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. 2023 Oct 30;13(1):18586.
doi: 10.1038/s41598-023-45878-7.

Moschus ameliorates glutamate-induced cellular damage by regulating autophagy and apoptosis pathway

Danni Xie #  1 , Caiyou Song #  1 , Tao Qin  1 , Zhenwei Zhai  2 , Jie Cai  2 , Jingyi Dai  2 , Tao Sun  3   4 , Ying Xu  5
Affiliations

Moschus ameliorates glutamate-induced cellular damage by regulating autophagy and apoptosis pathway

Danni Xie et al. Sci Rep. .

Abstract

Alzheimer's disease (AD), a neurodegenerative disorder, causes short-term memory and cognition declines. It is estimated that one in three elderly people die from AD or other dementias. Chinese herbal medicine as a potential drug for treating AD has gained growing interest from many researchers. Moschus, a rare and valuable traditional Chinese animal medicine, was originally documented in Shennong Ben Cao Jing and recognized for its properties of reviving consciousness/resuscitation. Additionally, Moschus has the efficacy of "regulation of menstruation with blood activation, relief of swelling and pain" and is used for treating unconsciousness, stroke, coma, and cerebrovascular diseases. However, it is uncertain whether Moschus has any protective effect on AD patients. We explored whether Moschus could protect glutamate (Glu)-induced PC12 cells from cellular injury and preliminarily explored their related action mechanisms. The chemical compounds of Moschus were analyzed and identified by GC-MS. The Glu-induced differentiated PC12 cell model was thought to be the common AD cellular model. The study aims to preliminarily investigate the intervention effect of Moschus on Glu-induced PC12 cell damage as well as their related action mechanisms. Cell viability, lactate dehydrogenase (LDH), mitochondrial reactive oxygen species, mitochondrial membrane potential (MMP), cell apoptosis, autophagic vacuoles, autolysosomes or autophagosomes, proteins related to apoptosis, and the proteins related to autophagy were examined and analyzed. Seventeen active compounds of the Moschus sample were identified based on GC-MS analysis. In comparison to the control group, Glu stimulation increased cell viability loss, LDH release, mitochondrial damage, loss of MMP, apoptosis rate, and the number of cells containing autophagic vacuoles, and autolysosomes or autophagosomes, while these results were decreased after the pretreatment with Moschus and 3-methyladenine (3-MA). Furthermore, Glu stimulation significantly increased cleaved caspase-3, Beclin1, and LC3II protein expression, and reduced B-cell lymphoma 2/BAX ratio and p62 protein expression, but these results were reversed after pretreatment of Moschus and 3-MA. Moschus has protective activity in Glu-induced PC12 cell injury, and the potential mechanism might involve the regulation of autophagy and apoptosis. Our study may promote research on Moschus in the field of neurodegenerative diseases, and Moschus may be considered as a potential therapeutic agent for AD.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The effect of Moschus and Glu on cell viability in PC12 cells. (a) The effect of different concentrations of Moschus on cell viability in PC12 cells. (b) The effect of different concentrations of Glu on cell viability in PC12 cells. *p < 0.05 and **p < 0.01 versus the control group.
Figure 2
Figure 2
The effect of Moschus on cell viability and LDH of PC12 cells after Glu stimulation. (a) The effect of Moschus on Glu-induced cell viability in PC12 cells. (b) The effect of Moschus on Glu-induced LDH release in PC12 cells. *p < 0.05 and **p < 0.01 versus Glu-induced group.
Figure 3
Figure 3
Effects of Moschus on Glu-induced cell apoptosis in PC12 cells. (a) Apoptosis was analyzed by flow cytometry using the Annexin V/PI double staining method. (b) Apoptosis was analyzed by laser confocal scanning microscopy using the Annexin V/PI double staining method. ***p < 0.001 versus the Glu-induced group.
Figure 4
Figure 4
Effects of Moschus on Glu-induced loss of membrane potential in PC12 cells. *p < 0.05 and **p < 0.01 versus the Glu-induced group.
Figure 5
Figure 5
Effect of Moschus on Glu-induced mitochondrial ROS levels. *p < 0.05, **p < 0.01, and ***p < 0.001 versus Glu-induced group.
Figure 6
Figure 6
Effects of Moschus on autophagy-related proteins and Glu-induced autophagy. (a) Effects of Moschus on Glu-induced Bclin1, p62, and LC3 protein expression by western blot analysis. 1: Control; 2: 20 mM Glu; 3: 3-MA + Glu; 4: Moschus-0.05 mg/ml + Glu; 5: Moschus-0.1 mg/ml + Glu. (b) Immunofluorescence staining of LC3 was performed. (c) The autolysosomes or autophagosomes were monitored by TEM assay. Black arrows: autophagosomes/autolysosomes, scale bars: 5 μm at × 3000, 2 μm at ×7000, and 500 nm at ×20,000. (d) The fluorescent dyes of MDC detected the autophagic vacuole. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the Glu-induced group.
Figure 6
Figure 6
Effects of Moschus on autophagy-related proteins and Glu-induced autophagy. (a) Effects of Moschus on Glu-induced Bclin1, p62, and LC3 protein expression by western blot analysis. 1: Control; 2: 20 mM Glu; 3: 3-MA + Glu; 4: Moschus-0.05 mg/ml + Glu; 5: Moschus-0.1 mg/ml + Glu. (b) Immunofluorescence staining of LC3 was performed. (c) The autolysosomes or autophagosomes were monitored by TEM assay. Black arrows: autophagosomes/autolysosomes, scale bars: 5 μm at × 3000, 2 μm at ×7000, and 500 nm at ×20,000. (d) The fluorescent dyes of MDC detected the autophagic vacuole. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the Glu-induced group.
Figure 7
Figure 7
The effect of Moschus on apoptosis-related proteins. The cleaved caspase-3, BAX, and Bcl-2 protein expressions were measured by western blot analysis. 1: Control; 2: 20 mM Glu; 3: 3-MA + Glu; 4: Moschus-0.05 mg/ml + Glu; 5: Moschus-0.1 mg/ml + Glu. *p < 0.05 versus the Glu-induced group.
Figure 8
Figure 8
The potential mechanism between Glu and Moschus in PC12 cells. (A) The potential mechanism involved in Glu-induced injury. (B) The mechanisms involved in the protective effect of Moschus in Glu-induced injury.
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