Di-Huang-Yi-Zhi herbal formula attenuates amyloid-β-induced neurotoxicity in PC12 cells

Abstract

Traditional Chinese medicine can be used for Alzheimer's disease management, such as the modern herbal formula Di-Huang-Yi-Zhi (DHYZ). In the present study, neuronal differentiated PC12 cells were used as a model to evaluate the effects of DHYZ against amyloid-β peptide 25-35 (Aβ_25-35) induced neurotoxicity, particularly regarding cell proliferation, apoptosis and related events. Following treatment with DHYZ, cell viability, cell membrane damage, apoptosis, mitochondrial membrane potential, cytochrome c release, caspase-3 activity and levels of reactive oxygen species in PC12 cells were detected. The results demonstrated that pretreatment with DHYZ significantly protected PC12 cells from Aβ_25-35-induced proliferation inhibition, lactate dehydrogenase release and apoptosis, as well as upregulating mitochondrial membrane potential and downregulating cytochrome c release and caspase-3 activation. DHYZ also inhibited the Aβ_25-35-induced reactive oxygen species generation in PC12 cells. These observations suggest that DHYZ protected PC12 cells from the Aβ-induced neurotoxicity.

Keywords: Di-Huang-Yi-Zhi; PC12 cells; amyloid-β; apoptosis; mitochondria; reactive oxygen species.

Figure 1.

Effect of DHYZ on the cell viability in Aβ-treated PC12 cells. Differentiated PC12 cells were pretreated with different concentrations of DHYZ for 1 h, followed by Aβ_25–35 treatment for 24 h. Cell viability was evaluated by CCK-8 assay. Data shown are representative of three independent experiments. ^#P<0.01, vs. non-treated group; **P<0.01 vs. Aβ_25–35 group. DHYZ, Di-Huang-Yi-Zhi; Aβ, amyloid-β; CCK-8, cell counting kit-8.

Figure 2.

Effect of DHYZ on the LDH release in Aβ-treated PC12 cells. Differentiated PC12 cells were pretreated with different concentrations of DHYZ for 1 h, followed by Aβ_25–35 treatment for 24 h. LDH release was determined by an LDH Cytotoxicity Assay kit, and is expressed as the fold of the control. Data are from three independent experiments. ^#P<0.01 vs. non-treated group; *P<0.05 and **P<0.01 vs. Aβ_25–35 group. DHYZ, Di-Huang-Yi-Zhi; Aβ, amyloid-β; LDH, lactate dehydrogenase.

Figure 3.

Effect of DHYZ on Aβ-induced apoptosis in PC12 cells. Differentiated PC12 cells were pretreated with different concentrations of DHYZ for 1 h, followed by Aβ_25–35 treatment for 24 h. Cell death was detected by ELISA. Results are expressed the as fold of the non-treated control, and were obtained from three separate experiments. ^#P<0.01 vs. non-treated group; *P<0.05 and **P<0.01, vs. Aβ_25–35 group. DHYZ, Di-Huang-Yi-Zhi; Aβ, amyloid-β.

Figure 4.

Effect of DHYZ on Aβ-induced MMP change in PC12 cells. Differentiated PC12 cells were pretreated with different concentrations of DHYZ for 1 h, followed by Aβ_25–35 treatment for 24 h. MMP was detected by JC-1 staining, and observed under a fluorescence microscope (magnification, ×200). DHYZ, Di-Huang-Yi-Zhi; Aβ, amyloid-β; MMP, mitochondrial membrane potential.

Figure 5.

Effect of DHYZ on Aβ-elicited Cyt-C release in PC12 cells. Differentiated PC12 cells were pre-incubated with different concentrations of DHYZ for 1 h, followed by Aβ_25–35 treatment for 24 h. Cyt-C concentration in the cytosol was determined by ELISA. Data are presented from three separate experiments. ^#P<0.01 vs. non-treated group; **P<0.01 vs. Aβ_25–35 group. DHYZ, Di-Huang-Yi-Zhi; Aβ, amyloid-β; Cyt-C, cytochrome c.

Figure 6.

Effect of DHYZ on caspase-3 activity in Aβ_25–35-treated PC12 cells. Differentiated PC12 cells were pretreated with different concentrations of DHYZ for 1 h, followed by Aβ_25–35 treatment for 24 h. Caspase-3 activity was detected using a kit, and is expressed as the fold of the control. Data shown are representative of three independent experiments. ^#P<0.01 vs. non-treated group; **P<0.01 vs. Aβ_25–35 group. DHYZ, Di-Huang-Yi-Zhi; Aβ, amyloid-β.

Figure 7.

Effect of DHYZ on Aβ_25–35-induced ROS generation in PC12 cells. Differentiated PC12 cells were pretreated with different concentrations of DHYZ for 1 h, followed by Aβ25–35 treatment for 24 h. The production of intracellular ROS was detected by DCFH-DA staining and quantified with a fluorescence microplate reader (excitation wavelength, 488 nm; emission wavelength, 525 nm). ROS production was expressed as the fold of the control group. Data shown are representative of three independent experiments. ^#P<0.01 vs. non-treated group; *P<0.05 and **P<0.01, vs. Aβ_25–35 group. DHYZ, Di-Huang-Yi-Zhi; Aβ, amyloid-β; ROS, reactive oxygen species; DCFH-DA, 2′,7′-dichlorofluorescin diacetate.