**3. Discussion**

PD is a neurodegenerative disease caused by both genetic and environmental factors. Neuroprotection therapeutic method caused interest in recently. Previous study found fucoidan extracted from brown seaweeds had neuron protective activity which related to its antioxidant activity [15]. Further studies found different fractions of fucoidan exhibited variety neuron protective activities. One fraction UF with higher uronic acid, lower sulfated group content and more complex monosaccharides exhibited the strongest activity. The neuron protective effect of UF may be mediated, in part, through antioxidant activity and the prevention of cell apoptosis [18]. The simplified depiction effect of UF on MPP<sup>+</sup>-induced cytotoxicity was summarized in Figure 7. UF treatment groups could decrease the cell apoptosis rate and increase the cell vitality. LY294002 could inactivate the PI3K–Akt pathway, thereby inhibiting cell proliferation and inducing apoptosis. The results found that the neuron protective effect of UF was alleviated when treated with LY294002. It is illustrated that the neuron protective effect of UF may relate with the PI3K–Akt pathway.

**Figure 7.** Effect of UF on MPP<sup>+</sup>-induced SH-SY5Y cells apoptosis.

The PI3K–Akt pathway plays an important role in the survival and maintenance of many neuronal function such as long-term potentiation and memory formation. Inhibition of PI3K activity can offset the ability of nerve growth factors to promote cell survival [8]. Chondroitin sulfate (CS) in the cell matrix can protect SH-SY5Y cells by activating the PI3K–Akt signaling pathway [6]. Nerve growth factor (NGF) is a peptide molecule that plays a nutritional role in nerve cells. In the nervous system, NGF can increase the tolerance of cells under oxidative stress, which is the main mechanism involves NGF-induced activation of the PI3K–Akt pathway. Studies have shown that the application of corresponding treatments after spinal cord ischemia can induce NGF to activate the PI3K–Akt pathway to inhibit neuronal apoptosis [20]. Seow et al. found crude polysaccharides extracted from *Lignosus rhinocerotis* could stimulate neurogenesis without stimulating the production of NGF in PC-12 cells [21]. We found UF had a backbone of alternating 4-linked GlcA and 2-linked Man with the first Man residue from the nonreducing end accidentally sulfated at C6. UF and CS both have GluA and sulfate group, so we suppose UF could combine with NGF and increase the expression of the NGF, then activation the PI3K–Akt pathway. Our study confirmed that UF treatment could increase the expression of NGF, the e ffect was alleviated when added LY294002, but not disappeared completely. From our data, we suppose UF exhibited protective e ffect against MPP<sup>+</sup>-induced SH-SY5Y cell apoptosis by activating PI3K–Akt pathway through reacting with NGF. The chemical composition and structure of the polysaccharide had relationship with the e ffect on the NGF, chondroitin sulfate and fucoidan could increase the expression of the NGF protein, however, polysaccharide extracted from *Lignosus rhinocerotis* mimics the neurogenic activity of NGF.

PI3K is one of the signal molecules involved in intracellular signal transduction. When cells are stimulated by stimulating factors such as NGF, the phosphorylation of PI3K is activated. The present results demonstrated that UF treatment could enhance the phosphorylation of PI3K first, and then promoted the phosphorylation of Akt. This e ffect was alleviated when combined with LY294002.

When phosphorylating the PI3K and Akt, it activates or inhibits its downstream target proteins Bad, Bcl-2, Bax, caspase-9, GSK-3, mTOR, nuclear transcription factors, etc., in turn regulate cell proliferation, di fferentiation, apoptosis and invasion [22]. The influence of UF on these anti-apoptotic and pro-apoptotic protein were analyzed. Our study showed that UF could partially inhibit MPP<sup>+</sup>-induced dysfunction of the Bax/Bcl-2 system and decrease the expression of P53 and GSK3β protein, then inhibited cell apoptosis. This impact was alleviated when adding LY294002 in UF treated groups. As an anti-apoptotic member of the Bcl-2 family, Bcl-2 can bind Bax to form Bax/Bcl-2 heterodimers, thereby, attenuating the pro-apoptotic e ffect of Bax. Habaike *et al*. found polysaccharides extracted from *Laricifomes o*ffi*cinalis* Ames could attenuating cell apoptosis, increasing the ratio of Bcl-2/Bax and inhibiting cytochrome C release from mitochondria to cytosol in PC12 cells [23]. An acid-soluble polysaccharide (GFAP) prepared from *Grifola frondosa* could upregulate the expressions of Bax in HCC cells and induced the cell apoptosis [24]. The UF is a heteropolysaccharide with uronic acid and sulfate group, we suppose it can react with Bax and e ffect the formation of Bax/Bcl-2 heterodimers. P53 and GSK3β are thought to be a key factor in the subsequent apoptotic processes among the pro-apoptotic proteins. UF can react with these two proteins directly and reduce the cell apoptosis in the H2O2-reduced cell model [25].

Caspase family such as caspase-3 (cas3), caspase-8 (cas8) and caspase-9 (cas9) are crucial checkpoint in cell commitment to apoptosis. Cas3 is a critical executor of apoptosis being responsible for the proteolytic cleavage of many key proteins, which damage initiates the cell death program. Yu et al. found an acid-soluble polysaccharide could trigger apoptosis of HCC cells through mitochondria apoptotic pathway in a caspases-dependent pattern [24]. It means the acid polysaccharide could react with caspases-dependent pattern, it could decrease or increase the expression of caspase protein, which confirmed by our results. The addition of UF significantly attenuated the expression of cas3 in MPP<sup>+</sup>-induced SH-SY5Y cells, confirming UF had e ffect in terms of apoptosis process. LY294002 used did not lead to a complete inhibition of initiation-programmed cell death by UF treated groups, suggesting that other pathways are also involved. Cas8 and cas9 have shown increase in MPP<sup>+</sup> and MPP++LY294002 groups simultaneously, UF treated groups could decrease this rising tendency. The observed changes may sugges<sup>t</sup> that UF could significantly decrease cas3, cas8 and casp9 activation, the consequence of which is apoptosis.

Our study showed that UF treatment could reversed the toxic effect of MPP<sup>+</sup> on SH-SY5Y cells by activation the PI3K–Akt pathway. Addition of LY294002 significantly inhibited the PI3K–Akt pathway active and enhanced DNA fragmentation in SH-SY5Y cells, UF treated groups could still alleviate the cell apoptosis. In the present study, we demonstrated for the first time that UF attenuated the MPP<sup>+</sup> induced apoptosis via reacting with NGF, Bax and cas3. Our data indicated that UF inhibited cell apoptosis by participating in PI3K–Akt pathway partially.
