The Chinese Herb Codonopsis pilosula Isolate Isorhapontigenin protects against oxidative stress injury by inhibiting the activation of PI3K/Akt signaling pathway

We investigated the effects of the Chinese herb Codonopsis pilosula isolate isorhapontigenin on antioxidant fac-tor and the PI3K/Serine/Akt signaling pathway in Parkin-son's disease. This research was, therefore, carried out to explore a possible protective mechanism of isorhamnetin in Parkinson's disease. The results support that isorhapontigenin could effectively inhibit isorhapontigenin restored myeloperoxidase + induced reduction of antioxidant levels. Also, 1-Methyl-4-phenylpyridine up-regulated the expression of phosphorylated-Akt, phosphorylated-PI3K, and phosphorylated mammalian target of ra-pamycin, while isorhapontigenin inhibited the expression of phosphorylated-Akt, phosphorylated-PI3K, and phosphorylated- mammalian target of rapamycin. Fur-thermore, LY294002 improved the antioxidant effect of isorhapontigenin in PC12 cells, and insulin-like growth fac-tor 1 inhibited the antioxidant effect of isorhapontigenin in PC12 cells. Our results support the finding that isorhamnetin enhanced the antioxidant effect induced by 1-Methyl-4-phenylpyridine in PC12 cells by suppressing the activation of the PI3K/Akt signaling pathway.


Introduction
Parkinson's disease (PD) is also known as a common neurodegenerative disease, which has become the second most prevalent disease of the nervous system (Armstrong, 2017). Screening new targets for the treatment of PD, and the development of new drugs that can be used for clinical prevention of PD have enormous social and economic benefits (Zucca et al., 2017). PD has been studied for decades, but the cause and the mechanisms are still unclear due to the complexity of its pathogenesis (Athauda et al., 2017). Current medicine used for the treatment of PD is mainly based on dopamine agonists, anticholinergics, and other drugs.
Studies have shown that many extracts of traditional Chinese medicine have protective effects on neurons and can antagonize the induced cytotoxicity by inhibiting oxidative stress and cell apop-tosis (Pan et al., 2011). Clinical practice has proven that traditional Chinese medicine can alleviate dyskinesia caused by levodopa drugs, relieve non-motor symptoms of PD patients, and improve patients' quality of life. Codonopsis pilosula is one of the most commonly used traditional Chinese medicines (Sun and Liu, 2008;Sun et al., 2010). Pharmacological studies have shown that Codonopsis pilosula has the function of regulating blood sugar levels, promoting hematopoietic function, reducing blood pressure, resisting hypoxia, resisting fatigue, boosting the immune system, regulating stomach contraction and anti-ulcer (Ng et al., 2004;Wang et al., 1996). Isorhamnetin (ISO) is a flavonoid isolated from Codonopsis pilosula. Studies have shown that flavonoid plays a critical role in regulating neurological function and neuroprotection in neurodegenerative diseases (Bakhtiari et al., 2017;Magalingam et al., 2015). Recent studies have also shown that ISO has many cardiovascular effects, such as anti-myocardial hypoxia, ischemia, relieving angina pectoris, anti-arrhythmia, anti-oxygen free radicals, and reducing serum cholesterol (Boeschsaadatmandi et al., 2011;Teng et al., 2006). However, the protective effect of isorhamnetin as a monomer on PD has not been investigated. PI3K/Akt signaling transduction pathway is involved in protein synthesis in the body (Díazserrano et al., 2018). It plays a critical role in regulating cell differentiation and apoptosis (Gui et al., 2018). Specific regulation of related sites of conduction pathways can prevent neuron apoptosis and promote neuron survival (Xiao et al., 2015).

Cell culture and grouping
The PC12 cell line (rat pheochromocytoma tumor) was obtained from Procell company (Wuhan, P. R. China). Cells were cultivated in RPMI-1640 medium supplemented with 10% FCS (Clark Bioscience, USA). Cells were split into the group of control, ISO, MPP+, and MPP++ISO. MPP+ was diluted to 400 mmol/L with a 1 : 10 gradient in the DMEM medium. Cells were plated into a 96-wells plate, and different concentrations of MPP+ were added. ISO was diluted to 1 mg/ml with a 1 : 10 gradient in DMEM. Then it was added in a 96-wells plate at the ratio of 1 : 100, and different concentrations of ISO were added.

Annexin V-FITC/PI staining
PC12 cells were washed and cultured with 5 µL of FITC Annexin V, 500 µL of binding buffer, and 5 µl of propidium iodide (PI). The apoptotic rate was determined using flow cytometry (FACS Calibur, USA). Experiments were carried out in Rieger et al. (2011).
Determination of the activity and content of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA) The cultured cells were lysed at 4 • C, followed by centrifugation to collect the supernatant. The activity and content were determined using GSH-Px, SOD, and MDA kits.

Statistical analysis
The data were analyzed using SPSS19.0 statistical software. The results were shown as mean ± standard deviation (SD). Multigroup comparisons were conducted using one-way ANOVA. Least Significant Difference (LSD) test was used for subsequent analysis. P < 0.05 indicated significant differences.

