*2.1. Chemical Analysis*

The chemical composition of FPS, DF (low molecular weight fucoidan) and UF are shown in Table 1. All these three samples were sulfated heteropolysaccharides; they were mainly made of fucose, uronic acid and sulfated groups. However, the exactly chemical composition were di fferent. The chemical composition of FPS and DF were nearly the same, however. The molecular weight of FPS was 10 times higher than DF. Among the three samples, UF had the highest uronic acid and lowest sulfated groups. The monosaccharides of three samples all contained fucose, galactose, glucose, rhamnose, mannose and xylose. Fucose was the mainly monosaccharide in all three samples, followed with galactose The structure of FPS, DF and UF were published by our group. The backbone of FPS and DF was similar: they were primarily made by (1 →3)-linked <sup>α</sup>-<sup>l</sup>-fucopyranose residues and a few (1 →4)- α-L-fucopyranose linkages. β-<sup>d</sup>-galactose and fucose were linked at the branch points at C-4 or C-2 of α-L-fucopyranose residues. Sulfate groups occupied at C-4 or C-2, sometimes C-2, 4 to fucose residues and C-3 and/or C-4 to galactose residues. The structure of UF was more complex. The backbone was made by 4-linked uronic acid and 2-linked mannose. The branch chain was composed of 1–3-linked fucose, 1–6-linked galactose and 1–4-linked glucuronic acid. The sulfate group was connected to the C-6 position of mannose. and C-4 or C-2 position of fucose [19].

**Table 1.** The yield (% compared with the dry *S. japonica*), chemical composition (% dry weight) and molecular weight of fucoidan (FPS), DF and its fraction (UF) isolated from *S. japonica.*


aFuc: fucose; Gal: galactose; Man: mannose; Glc: glucose; Rha: rhamnose; Xyl: xylose.

#### *2.2. Protective E*ff*ect of UF on MPP*<sup>+</sup>*-Induced Neurotoxicity in SH-SY5Y Cells*

The results of protective effect of UF on MPP<sup>+</sup>-induced neurotoxicity in SH-SY5Y cells are shown in Figure 1. The exposure of the SH-SY5Y cells to 100-μM MPP<sup>+</sup> significantly reduced cell viability to 60% compared with that of the normal cells. In contrast, pretreatment with different concentrations of UF reversed the decreased cell viability induced by MPP<sup>+</sup>. Co-incubation of UF at the dose of 500 μg/mL and 800 μg/mL increased the cell viability more than 30% compared with the MPP group. When exposed with PI3K inhibitor LY294002, the cell viability decreased in all groups compared with the NC group. The cell viability of NCLY and MPPLY group was nearly 80% and 32% compared with the NC group. Pretreatment with UF increased the cell viability to 55%—especially at the dose of 800 μg/mL.

**Figure 1.** Effect of the samples on the neuronal cell viability induced by MPP<sup>+</sup>. # Vs NC *p* < 0.05; ## Vs NC *p* < 0.01; ### Vs NC *p* < 0.001; \* Vs MPP *p* < 0.05; \*\* Vs MPP *p* < 0.01; \*\*\* Vs MPP *p* < 0.001; ˆ Vs MPPLY *p* < 0.05; ˆˆ Vs MPPLY *p* < 0.01; ˆˆˆ Vs MPPLY *p* < 0.001.

