The Combination of Salidroside and Hedysari Radix Polysaccharide Inhibits Mitochondrial Damage and Apoptosis via the PKC/ERK Pathway

Background Beta-amyloid (Aβ) peptide is a widely recognized pathological marker of Alzheimer's disease (AD). Salidroside and Hedysari Radix polysaccharide (HRP) were extracted from Chinese herb medicine Rhodiola rosea L and Hedysarum polybotrys Hand-Mazz, respectively. The neuroprotective effects and mechanisms of the combination of salidroside and Hedysari Radix polysaccharide (CSH) against Aβ25–35 induced neurotoxicity remain unclear. Objective This study aims to investigate the neuroprotective effects and pharmacological mechanisms of CSH on Aβ25–35-induced HT22 cells. Materials and Methods HT22 cells were pretreated with various concentrations of salidroside or HRP for 24 h, followed by exposed to 20 μm Aβ25–35 in the presence of salidroside or RHP for another 24 h. In a CSH protective assay, HT22 cells were pretreated with 40 μm salidroside and 20 μg/mL HRP for 24 h. The cell viability assay, cell morphology observation, determination of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and cell apoptosis rate were performed. The mRNA expression of protein kinase C-beta (PKCβ), Bax, and Bcl-2 were measured by qRT-PCR. The protein expression levels of cleaved caspase-3, Cyt-C, PKCβ, phospho-ERK1/2, Bax, and Bcl-2 were measured by Western blot. Results CSH treatment increased cell viability, MMP, and decreased ROS generation in Aβ25–35-induced HT22 cells. PKCβ and Bcl-2 mRNA expression were elevated by CSH while Bax was decreased. CSH increased the protein expression levels of PKCβ, Bcl-2, and phospho-ERK1/2, and decreased those of Bax, Cyt-C, and cleaved caspase-3. Conclusions CSH treatment have protective effects against Aβ25–35-induced cytotoxicity through decreasing ROS levels, increasing MMP, inhibiting early apoptosis, and regulating PKC/ERK pathway in HT22 cells. CSH may be a potential therapeutic agent for treating or preventing neurodegenerative diseases.


Introduction
Alzheimer's disease (AD) has an increasing number of patients, especially the elderly. Nowadays, AD affects approximately 40 million people and is expected to impact 135 million people by 2050 [1]. AD has imposed a heavy economic burden, which costs 0.65% of global gross domestic product (GDP) [2]. e clinical manifestations of AD include short-term memory difficulties, abnormal behavioral function (language expression, visuospatial processing, and execution), and personality changes. e neuropathologies of AD are characterized by amyloid-β (Aβ) deposition, neurofibrillary tangles (NFTs), which cause neuronal dysfunction, synapses loss in the hippocampus, and temporal cortex.
Neuronal apoptosis has been postulated as a possible explanation for the etiology of AD. Neurons displayed apoptotic characteristics, including apoptotic mitochondrial changes during AD progression [3]. Alteration in apoptosisrelated factors may result in neurodegenerative diseases [4]. HT22 mouse hippocampal neuronal cells were usually used for underlying therapeutic mechanisms of neurodegenerative illness in place of primary neuronal cultures, and better mimic the pathological changes of AD neurons [5,6]. erefore, Aβ 25-35 -induced HT22 cells were used to explore the neuroprotective effects in this study [7].
Although there are many methods for treating AD [8], natural products are still an effective alternative treatment [9]. Salidroside extracted from the traditional Chinese medicine (TCM) Rhodiola rosea L. It has been shown that salidroside protects PC12 cells against the toxicity and apoptosis caused by Aβ 1-42 through activating AKT and ERK1/ 2 pathways [10]. Hedysari Radix polysaccharide (HRP) is the major bioactive component derived from Hedysarum polybotrys Hand-Mazz. Studies have found that HRP attenuate Aβ-induced cell injury and improve the learning and memory in AD rats [11]. However, the neuroprotective effects and pharmacological mechanisms of the combination of salidroside and Hedysari Radix polysaccharide (CSH) against AD are unclear.

Measurement of ROS Generation.
e reduction of oxidative stresses could reduce the onset of AD [13]. HT22 cells were seeded into 6-well plates and cultured as previously described. Cells were incubated at 37°C with 10 μm DCFHDA for 30 min and washed 3 times. e level of ROS was detected by a fluorescence microscope (Olympus IX 53, Tokyo, Japan).

Determination of Mitochondrial Membrane Potential (MMP).
e decreased MMP is a characteristic in the early stage of cell apoptosis [14]. e Aβ-mediated cytotoxicity and CSH intervention on MMP in HT22 cells were evaluated by JC-1 fluorescence. HT22 cells were cultured as previously described and stained with 10 μg/mL JC-1 for 20 min at 37°C, after washed 3 times, a fluorescence microscope was used to observe the changes of MMP. With a typical negative membrane potential, JC-1 dye diffused into cells and entered mitochondria. e dye was subsequently collected in healthy mitochondria and fluoresces red. JC-1 remained distributed in the cell as a monomer and fluoresced green if MMP was disturbed.