The cytotoxicity of MPP+
As shown in Fig. 1A, MPP+ reduced the growth rate of PC12 tumor cells, and the cell viability gradually decreased with the increase of dose and time. At 24, 48, and 72 h, the IC50 values of PC12 cells were 1,274.56, 203.48, and 81.25 µM, respectively. Therefore, 203 µM and 48 h were selected as the working concentration and incubation period for the intervention drug of MPP+ in the following experiments. Whether MPP+ could induce apoptosis was further investigated. As shown in Fig. 1B, contrasted with the control group, the proportion of apoptotic cells treated with 1/2 MPP+, 1 MPP+, 2 MPP+ was increased to 10.5 %, 15.2 %, and 30.9 %, respectively. These data suggest that MPP+ increases the apoptotic rate of PC12 tumor cells.

ISO suppressed cytotoxicity induced by MPP+
In Fig. 2A, there is a statistically significant difference between groups determined by one-way ANOVA (F(6, 14) = 9.321, P = 0.000). The LSD post-hoc test shows that the viability of PC12 cells pretreated with different concentrations of ISO was increased, and the concentration of 64 µM ISO had the best protective effect on PC12 cells (0.71 ± 0.04, P = 0.000), contrasted with the control group (0.45 ± 0.03). Therefore, 64 µM was selected as the intervention concentration of ISO for the following experiments. Also, compared to the control group, MPP+ increased the apoptosis rate of PC12 cells, while ISO+MPP+ decreased the apoptosis rate (Fig. 2B), indicating that ISO has a protective effect on MPP+-induced apoptosis of PC12 cells.

Discussion
The effects of isorhamnetin on antioxidant factors and the PI3K/Akt signaling pathway in Parkinson's cell model was investigated. MPP+ was applied to PC12 cells for 48 hrs to establish a model of PD cell injury in vitro. The cell viability and apoptosis were measured, and it showed that ISO pretreatment increased the survival rate of MPP+-induced damage in PC12 cells, and decreased the apoptotic rate of PC12 cells.
Free radical scavenging systems in the body include SOD, catalase, MDA, and GSH-Px (Wang et al., 2011). The level of SOD activity indirectly reflects the body's ability to scavenge oxygen free radicals. GSH-Px is an important peroxide-degrading enzyme that catalyzes the reaction of reduced GSH to hydroperoxides and protects cell membrane structure and mitochondrial function (Bao et al., 2013). The content of MDA can reflect the rate of lipid peroxidation (Laguna et al., 2015). Studies have shown that lipid peroxidation selectively increases in the nigrostriatal cells of a PD patient (Ahlskog, 2005), in which the levels of GSH and GSH-Px were decreased, while the levels of MDA were raised.
Oxidative stress is well documented in PD (Li et al., 2014). Oxidative stress has long been considered to play a critical role in the pathogenesis of AD. Some antioxidants rich natural extracts are believed to be neuroprotective, and therefore may have cognitive benefits in aging and neurodegenerative diseases (Rabadiya et al., 2012). For example, Ingale and Kasture (2017) found that the flower butanol extract improved PD by improving antioxidant capacity.
Our research revealed that ISO raised the activity of SOD and GSH-Px in PC12 cells, and reduced the content of MDA, which was consistent with previous studies (Dong et al., 2015). It showed that ISO could increase the activity of the antioxidant system of the PD cell model to different degrees, reduce lipid peroxidation, improve the antioxidant effect, and have a neuroprotective role.
Studies have shown close relation to Chinese medicine action with the regulation of the PI3K/AKT-mTOR signaling pathway . Many pharmacological compounds have proved to have a neuroprotective role in oxidative stress by activating the PI3K/Akt pathway (Zhang et al., 2016). For example, Zhang et al. (2016) found that salidroside (SAL) could protect cell apoptosis induced by 1-methyl-4-phenylpyridine by regulating PI3K/Akt/GSK3β pathway, and Baicalein can significantly inhibit the damage of 6-O-HDA to MES23.5 neurons. Its mechanism might involve the activation of the PI3K/Akt pathway .
We found that ISO down-regulated the expression of p-PI3K, p-Akt, and p-mTOR in MPP+-induced PC12 cells. Also, LY294002 inhibited the PI3K/Akt/mTOR signaling pathway, while IGF-1 activated the PI3K/Akt/mTOR signaling pathway. It was also found that activation of the PI3K/Akt/mTOR signaling pathway increased oxidative stress levels. The antioxidant factors were significantly increased after adding LY294002. Hence, combining our present findings with previous studies (Xu et al., 2013), it is demonstrated that the anti-apoptotic protective mechanism of ISO on MPP+ induced PC12 cells is through PI3K/Akt signaling pathway.

Conclusions
The anti-apoptotic protective mechanism of ISO on MPP+ induced PC12 cells by reducing oxidative stress and inhibiting the PI3K/Akt signaling pathway. This would provide an experimental basis for the clinical application of ISO in the treatment of PD, and a theoretical basis for the future development of ISO. This finding will also provide insight into the pathogenesis of PD and provide an experimental basis of its clinical prevention.