Allicin Attenuated Advanced Oxidation Protein Product-Induced Oxidative Stress and Mitochondrial Apoptosis in Human Nucleus Pulposus Cells

Abstract

Intervertebral disc degeneration (IDD) is one of the most common chronic degenerative musculoskeletal disorders. Oxidative stress-induced apoptosis of the nucleus pulposus (NP) cells plays a key role during IDD progression. Advanced oxidation protein products (AOPP), novel biomarkers of oxidative stress, have been reported to function in various diseases due to their potential for disrupting the redox balance. The current study is aimed at investigating the function of AOPP in the oxidative stress-induced apoptosis of human NP cells and the alleviative effects of allicin during this process which was known for its antioxidant properties. AOPP were demonstrated to hamper the viability and proliferation of NP cells in a time- and concentration-dependent manner and cause cell apoptosis markedly. High levels of reactive oxygen species (ROS) and lipid peroxidation product malondialdehyde (MDA) were detected in NP cells after AOPP stimulation, which resulted in depolarized mitochondrial transmembrane potential (MTP). Correspondingly, higher levels of AOPP were discovered in the human degenerative intervertebral discs (IVD). It was also found that allicin could protect NP cells against AOPP-mediated oxidative stress and mitochondrial dysfunction via suppressing the p38-MAPK pathway. These results disclosed a significant role of AOPP in the oxidative stress-induced apoptosis of NP cells, which could be involved in the primary pathogenesis of IDD. It was also revealed that allicin could be a promising therapeutic approach against AOPP-mediated oxidative stress during IDD progression.

Figure 1

AOPP treatment inhibited human NP cell viability and proliferation in a dose- and time-dependent manner. (a) The human NP cells were treated with AOPP (0, 100, 200, and 400 μg/ml) for 24 h, and 0 μg/ml group served as a control. The cell viability of each group was detected by a CCK-8 assay. (b) The human NP cells were treated with AOPP (0-400 μg/ml) for 24 h, and 0 μg/ml group served as a control. The cell proliferation was determined using EdU staining combined with DAPI staining for the nuclei under fluorescence microscope, with the EdU positive cells quantitated. Original magnification: ×200. (c) The human NP cells were treated with 400 μg/ml AOPP for 0 h, 2 h, 6 h, 12 h, and 24 h, and 0 h group served as a control. The cell viability of each group was detected by a CCK-8 assay. (d) The human NP cells were treated with 400 μg/ml AOPP for 0 h, 2 h, 6 h, 12 h, and 24 h, and 0 h group served as a control. The cell proliferation was determined using EdU staining combined with DAPI staining for the nuclei under fluorescence microscope, with the EdU positive cells quantitated. Original magnification: ×200. Data were represented as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01 versus the control group, n = 3.

Figure 2

Effects of allicin on the human NP cell viability and proliferation. (a) The human NP cells were treated with allicin (0, 5, 10, 20, and 40 μM) for 24 h, and 0 μM group served as a control. The cell viability of each group was detected by a CCK-8 assay. ^∗p < 0.05 versus the control group, n = 3. (b) The human NP cells were treated with allicin (0-40 μM) for 24 h, and 0 μM group served as a control. The cell proliferation was determined using EdU staining combined with DAPI staining for the nuclei under fluorescence microscope, with the EdU positive cells quantitated. Original magnification: ×200. ^∗p < 0.05 versus the control group, n = 3. (c) The human NP cells pretreated by allicin (0, 5, 10, and 20 μM) were treated with 400 μg/ml AOPP, and the cell viability of each group was examined by the CCK-8 assay. ^#p < 0.05 versus the control group, ^∗p < 0.05 versus the AOPP alone treatment group, n = 3. (d) The cell proliferation of each group was determined using EdU staining under fluorescence microscope, with the EdU positive cells quantitated. Original magnification: ×200. Data were represented as mean ± SD. ^##p < 0.01 versus the control group, ^∗p < 0.05 versus the AOPP alone treatment group, n = 3.

Figure 3

Allicin treatment alleviated AOPP-induced human NP cell apoptosis. (a) The human NP cells were pretreated with various concentrations of allicin (0, 5, 10, and 20 μM) for 2 h, followed by stimulation with 400 μg/ml AOPP for 24 h. And the rate of cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI dual staining. The proportion of apoptotic cells in the first and fourth quadrant was measured for analysis. (b–h) The protein levels of Bax, Bcl-2, c-caspase 3, c-caspase 9, mitochondrial Cyt-c, and cytoplasmic Cyt-c were determined using Western blotting analysis (b) and quantified in (c–h). (i) Representative images of immunofluorescence staining for cleaved caspase-3 in each group, with the relative fluorescence intensity quantified. Original magnification: ×400. Data were represented as mean ± SD. ^##p < 0.01 versus the control group; ^∗p < 0.05 and ^∗∗p < 0.01 versus the AOPP alone treatment group, n = 3.

