S. choloroleuca, S. mirzayanii and S. santolinifolia protect PC12 cells from H(2)O (2)-induced apoptosis by blocking the intrinsic pathway

Abstract

Several studies have shown that neuronal cell death due to apoptosis is the major reason for cognitive decline in Alzheimer's disease. In this study, we report the anti-apoptotic effects of three Salvia species from Iran-S. choloroleuca, S. mirzayanii and S. santolinifolia-against H(2)O(2)-induced cytotoxicity in neuron-like PC12 cells. We showed that these antioxidant species could interfere with the intrinsic pathway of apoptosis by attenuating Bax/Bcl-2 ratio, decreasing outer mitochondrial membrane break and decreasing cytochrome c release to cytoplasm. Interestingly, we found that these species were able to replenish reduced glutathione level which affects cellular redox status and cytochrome c activity. Moreover, the decreased level of caspase-3, the executioner caspase, resulted in decrease of PARP-1 cleavage. Anti-apoptotic effects of these species along with their antioxidant effects, may represent a promising approach for treatment of neurodegenerative diseases.

Fig. 1

Effect of Salvia species on H₂O₂-induced disruption of neurite outgrowth in differentiated PC12 cells. a The criteria of PC12 differentiation is shown on two neurons (left image) of a sample image. The letter ‘P’ on right image indicates the primary neuritis of neuron 1. The two ways arrow shows the length of a neurite, extent elongated and membrane-enclosed protrusions of cytoplasm. The circle on the right image shows the cell body of neuron 1. Neurite width is not equal in all parts of neurons, thus the average neurite width must be calculated by dividing cell body area to average neurite length. The arrows show to bipolar cells. The letter ‘N’ indicates the nodes, the sites at which individual neurites branched or separate neurites contacted each other. b NGF-differentiated PC12 cells were pretreated with different concentrations of Salvia (10, 25, 50 and 100 μg/mL) in the presence of H₂O₂ (150 μM). Criteria were quantified at 24 h; c cell body area; d average neurite length; e average neurite width; f primary neurites per cell; g percent of bipolar cells and h the ratio of nodes to primary neurites. Each value indicates the mean ± SEM from three independent experiments and three replicates (n = 3). ^#P < 0.05; ^##P < 0.01 significantly different from control cells. *P < 0.05; **P < 0.01 significantly different from H₂O₂-treated cells

Fig. 1

Effect of Salvia species on H₂O₂-induced disruption of neurite outgrowth in differentiated PC12 cells. a The criteria of PC12 differentiation is shown on two neurons (left image) of a sample image. The letter ‘P’ on right image indicates the primary neuritis of neuron 1. The two ways arrow shows the length of a neurite, extent elongated and membrane-enclosed protrusions of cytoplasm. The circle on the right image shows the cell body of neuron 1. Neurite width is not equal in all parts of neurons, thus the average neurite width must be calculated by dividing cell body area to average neurite length. The arrows show to bipolar cells. The letter ‘N’ indicates the nodes, the sites at which individual neurites branched or separate neurites contacted each other. b NGF-differentiated PC12 cells were pretreated with different concentrations of Salvia (10, 25, 50 and 100 μg/mL) in the presence of H₂O₂ (150 μM). Criteria were quantified at 24 h; c cell body area; d average neurite length; e average neurite width; f primary neurites per cell; g percent of bipolar cells and h the ratio of nodes to primary neurites. Each value indicates the mean ± SEM from three independent experiments and three replicates (n = 3). ^#P < 0.05; ^##P < 0.01 significantly different from control cells. *P < 0.05; **P < 0.01 significantly different from H₂O₂-treated cells

Fig. 2

Analysis of apoptosis by using AO/EB double staining. a The cells were exposed to different concentrations of Salvia for 24 h followed by exposure to 150 μM of H₂O₂ for 24 h. The morphological patterns of apoptotic cells are described in the text. b The number of stained cells was counted in 10 randomly selected fields. Viability was calculated as the percentage of living cells in treated cultures compared to those in control cultures. *Significantly different from untreated cells. ^#Significantly different from H₂O₂-treated cells (** or ^##P < 0.01 and *** or ^###P < 0.001)

Fig. 3

Analysis of apoptosis by using Hoechst staining. The cells were exposed to different concentrations of Salvia for 24 h followed by exposure to 150 μM of H₂O₂ for 24 h. The morphological patterns of apoptotic cells are described in the text. All experiments were repeated three times and the number of stained cells was counted in 10 randomly selected fields