Cell Apoptotic Morphological
Assay. HT22 cells were cultured as previously described. Hoechst 33342 staining solution was made in accordance with the manufacturer's specified methodology, and 5 μL Hoechst 33342 was added to 1 mL staining buffer and mixed well. 500 μL Hoechst 33342 was incubated and protected from light. Cell apoptotic morphology was observed by a fluorescence microscope. Evidence-Based Complementary and Alternative Medicine 2.8. Detection of the Cell Apoptosis Rate. Neuronal cell injury and loss contribute to Aβ-induced apoptosis [15]. erefore, cellular apoptosis was analyzed to confirm that CSH had a neuroprotective effect to antagonize Aβ neurotoxicity. HT22 cells were cultured as previously described and seeded in 6-well plates. Cells were harvested and resuspended in 100 μL of binding buffer. Samples were incubated with 5 μL Annexin-V-FITC and 5 μL PI for 15 min at room temperature. 400 μL of binding buffer was added, gently mixed. Samples were analyzed with flow cytometry (Beckman Coulter Cytoflex, USA).

Quantitative Real-Time PCR (qRT-PCR).
Total RNA was extracted using Trizol reagent according to the manufacturer's instructions. Total RNA was reverse transcribed to cDNA and amplified and analyzed using the ChamQ SYBR ® qPCR Master Mix kit and a LightCycler96 PCR instrument (Roche, Mannheim, Germany). e mRNA expression of protein kinase C-beta (PKCβ), Bcl-2, and Bax were analyzed using the 2 −ΔΔCt method and β-actin was set as internal reference. e primer sequences used are shown in Table 1.
2.11. Statistical Analysis. All statistical analyses were performed by SPSS 24.0. Results are expressed as mean-± standard deviation (SD). Comparisons among groups were analyzed using one-way analysis of variance (ANOVA) with the Tukey's posthoc multiple comparisons test. P < 0.05 was considered statistically significant.

CSH Attenuated Aβ 25-35 -Induced Cytotoxicity in HT22
Cells. HT22 cells were exposed to different concentrations  . e cell viability of CSH treatment was higher than that of 40 μm salidroside treatment or 20 μg/mL HRP treatment (P < 0.05). e results indicated that CSH attenuate Aβ 25-35 -induced cytotoxicity in HT22 cells.
e results suggested that CSH protects Aβ 25-35 -induced cell morphology damage.

CSH Decreased ROS Production and Inhibited Cell Early
Apoptosis. ROS production was increased in the Aβ 25-35 group compared to the control group (P < 0.01). e levels of ROS were decreased after CSH treatment (Figures 3(a) and 3(b)). ese results suggested that CSH could significantly protect HT22 cells from oxidative damage. e cell apoptotic morphology of HT22 cells identified by Hoechst 33342 staining are shown in Figure 3(c). e control group showed light blue staining. Compared to the control group, cells in the Aβ 25-35 group showed shrinking nuclei and bright blue staining (P < 0.01). Compared to the Aβ 25-35 group, bright blue cells decreased in the CSH group (P < 0.01). As shown in Figure 3(d), the apoptotic rate was analyzed by flow cytometry. e apoptosis rate was the sum of early and late apoptosis. Compared to the Aβ 25-35 group, the early apoptosis rate was significantly decreased in the CSH group (P < 0.01) (Figure 3(e)). ere were no significant differences in the late apoptosis rate between the CSH and Aβ [25][26][27][28][29][30][31][32][33][34][35] groups. e percentage of apoptotic cells was scored and depicted graphically as shown in Figure 3  e results showed that the mitochondrial function of HT22 cells was significantly damaged by Aβ 25-35 but restored by CSH. CSH inhibited Aβ 25-35 -induced apoptosis and exert a protective effect via maintaining high MMP.

CSH Regulated the mRNA Levels of PKCβ, Bcl-2, and Bax.
e mRNA levels of PKCβ, Bcl-2, and Bax in HT22 cells were determined by a qRT-PCR assay. Compared with the control group, the Bax mRNA level was increased in the Aβ 25-35 group, but decreased in the CSH group (P < 0.05). In the Aβ 25-35 group, PKCβ and Bcl-2 mRNA levels were decreased while they were higher in the CSH group (P < 0.05) (Figure 5(a)). ese results indicated that CSH can inhibit apoptosis primarily through the intrinsic-apoptotic pathway in Aβ 25-35 -induced HT22 cells.

