Objective: To investigate the effect of manganese chloride (MnCl₂) or 1-methyl-4-phenylpyridinium (MPP ⁺) on oxidative stress and autophagy in human neuroblastomaSK-N-SH cells and the mechanism of the neurotoxicity of manganese. Methods: SK-N-SH cells were treated with MnCl₂ or MPP⁺ at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, and 2.0 mmol/L for 24 hours, and MTT assay was used to measure cell viability. The cells weretreated with MnCl₂ or MPP⁺ at doses of 0.125, 0.25, and 0.5 mmol/L for 24 hours, and flow cytometry was used to measure the content of reactive oxygen species (ROS) in cells, a laser scanning confocal microscope was used to observe autophagosome in cells, and Western blot was used to measure the expression of autophagy-related proteins P62 and LC3-II/LC3-I. Results: Compared with the control group, the 0.0625-2.0 mmol/L MnCl₂ and 0.125-2.0 mmol/L MPP ⁺ treatment groups had significant reductions in the viability of SK-N-SH cells, and the 0.25-2.0 mmol/L MnCl₂ treatment groups had significantly lower viability than the groups treated with the same doses of MPP⁺ (all P<0.05) . Compared with the control group, the 0.125-0.25 mmol/L MnCl₂ and 0.125-0.5 mmol/L MPP⁺ treatment groups had significant increases in the content of ROS, and the 0.25-0.5 mmol/L MPP⁺ treatment groups had significantly higher content of ROS than the groups treated with the same doses of MnCl₂ (all P<0.05) . Compared with the control group, the 0.25-0.5 mmol/L MnCl₂ andMPP⁺ treatment groups had significant increases in autophagy-related proteins LC3-II/LC3-I and significant reductions in P62 expression; the 0.125-0.5 mmol/L MPP⁺ treatment groups had significantly higher LC3-II/LC3-I than the groups treated with the same doses of MnCl₂, and the 0.125 and 0.25 mmol/L MPP ⁺ treatment groups had significantly lower P62 expression than the groups treated with the same doses of MnCl₂ (all P<0.05) . Conclusion Both MnCl₂ and MPP⁺ can induce oxidative stress and autophagy in SK-N-SH cells, and MPP⁺ has a significantly greater inductive effect on autophagy of SK-N-SH cells than MnCl₂.

Objective:To investigate the mechanism of polygonatum and astragalus compound(PA)in inhibiting the progression of lung adenocarcinoma. Methods:CCK-8 assay was used to assess the inhibitory rate of proliferation in A549 and H1299 cells treated with PA at different concentrations and to calculate the half maximal inhibitory concentration(IC50).C57BL/6 mice(KRASG12D/+;TP53flox/flox)were treated with adenovirus carrying Cre enzyme via nasal inhalation to establish a mouse model of primary lung adenocarcinoma.The model mice were fed with PA-containing diet to directly observe the effect of PA on the lung adenocarcinoma tissue.Immunohistochemical staining was used to examine the pathological status of the lung tissue.Bioinformatics analysis indicated that PA affects the progression of lung adenocarcinoma through the apelin-peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC1α)-mitochondrial brown fat uncoupling protein 1(UCP1).Real-time quantitative PCR and Western blotting analysis were used to study the effect of PA on the mRNA and protein expression levels of apelin-PGC1α-UCP1 signaling pathway related genes.An ATP detection kit and flow cytometry were used to evaluate the effect of PA on the ATP and mitochondrial ROS production,respectively,in A549 and H1299 cells.siUCP1 was used to silent the expression of UCP1 while Z160 was used to induce UCP1 overexpression in A549 and H1299 cells,and the changes in ATP and mitochondrial ROS production were examined to further investigate whether PA acts on apelin-PGC1α-UCP1 signaling pathway to affect the progression of lung adenocarcinoma. Results:PA could obviously inhibit the proliferation of A549 and H1299 cells with the IC50 values of 10.66 mg/mL for A549 cells and 9.66 mg/mL for H1299 cells.In the mouse primary lung adenocarcinoma model,PA could effectively inhibit the growth of tumor,downregulate apelin-PGC1α-UCP1 signaling pathway and inhibit the expression of lung adenocarcinoma-promoting gene UCP1.In A549 and H1299 cells,PA could significantly inhibit the expression of apelin,PGC1α and UCP1(P＜0.05),promote the production of ATP(P＜0.000 1)and ROS,restore mitochondrial oxidative phosphorylation,and inhibit aerobic glycolysis(P＜0.01).UCP1 silencing could increase the production of ATP(P＜0.01)and mitochondrial ROS and decrease the expression of key glycolysis enzymes hexokinase 2(HK2)and pyruvate kinase isozyme type M2(PKM2)(P＜0.05).Increasing the expression of UCP1 could reduce the ATP production(P＜0.01)and mitochondrial ROS generation in cells while increase the expression of HK2 and PKM2(P＜0.05).Treating cells with PA and Z160 simultaneously(PA+Z160)could reverse the inhibitory effect of PA on the ATP production and glycolysis of tumor cells(P＜0.05). Conclusion:PA can downregulate the apelin-PGC1α-UCP1 signaling pathway,inhibit mitochondrial uncoupling,restore mitochondrial oxidative phosphorylation,inhibit aerobic glycolysis,reverse the Warburg effect,and thus inhibit lung adenocarcinoma progression.