ABSTRACT
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.
ABSTRACT
Objective To identify the anatomic advantages of pre-contouring long helical plate of proximal humerus internal locking system (PHILOS) aided by 3D-printing technique in minimally invasive plate oseoynthesis (MIPO) for metadiaphyseal fractures of the proximal humerus.Methods Firstly in a cadaveric study,12 fresh frozen samples of upper extremity were divided into 2 equal groups:6 in a Synbone group and 6 in a 3D-printing group.Long 10-hole PHILOS plates were pre-contoured helically according to Synbone humerus models in the Synbone group but according to 3D-printing humerus models in the 3D-printing group.All these helical plates were fixed onto the cadaveric humerus using MIPO technique respectively.The horizontal distances between the radial nerve and the lateral side of the plate were measured at the point where the radial nerve penetrates the muscular septum,at the 6th hole on the plate and at the distal end of the plate;the distance between the medial side of the distal plate and the upper arm neurovascular bundle was also measured.Finally,the full lengths of all these cadaveric humeri were measured.Secondly,on the basis of the anatomic study,16 patients with metadiaphyseal fracture of the proximal humerus were treated at Department of Orthopedic Surgery,The Sixth People's Hospital of Shanghai from February 2013 to March 2016,using the same MIPO technique and the same long 10-hole PHILOS plates pre-contoured helically on the 3D printing models which were mirrored on the humerus on the healthy side of the patients.They were 4 men and 12 women,aged from 59 to 87 years (mean,71.0 years).By the AO/OTA classification,10 cases were type B and 6 type C.The fracture line involved the proximal humerus in 7 cases.Postoperative complications were followed up.At the final follow-up,the shoulder function was assessed according to the Constant-Murley score and the elbow function according to the Mayo Elbow Performance Score (MEPS).Results By the anatomic measurement,the full length of cadaveric humerus averaged 30.17 ± 1.93 cm in the Synbone group and 29.75 ± 2.17cm in the 3D-printing group,showing no significant difference (P ＞ 0.05).The horizontal distances between the radial nerve and lateral side of the plate at the point where the radial nerve penetrates the muscular septum,at the 6th hole on the plate and at the distal end of the plate in the Synbone and 3D-printing groups were,respectively,11.50 ± 0.92 mm versus 17.87 ± 1.88 mm,6.90 ± 1.78 mm versus 14.83 ± 1.50 mm,and 5.14 ± 1.14 mm versus 8.36 ± 1.27 mm,all showing significant differences between the 2 groups (P ＜ 0.05％).The distance between the medial side of the distal plate and the upper arm neurovascular bundle was 6.25 ± 0.95 mm in the Synbone group and 6.88 ± 1.21 mm in the 3D-printing group,showing an insignificant difference between the 2 groups (P ＞ 0.05).The clinical observation found no iatrogenic nerve injury.The 16 patients were followed up for an average of 25.3 months (range,from 24 to 38 months).All fractures got united uneventfully.At the final follow-up,their Constant-Murley scores averaged 77.6 points and their MEPS 96.5 points.Conclusion The risk of iatrogenic injury to the radial nerve can be lowered when 3D-print technique is applied for helical pre-contouring of long PHILOS plate in the MIPO for metadiaphyseal proximal humeral fractures,leading to satisfactory clinical results.