What happens when a traditional animal medicine meets a chemical model of Alzheimer’s-related cell injury? In this study, Moschus reduced several signs of glutamate damage in cultured PC12 cells. Alzheimer’s disease is a progressive disease that initiates with mild memory loss and may progress to full impairment of cognitive and executive functioning.

One in three elderly people dies from Alzheimer’s disease or another form of dementia. Glutamate is one of the primary excitatory neurotransmitters in the central nervous system, but excitatory neurotoxicity could emerge when glutamate levels are significantly enhanced.

Normal levels of glutamate play an essential role in learning and memory, while abnormally high glutamate levels could lead to over-excitation of the nerve cell, causing cell damage or death. Glutamate-induced cytotoxicity is associated with autophagic cell death, and inhibited autophagy attenuates glutamate-induced neuronal death.

The study found that glutamate toxicity to PC12 cells occurred dose-dependent, with the half-maximal inhibitory concentration appearing at twenty millimolar glutamate for twenty-four hours. Moschus is one of the rarest and most valuable animal medicines in traditional Chinese medicines, derived from dry secretions of mature male musk deer.

Modern pharmacology suggests that Moschus has neurological, cardio-cerebrovascular, anti-oxidant, anti-apoptotic, anti-inflammatory, and immune actions. However, the neuroprotective effect and the underlying neuroprotective mechanism of Moschus on glutamate-injured PC12 cells remain unclear.

Apoptosis is mediated by caspase proteases, while the antiapoptotic B-cell lymphoma 2 protein and the proapoptotic Bax protein are essential effectors of apoptosis. Autophagy is the process by which intracellular constituents are degraded in the lysosome, and it allows the cell to adapt to changing environmental conditions and eliminate damaged organelles.

Beclin 1, SQSTM1 or p62, and LC3 are markers of autophagy, with Beclin 1 serving as an essential protein for initiating autophagy. Three-methyladenine was commonly used as an autophagy inhibitor, preventing autophagy by inhibiting autophagosome formation at an early stage.

The PC12 cells were selected as the Alzheimer’s disease cellular model to investigate whether Moschus possesses protective capacity against glutamate-induced cell injury and to preliminarily explore the related action mechanisms. That gives the experiment a focused question: does Moschus protect this glutamate-injured cell model, while the researchers preliminarily investigate the related mechanisms behind any protective effect?

Moschus was weighed to fifty milligrams in a five-milliliter measuring flask, diluted with absolute ethanol to a ten-milligram-per-milliliter standard solution, and then filtered through a zero point two two micrometer membrane. The supernatant was analyzed by gas chromatography–mass spectrometry, and the sample ingredients were searched using the NIST fourteen point L library.

Highly differentiated PC12 cells were cultured in RPMI-1640 medium containing ten percent fetal bovine serum and antibiotics, in an incubator with a five-percent carbon dioxide atmosphere at thirty-seven degrees Celsius. PC12 cells could be passaged or seed-plated when eighty to ninety percent confluence was reached.

PC12 cells were treated with different concentrations of Moschus, from zero point zero five to zero point four milligrams per milliliter, and glutamate, from five to forty millimolar, for twenty-four hours. Based on concentration screening, cells were pretreated with zero point zero five or zero point one milligrams per milliliter Moschus, or five millimolar three-methyladenine, for twenty-four hours, then incubated with twenty millimolar glutamate for another twenty-four hours.

Cell viability was measured with the CCK-8 kit, absorbance was evaluated at four hundred fifty nanometers, and cell viability was calculated according to the kit instructions. The chemical components in the Moschus samples were determined by gas chromatography–mass spectrometry and searched in the NIST standard library.

Chromatographic peaks with a matching degree higher than eighty were selected and compared with relevant literature to confirm their chemical composition further. Seventeen chemical ingredients were identified by gas chromatography–mass spectrometry, and the result is shown in Table 1.