Fig. 4

Intracellular ROS levels in PC12 cells pretreated with Salvia species. The PC12 cells were incubated 24 h with or without S. choloroleuca, S. mirzayanii and S. santolinifolia (10, 25, 50 and 100 μg/mL) and then exposed to H₂O₂ (150 μM) for 24 h. Intracellular levels of ROS were measured by using DCFH-DA. The mean of three independent experiments is shown. *Significantly different from control cells. ^#Significantly different from H₂O₂-treated cells (* or ^#P < 0.05, ** or ^##P < 0.01)

Fig. 5

Bax and Bcl-2 levels in PC12 cells pretreated with Salvia species. a PC12 cells were pretreated with (10, 25, 50 and 100 μg/mL) of Salvia species, for 24 h and then exposed to H₂O₂ (150 μM) for 24 h. 20 μg proteins were separated on SDS-PAGE, Western blotted, probed with anti-Bax and/or anti-Bcl-2 antibodies and reprobed with anti-β-actin antibody (one representative Western blot is shown; n = 3). b The densities of the corresponding bands in the presence of S. choloroleuca, S. mirzayanii, S. santolinifolia, were measured and the ratio was calculated. The mean of three independent experiments is shown. *Significantly different from untreated cells. ^#Significantly different from H₂O₂-treated cells (* or ^#P < 0.05, ** or ^##P < 0.01)

Fig. 6

Effect of Salvia species on MMP level. PC12 cells were incubated 24 h with or without S. choloroleuca, S. mirzayanii and S. santolinifolia (10, 25, 50 and 100 μg/mL) and then exposed to H₂O₂ (150 μM) for 24 h. MMP level was measured with Rhodamine 123. The mean of three independent experiments is shown. *Significantly different from control cells. ^#Significantly different from H₂O₂-treated cells (* or ^#P < 0.05, ** or ^##P < 0.01)

Fig. 7

Cytochrome c levels in PC12 cells pretreated with Salvia species. a PC12 cells were pretreated with (10, 25, 50 and 100 μg/mL) of Salvia species for 24 h and then exposed to H₂O₂ (150 μM) for 24 h. Twenty μg proteins were separated on SDS-PAGE, western blotted, probed with anti- Cytochrome c antibody and reprobed with anti-β-actin antibody (one representative Western blot is shown; n = 3). b The densities of the corresponding bands in the presence of S. choloroleuca, S. mirzayanii, S. santolinifolia, were measured and the ratio was calculated. The mean of three independent experiments is shown. *Significantly different from untreated cells. ^#Significantly different from H₂O₂-treated cells (* or ^#P < 0.05, ** or ^##P < 0.01)

Fig. 8

Caspase-3 levels in PC12 cells pretreated with Salvia. Cells were pretreated with Salvia species (10, 25, 50 and 100 μg/mL) for 24 h and then exposed to H₂O₂ (150 μM) for 24 h. Twenty μg proteins were separated on SDS-PAGE, Western blotted, probed with anti-caspase antibody and reprobed with anti-β-actin antibody (one representative western blot is shown; n = 3). b The densities of the corresponding bands in the presence of S. choloroleuca, S. mirzayanii, S. santolinifolia, were measured and the ratio was calculated. The mean of three independent experiments is shown. *Significantly different from untreated cells. ^#Significantly different from H₂O₂-treated cells (* or ^#P < 0.05, ** or ^##P < 0.01)

Fig. 9

Western blot analysis to measure the effects of Salvia species on nuclear levels of PARP-1 in PC12 cells. The cells were pretreated with (10, 25, 50 and 100 μg/mL) of Salvia species for 24 h and then exposed to H₂O₂ (150 μM) for 24 h. a Twenty μg of nuclear extract was separated on SDS-PAGE, Western blotted, probed with anti-PARP antibody and reprobed with anti-β-actin antibody (one representative Western blot is shown; n = 3). The densities of PARP bands in the presence of bS. choloroleuca, cS. mirzayanii, dS. santolinifolia, were measured and the ratio was calculated. The mean of three independent experiments is shown. *Significantly different from untreated cells. ^#Significantly different from H₂O₂-treated cells (* or ^#P < 0.05, ** or ^##P < 0.01)