Objective:To investigate the effect of dihydroartemisinin (DHA) and gasdermin E(GSDME) on the proliferation, metastasis and pyroptosis of laryngeal cancer cells as well as its related mechanisms.Methods:Human laryngeal squamous cell cancer Hep-2 cells were taken and divided into 4 groups: the blank group (untreated Hep-2 cells), DHA group (Hep-2 cells treated with 50 μmol/L DHA), GSDME-siRNA group (Hep-2 cells transfected with GSDME-siRNA), and DHA+GSDME-siRNA group (Hep-2 cells treated with 50 μmol/L DHA and transfected with GSDME-siRNA). Methyl thiazolyl tetrazolium (MTT) method was used to detect the effect of DHA on the proliferation ability of Hep-2 cells, and the cell proliferation inhibition rate and half inhibitory concentration ( IC50) were calculated. Flow cytometry was used to detect the pyroptosis rate, Transwell assay was used to detect cell invasion ability and Western blot was used to detect the relative expression levels of GSDME, caspase-3, hexokinase Ⅱ (HK-Ⅱ), cyclophilin D, and voltage-dependent anion channel (VDAC) proteins. Results:The cell proliferation inhibition rates of Hep-2 cells treated with 10, 20, 40, 80, 160 μmol/L DHA for 48 h were higher than those treated with the corresponding concentration for 24 h (all P < 0.05). The IC50 values of Hep-2 cells treated by DHA for 24 h and 48 h were 57.20 μmol/L and 43.50 μmol/L, respectively, and thus 50 μmol/L DHA was selected for subsequent experiments. The pyroptosis rate was (6.5±0.8)%, (22.7±2.5)%, (3.1±0.6)% and (7.0±1.0)%, respectively in the blank group, DHA group, GSDME-siRNA group, and DHA+GSDME-siRNA group, and the difference was statistically significant ( F = 221.20, P < 0.05). The number of invasive cells was (153±14), (95±10), (205±16), and (148±16), respectively in the blank group, DHA group, GSDME-siRNA group, and DHA+GSDME-siRNA group, and the difference was statistically significant ( F = 56.89, P < 0.05). The results of Western blot showed that the relative expression levels of GSDME and caspase-3 in DHA group were higher than those in the blank group (both P < 0.05); the relative expression levels of GSDME and caspase-3 in GSDME-siRNA group were lower than those in DHA group (both P < 0.05); the relative expression levels of GSDME and caspase-3 in DHA+GSDME-siRNA group were higher than those in GSDME-siRNA group (both P < 0.05); the relative expression levels of HK-Ⅱ, cyclophilin D, and VDAC in DHA group were lower than those in the blank group (all P < 0.05); the relative expression levels of HK-Ⅱ, cyclophilin D, and VDAC in GSDME-siRNA group were higher than those in DHA group (all P < 0.05); the relative expression levels of HK-Ⅱ, cyclophilin D, and VDAC in DHA+GSDME-siRNA group were lower than those in GSDME-siRNA group (all P < 0.05). Conclusions:Dihydroartemisinin can increase the pyroptosis of laryngeal cancer cells and reduce cell proliferation and metastasis ability. The mechanism may be related to the inhibition of mitochondrial HK-Ⅱ expression.

Objective:To explore the clinical efficacy of disease-modifying drug nusinersen on children with spinal muscular atrophy.Methods:The baseline and longitudinal clinical data of 15 children who were treated with nusinersen in the Children′s Hospital, Zhejiang University School of Medicine from October 2019 to October 2021 were retrospectively collected. The general data (gender, age, genotype, and clinical classification, etc.), motor function, nutritional status, scoliosis and respiratory function were analyzed. Wilcoxon rank-sum test was used for comparing multi-system conditions before and after treatment.Results:The age of 15 cases (7 males, 8 females) was 6.8 (2.8, 8.3) years, with 2 cases of type 1, 6 cases of type 2, and 7 cases of type 3 respectively, and the course of disease was 55.0 (21.0, 69.0) months. After 9.0 (9.0, 24.0) months of treatment, the motor function scale evaluations of the Hammersmith neurological examination section 2 (13.0 (7.0, 23.0) vs. 18.0 (10.0, 25.0) scores, Z=-2.67, P=0.018) of 15 children, the Hammersmith functional motor scale expanded (38.0 (18.5, 45.5) vs. 42.0 (23.0, 51.0) scores, Z=-2.38, P=0.018), and the revised upper limb module (27.0 (19.5, 32.0) vs. 33.0 (22.5, 35.5) scores, Z=-2.52, P=0.012) of children with type 2 and 3 had significantly improved. Thirteen patients achieved clinically significant motor function improvement, and 2 of them had kept stable scale scores. Subjective reports also indicated that the muscle strength and daily exercise ability of these children improved after treatment, and no serious adverse reactions were reported. Supplemented by the multi-disciplinary team management, the levels of some indicators such as Cobbs angle of scoliosis and forced vital capacity all had significantly improved (all P<0.05). Conclusions:Nusinersen can improve the motor function of patients with 5q spinal muscular atrophy, which is also proved safe to be used in children. The drug treatment supplemented by the multi-disciplinary team management is helpful to improve the multi-system function of the children with spinal muscular atrophy.