Table one lists seventeen chemical ingredients identified in the Moschus samples by gas chromatography–mass spectrometry, with retention time, molecular formula, CAS number, relative molecular mass, and library similarity. The authors report selecting chromatographic peaks with matching degrees above eighty through the NIST standard library, then consulting relevant literature to support identification.

This chemical profile matters because it defines the composition of the Moschus solutions used in the subsequent cellular experiments. The screening drug concentrations of Moschus and glutamate were evaluated using the CCK-8 kit. The half-maximal inhibitory concentration of Moschus was zero point two eight eight five milligrams per milliliter; cell viability at zero point zero five and zero point one milligrams per milliliter had no significant difference, while viability at zero point two, zero point three, and zero point four milligrams per milliliter was significantly decreased.

Figure one uses a CCK-eight assay to screen Moschus and glutamate concentrations by measuring PC12 cell viability relative to control. In panel A, the Moschus IC fifty is reported as zero point two eight eight five milligrams per milliliter, with significant decreases at zero point two, zero point three, and zero point four milligrams per milliliter.

Panel B reports a glutamate IC fifty of fifteen point eighty-one millimolar and shows concentration-associated viability loss, supporting the selection of zero point zero five and zero point one milligrams per milliliter Moschus for subsequent protection experiments.

Cell viability dramatically decreased after glutamate stimulation compared with the control group, while pretreatment with Moschus and three-methyladenine notably increased cell viability. LDH release increased by one point three seven three-fold after glutamate stimulation compared with the control group, while pretreatment with Moschus and three-methyladenine notably reduced LDH release.

These results showed that Moschus might be protective against glutamate-induced cellular injury in PC12 cells. Figure two compares PC12 cells across control, glutamate-stimulated, and pretreatment conditions with Moschus or three-MA. In panel A, glutamate stimulation is associated with reduced cell viability, while Moschus and three-MA pretreatment are associated with increased viability relative to the glutamate group.

Panel B shows glutamate-associated LDH release, a marker of cellular membrane damage, with significance markers indicating differences versus the glutamate-induced group. Flow cytometry was used to measure the apoptotic rate of PC12 cells and explore whether Moschus affects glutamate-induced cell apoptosis.

The early apoptosis rate markedly increased after glutamate stimulation compared with the control group, while pretreatment with Moschus and three-methyladenine significantly decreased the early apoptosis rate. Annexin V and PI fluorescence were most apparent after glutamate stimulation and were inhibited after pretreatment with Moschus and three-methyladenine.

These results indicated that Moschus might protect PC12 cells from glutamate-induced cell apoptosis. Figure three examines apoptosis in PC12 cells using Annexin V and PI staining, measured by flow cytometry in panel a and confocal microscopy in panel b.

Glutamate stimulation produces the strongest apoptotic signal, while pretreatment with three-MA or Moschus is associated with reduced early-apoptosis percentages and weaker fluorescence signals. The figure matters because it provides complementary quantitative and imaging evidence that Moschus modulates glutamate-induced cell death.

Damaged mitochondria generate excess reactive oxygen species, leading to a reduction of mitochondrial membrane potential. In glutamate-stimulated PC12 cells, the mitochondrial reactive oxygen species peak moved significantly to the right, indicating increased generation, while Moschus and three-methyladenine shifted the peak to the left.

The result showed that Moschus protects PC12 cells against glutamate-induced cell injury by inhibiting the accumulation of mitochondrial reactive oxygen species. Figure five measures mitochondrial ROS with MitoSOX Red in PC12 cells. The flow-cytometry distributions and fluorescence-intensity bars show a Glu-associated increase, while three-MA and Moschus treatments are accompanied by left-shifted peaks and reduced fluorescence relative to the Glu-induced group, with significance markers ranging from one to three asterisks.

This matters because excessive mitochondrial ROS is linked to mitochondrial dysfunction and can contribute to apoptosis. LC3, Beclin 1, and p62 are considered indicators of autophagy. Glutamate stimulation increased Beclin 1 and LC3 two protein expression but reduced p62 protein expression, while pretreatment with Moschus and three-methyladenine reduced Beclin 1 and LC3 two expression and increased p62 expression.

