after intervention, the expression of intracellular ferroptosis marker protein glutathione peroxidase 4 (GPX4) was detected by Western blot. Intervention of HK-2 cells with ferroptosis inducer Erastin and ferroptosis inhibitor ethyl 3-amino-4-cyclohexylaminobenzoate (Ferrostatin-1, Fer-1) to regulate intracellular ferroptosis Level. HK-2 cells were divided into 4 groups: normal control group (NC; no intervention treatment, cultured in complete medium only); calcium oxalate crystal stimulation group (CaOx; cultured in complete medium containing 4.0 mmol/L CaOx crystals); calcium oxalate crystals + erastine treatment group (CaOx+ Erastin; cultured in complete medium containing 10.0 μmol/L erastine and 4.0 mmol/L calcium oxalate crystals); calcium oxalate crystals + Fer-1 Treatment group (CaOx+ Fer-1; cultured in complete medium containing 1.0 μmol/L Fer-1 and 4.0 mmol/L calcium oxalate crystals). After 24 hours, the expression of ferroptosis-related protein GPX4, long-chain fatty acyl-CoA synthase 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) in HK-2 cells was analyzed by western blot and immunofluorescence techniques; the content of glutathione in HK-2 cells was detected; DCFH-DA fluorescence staining was used to observe the expression of reactive oxygen species (ROS) in HK-2 cells. The adhesion of calcium oxalate in HK-2 cells in each group was observed by light microscope, and the nuclear damage of HK-2 cells was detected by DAPI staining.Results:The expression levels of GPX4 in cells in the concentration gradient of 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L were5.67±1.05, 5.60±0.02, 4.99±0.94, 4.82±0.93, 4.50±0.70, 4.14± 0.53, 0.97±0.53. The expression difference of GPX4 between the 4.0 mmol/L group and the 0 mmol/L group was statistically significant ( P=0.026). 4.0 mmol/L was selected as the optimal concentration to intervene the cells. The expression levels of GPX4 in the time gradient (0, 3, 6, 9, 12, 24, 48 h) cells were 11.73±1.29, 11.68±1.32, 11.72±1.30, 10.97±1.28, 10.63±1.21, 8.79±1.10, 8.03±1.06. The expression difference of GPX4 between the 24h intervention group and the 0h intervention group was statistically significant( P=0.090), so 24h was chosen as the optimal intervention time for calcium oxalate crystals. Compared with the NC group, the CaOx+ Erastin group had higher expression of ACSL4 (9.71±0.68 vs. 3.96±0.17, P<0.01); SLC7A11 (5.76±1.31 vs. 9.18±1.54, P=0.001) and GPX4 (3.61±0.25 vs. 9.26±0.13, P<0.01) the expression level decreased. Compared with the CaOx group, the CaOx+ Fer-1 group had higher protein expression levels of GPX4 (7.52±0.23 vs. 3.61±0.25, P<0.01), SLC7A11 (7.85±1.34 vs. 5.76±1.31, P=0.012), ACSL4 (5.84 ±0.62 vs. 9.71±0.68, P=0.002) protein expression was significantly decreased. Compared with CaOx group, CaOx+ Erastin group had significantly lower protein expression of GPX4 (2.71±0.18 vs. 3.61±0.25, P=0.001), SLC7A11 (3.82±1.60 vs. 5.75±1.31, P=0.017), ACSL4(11.15±0.44 vs.9.71±0.68, P<0.01) protein expression increased. The results of glutathione determination showed that compared with the NC group, the glutathione content in the CaOx group was significantly reduced [(53.38±3.53) mmol/L vs. (81.88±4.02) mmol/L, P<0.01]. Compared with the CaOx group, the CaOx+ Fer-1 group had significantly higher glutathione content [(68.26±4.55)mmol/L vs. (53.38±3.53)mmol/L, P=0.001]. Compared with the CaOx group, the glutathione content was decreased [(38.22±2.95)mmol/L vs.(53.38±3.53)mmol/L, P=0.01]. The results of DCFH-DA fluorescence staining showed that compared with the NC group (63.36±5.17 vs. 22.72±3.73, P<0.01), the CaOx group had a significantly higher fluorescence intensity, Compared with the CaOx group (45.32±4.33 vs. 63.36±5.17, P=0.002), the fluorescence intensity of cells in the CaOx+ Fer-1 group was significantly weakened, Compared with the CaOx group (82.38±6.25 vs.63.36±5.17, P=0.002), the fluorescence intensity of the cells in the CaOx+ Erastin group was significantly increased. The results of immunofluorescence showed that the CaOx group was significantly weakened compared with the NC group (31.63±2.86 vs. 50.36±4.23, P<0.01), and the CaOx+ Fer-1 group was significantly weakened compared with the CaOx group (39.89±3.35 vs. 31.63±2.86), P=0.038), the fluorescence intensity of cells in the CaOx+ Fer-1 group was significantly enhanced, the CaOx+ Erastin group was compared with the CaOx group (23.36±3.74 vs. 31.63±2.86, P=0.022), the cell fluorescence in the CaOx+ Erastin group was The intensity is significantly reduced. DAPI staining to calculate the damage ratio of each group of nuclei: NC group (2.85%), CaOx group (11.96%), CaOx+ Fer-1 group (8.76%), CaOx+ Erastin group (16.27%). Conclusion:CaOx crystals can induce ferroptosis in HK-2 cells by increasing the level of oxidative stress in HK-2 cells.