Discussion
AD is mediated by multiple factors, with many theories of pathogenesis remaining unconfirmed [16]. Formed by aggregation of Aβ monomer and hyperphosphorylation of microtubule-associated protein Tau (p-Tau), Aβ plaques and neurofibrillary tangles (NFTs) were considered to be critical biomarkers in AD brains [17]. Apoptosis is a crucial point in Aβ-induced cytotoxicity, which includes promoting mitochondrial division, increasing intracellular ROS levels, and disrupting MMP [18]. us, the alleviation of Aβ-induced neuronal apoptosis seems to be a potentially curative treatment for AD [19].
is study showed that CSH Deeply integrating theory and practice, TCM has shown promising efficacy in miscellaneous disorders for over 2,000 years [20], which has opened up new avenues to discover effective drugs and compounds for neurodegeneration, certainly including AD [21][22][23]. Salidroside, the main active ingredient in Rhodiola rosea L, was thought to help with mental and behavioral diseases when treated before the experimental injury [24]. Previous studies showed that salidroside contributed to relieving oxidative damage, decreasing TNF-α, IL-6 expression, and reducing hippocampus neuronal apoptotic rates in the AD mice model [25]. Salidroside had protective effects and alleviated PC12 cells damage induced by Aβ 1-40 through the NAMPT signaling pathway [26]. Salidroside decreased the deposition of Aβ, and attenuated Aβ-mediated neurotoxicity by upregulating the PI3K/Akt/mTOR pathway in APP/PS1 mice [27,28].
ese results were consistent with the apoptotic effects of   Figure 6.
Neuronal loss and apoptosis were considered major mechanisms of cell death, which had an important implication on neurodegenerative diseases, notably AD [38,39]. Apoptosis usually shows typical morphological features, chromatin condensation, such as nuclei shrinkage, nuclei fragmentation, and apoptosis body formation [40]. Both Bax and Bcl-2 are pivotal regulators in the intrinsic apoptosis pathway [41]. Bax could influence mitochondria-mediated cell death by three mechanisms. e first is to cleave Bax by cysteine protease [42]. e second was aided by an increased Bax/Bcl-2 ratio. e mitochondrial apoptotic pathway is driven by inhibitory interaction between Bax and Bcl-2 family [43]. e third way is related to translocated Bax to mitochondria so that mitochondrial permeability could be enhanced before apoptosis.
Similar to Bax, Bcl-2 regulates cell apoptosis via its activation in mitochondria and regulates the beginning of apoptosis [44]. Recent research showed that this antiapoptosis function was associated with the regulation of intracellular Ca 2+ signaling. Aberrant Ca 2+ signaling resulted in the dysregulated Bcl-2-Ca 2+ signaling axis, ultimately accelerating AD pathology [45]. Ca 2+ dysregulation and activated p38K caused increased ROS and decreased Bcl-2 levels in AD patients, leading to apoptosis [46]. In the present study, the mRNA and protein levels of Bax and Bcl-2 after CSH treatment were used as an assessment to detect Aβ 25-35 -induced HT22 cells apoptosis. CSH significantly decreased the protein expression and mRNA levels of Bcl-2 while increased that of Bcl-2 in Aβ 25-35 -induced HT22 cells.
PKCβ belongs to the kinase C family and can negatively regulate the mitochondrial energy. Contributing significantly to cell proliferation, differentiation, and apoptosis, PKCβ attenuated mitochondrial energy and reduced autophagy. e process was governed by multiple cell signaling pathways and diverse functions, including apoptosis induction, B-cell activation, and endothelial cell proliferation [47,48]. Previous studies showed that PKCβ could regulate neuronal function, which is associated with anxiety-related stress and contributes to conflict behaviors [49]. e precise molecular mechanisms underlying PKCβ-mediated AD development remain obscure. Low expressions of PKCβ was likely an underlying etiology of AD, potentially involving FccR-mediated phagocytosis and the MAPK pathway [50]. ERK1/2 can regulate long-term neuronal plasticity and survival [51]. ERK1/2 expression can also be influenced by PKCβ protein, protecting cells from Aβ-induced apoptosis. ERK1/2 has the potential to act as the target of Ca 2+ signaling and was involved in granulosa cell apoptosis [52]. As a diagnostic marker, ERK1/2 displayed elevation in AD patients compared to healthy control, although it happens later than accumulated t-Tau and p-Tau [53]. In vitro and in vivo results proved that the ERK1/2 pathway would be activated by excitotoxic injury, exerting protective actions against damage, and neural loss [54]. e activation of the ERK1/2 induced cortical neuron apoptosis [55], and caused multiple changes of functional plastic in neuronal cells [56]. Both in clinical cases and AD mouse models, activated p38 MAPK has been observed in early-AD brain [57]. In this study, CSH treatment improved PKCβ and phospho-ERK1/2 levels in mRNA and protein expression, which further confirmed the antiapoptotic effects of CSH.

Conclusion
In conclusion, the present study indicate that CSH treatment have protective effects against Aβ 25-35 -induced cytotoxicity in HT22 cells. CSH treatment increased cell viability through decreasing ROS levels, increasing MMP, inhibiting early apoptosis, and regulating the PKC/ERK pathway. e results of our study suggest that CSH may be a potential therapeutic agent for treating or preventing neurodegenerative diseases.
Data Availability e data of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Sixia Yang and Linshuang Wang have contributed equally to this work. Evidence-Based Complementary and Alternative Medicine 9