The number of LC3-positive vesicles increased significantly in the glutamate-stimulated group and decreased after pretreatment with three-methyladenine and Moschus. Figure six tracks Glu-induced autophagy using protein blots and LC3 immunofluorescence.

Glu increases LC3 and Beclin one signals while reducing p62, and both three-MA and Moschus alter these markers toward the control pattern. The reduced LC3 staining after Moschus pretreatment supports the same interpretation, suggesting that Moschus may lessen Glu-associated cellular injury by regulating autophagy.

BAX is used as a pro-apoptotic protein to participate in apoptosis, and cleaved caspase-3 is considered an early apoptotic marker. Glu stimulation increased cleaved caspase-3 protein expression and decreased the Bcl-2-to-BAX ratio, while pretreatment with Moschus and three-methyladenine reduced cleaved caspase-3 expression and increased the Bcl-2-to-BAX ratio.

The data demonstrated that Moschus might protect PC12 cells against glutamate-induced injury through regulating the apoptosis pathway. Panels c and d assess autophagy in PC12 cells using transmission electron microscopy and MDC fluorescence. After twenty millimolar Glu stimulation, the electron micrographs show autolysosomes or autophagosomes, including double-membrane vesicles, while the MDC images show intracellular fluorescent signal; pretreatment with three-MA or Moschus is accompanied by fewer visible autophagic structures and reduced MDC fluorescence.

These complementary readouts support the authors’ conclusion that Moschus may alleviate Glu-induced autophagic impairment. Figure seven measures apoptosis-related proteins using western blots across control, glutamate-induced, three-MA, and two Moschus treatment groups.

It reports cleaved caspase-three, BAX, and Bcl-two, with GAPDH or beta-actin as loading controls; asterisks indicate p less than zero point zero five versus the glutamate-induced group. Because cleaved caspase-three and BAX mark pro-apoptotic signaling, while Bcl-two is anti-apoptotic, these results support the authors’ investigation of whether Moschus’s neuroprotective effect involves apoptosis regulation.

Previous studies suggest that autophagy could participate in the pathogenesis of Alzheimer’s disease, and various stress pathways could elicit autophagy and apoptosis. Unlike apoptosis, autophagy provides energy and nutrients to promote cell survival by degrading cytoplasmic components in lysosomes.

The potential mechanism of the effect of Moschus and glutamate in PC12 cells is shown in Figure 8. Figure eight presents a proposed mechanism for glutamate, or Glu, injury and Moschus protection in PC12 cells. Panel A links Glu exposure to mitochondrial changes, reactive oxygen species, DNA damage, apoptosis-related signaling, and autophagy markers.

Panel B shows how Moschus solution is proposed to modify these pathways, including mitochondrial stress, Bcl-two and Beclin-one signaling, caspase activation, apoptosis, and autophagosome formation. The figure matters because it integrates the study’s cellular observations into a mechanistic model rather than presenting an isolated pathway.

Modern pharmacology suggests that Moschus possesses neuroprotective, anti-apoptotic, anti-oxidant, and immunity-enhancing biological activities. Seventeen chemical compounds from the Moschus sample were identified through gas chromatography–mass spectrometry analysis.

Muscone, Prasterone-3-sulfate, Cholesta-3,5-diene, three alpha-hydroxy-five beta-androstan-seventeen-one, and Androstane-three-seventeen-dione, five beta, possess protective effects on the central nervous system. Muscone dramatically alleviated glutamate-induced apoptosis and oxidative stress in PC12 cells.

In summary, the study manifested that Moschus might possess a neuroprotective effect in glutamate-induced neurotoxicity. In-depth studies on the multiple components of Moschus might provide a potential target for preventing and treating Alzheimer’s disease. In this cell model, Moschus reduced glutamate-associated injury, mitochondrial stress, apoptosis, and autophagy-related changes.

The findings support further research, but they do not yet establish a treatment for Alzheimer’s disease.