Figure 4

Allicin treatment inhibited AOPP-induced oxidative stress and mitochondrial dysfunction of human NP cells. (a, b) The human NP cells were pretreated with different concentrations of allicin (0, 5, 10, and 20 μM) for 2 h, before treatment with 400 μg/ml AOPP for 24 h. The intracellular ROS levels of the NP cells for each group were detected by ROS-specific fluorescent probe DHE and measured by subsequent flow cytometry analysis. Representative peak charts of flow cytometry and relative quantitative analysis were shown. (c) The intracellular MDA levels (as a marker of lipid peroxidation) of human NP cells were examined by a commercial kit. (d, e) The mitochondrial membrane potential of human NP cells in each group was examined by JC-1 staining and measured by subsequent flow cytometry analysis. The quantitative analysis of the ratio of red fluorescence (y axis) to green fluorescence (x axis) and representative scatter plots of flow cytometry were shown. Data were represented as mean ± SD. ^#p < 0.05 and ^##p < 0.01 versus the control group; ^∗p < 0.05 and ^∗∗p < 0.01 versus the AOPP alone treatment group, n = 3.

Figure 5

Effects of allicin on AOPP-induced MAPK pathway activation. (a) The human NP cells were pretreated with various concentrations of allicin (0, 5, 10, and 20 μM) for 2 h, followed by stimulation with 400 μg/ml AOPP for 24 h. The protein levels of ERK, phosphorylated ERK, p38, phosphorylated p38, JNK, and phosphorylated JNK were determined using Western blotting analysis. (b–d) Immunoblot bands corresponded to (b) p-ERK, (c) p-p38, and (d) p-JNK were quantified by densitometric analysis and normalized to their corresponding total kinase. Data were represented as mean ± SD. ^##p < 0.01 versus the control group; ^∗p < 0.05 and ^∗∗p < 0.01 versus the AOPP alone treatment group, n = 3.

Figure 6

Allicin alleviated AOPP-induced oxidative stress and mitochondrial dysfunction via p38-MAPK pathway in human NP cells. (a) The human NP cells were pretreated with allicin (10 μM) or p38-MAPK inhibitor SB202190 (10 μM) for 2 h, then treated with AOPP (400 μg/ml) for 24 h. The cell proliferation was determined using EdU staining combined with DAPI staining for the nuclei under fluorescence microscope, with the EdU positive cells quantitated. Original magnification: ×200. (b) Representative images of immunofluorescence staining for cleaved caspase-3 in each group, with the relative fluorescence intensity quantified. Original magnification: ×400. (c) The intracellular ROS levels of the NP cells for each group were detected by ROS-specific fluorescent probe DHE and measured by subsequent flow cytometry analysis. (d) The mitochondrial membrane potential of human NP cells in each group was examined by JC-1 staining and measured by subsequent flow cytometry analysis. Representative scatter plots of flow cytometry and the quantitative analysis of red fluorescence to green fluorescence ratio were shown. Data were represented as mean ± SD. ^#p < 0.05 and ^##p < 0.01 versus the control group; ^∗p < 0.05 and ^∗∗p < 0.01 versus the AOPP alone treatment group, n = 3.

Figure 7

Allicin alleviated AOPP-induced oxidative stress and mitochondrial dysfunction via p38-MAPK pathway in human NP cells. (a) The human NP cells were pretreated with allicin (10 μM) or allicin (10 μM) in combination with p38-MAPK activator Dehydrocorydaline chloride (Dc, 500 nM) for 2 h, then treated with AOPP (400 μg/ml) for 24 h. The cell proliferation was determined by EdU staining combined with DAPI staining for the nuclei under fluorescence microscope, with the EdU positive cells quantitated. Original magnification: ×200. (b) The cell apoptosis rate was detected by flow cytometry with Annexin V-FITC/PI dual staining. The proportion of apoptotic cells in the first and fourth quadrant was measured for analysis. (c) The intracellular ROS levels for each group were detected by ROS-specific fluorescent probe DHE and measured by subsequent flow cytometry analysis. (d) The mitochondrial membrane potential of human NP cells in each group was examined by JC-1 staining and measured by subsequent flow cytometry analysis. Data were represented as mean ± SD. ^##p < 0.01 versus the control group, ^∗p < 0.05 and ^∗∗p < 0.01 versus the AOPP alone treatment group, and ^&p < 0.05 and ^&&p < 0.01 versus the AOPP+allicin treatment group, n = 3.