To determine whether the survival rate of SH-SY5Y neurons increased by UF treatment was related to cell apoptosis, cells were stained with the DNA dye Hoechst 33342/PI to visualize nuclear morphology (Figure 2). The results showed that incubation with MPP<sup>+</sup> could cause SH-SY5Y cells apoptosis—including the degeneration of neuritis and shrinkage of cell bodies—as well as fragmentation and condensation of nuclei. Without exposure to MPP<sup>+</sup>, SH-SY5Y cells exhibited normal cellular morphology. Different doses of UF administration groups could reduce the apoptosis and death rate of SH-SY5Y cells (500 μg/mL and 800 μg/mL, respectively, *p* < 0.01), which indicated that the protective effect of UF on SH-SY5Y cells was related to reducing the apoptosis of SH-SY5Y cells. To determine whether the apoptosis of SH-SY5Y cells caused by MPP<sup>+</sup> was related to the PI3K/AKT pathway, we added PI3K/AKT pathway inhibitor LY294002 during cell culture [7,8]. The degree of apoptosis and death rate of SH-SY5Y cells in MPPLY group increased significantly compared with NC group. Different concentrations of UF administration groups could reduce the apoptosis and death rate. The MPP<sup>+</sup>-induced apoptosis rate was 37.6%; the addition of LY294002 increased the frequency of apoptosis rate to 51.5%. UF at 800 μg/mL greatly reduced MPP<sup>+</sup>-induced apoptosis to 11.1%. With the addition of LY294002, the effect of UF at 800 μg/mL on MPP<sup>+</sup>-induced apoptosis was 28.7%. The apoptosis rate was different when cells were pretreatment with UF alone or together with LY294002 (11.1% versus 28.7%, *p* < 0.001). These data sugges<sup>t</sup> that the protective effect of UF on SH-SY5Y cells was partly related to the PI3K/AKT pathway.

**Figure 2.** *Cont*.

**Figure 2.** Nuclear morphology of MPP<sup>+</sup> and UF treated SH-SY5Y cells for 48 h. (**a**) Protective effects of UF on MPP<sup>+</sup>-induced cell apoptosis rate %; (**b**) protective effects of UF on MPP<sup>+</sup>-induced cell Death rate % (**c**) Data are expressed as percentages and represent the mean ± SD of three separate experiments in which at least 200 cells were counted per one treatment group. # Vs NC *p* < 0.05; ## Vs NC *p* < 0.01; ### Vs NC *p* < 0.001; \* Vs MPP *p* < 0.05; \*\* Vs MPP *p* < 0.01; \*\*\* Vs MPP *p* < 0.001; ˆ Vs MPPLY *p* < 0.05; ˆˆ Vs MPPLY *p* < 0.01; ˆˆˆ Vs MPPLY *p* < 0.001; Scale bar in the picture is 50 in length.

#### *2.3. UF E*ff*ect on the Expression of PI3K, Akt and Its Phosphorylation*

Figure 3 summarizes the effect of the samples on the phosphorylation of PI3K and Akt proteins (). The immunochemistry results showed that MPP<sup>+</sup> treatment decreased the phosphorylation of PI3K and Akt and inhibited the activation of PI3K/AKT pathway. Different concentrations of UF administration groups promoted the phosphorylation of PI3K and Akt, thereby activating the PI3K/AKT pathway (Figure 3b,c). The ratio of pAkt/tAkt and pPI3K/tPI3K were analyzed; the two ratios were lower in MPP group than in NC group. Different doses of UF treated increased the two ratios, respectively. We also examined whether the PI3K inhibitor LY294002 could inhibit the cytoprotective effect of UF. After incubation with LY294002, the degree of phosphorylated PI3K and Akt decreased significantly compared with NC group. Different concentrations of UF administration groups increased the phosphorylated PI3K and Akt level. Comparing the groups UF1 and UF1LY, UF2 and UF2LY, UF3 and UF3LY, the phosphorylated PI3K and Akt increased rates in UF1LY, UF2LY and UF3LY groups were lower than in the UF1, UF2 and UF3 groups, respectively. The results sugges<sup>t</sup> that UF activated the PI3K/AKT pathway to inhibit the apoptosis of neuron cells and additional LY294002 alleviated UF neuron protective, but not completely. We tested the pAkt and pPI3K protein expression using western blotting to confirm. As we expected, results showed that the expression of pAkt and pPI3K was decreased in MPP group compared with the NC group. UF treatment groups reversed the MPP<sup>+</sup> induced pAkt and pPI3K decrease markedly. After incubation with LY294002, the expression of pAkt and pPI3K were lower than the groups without treated with LY294002, respectively. Treatment with UF and positive drug MA increased the expression of pAkt and pPI3K at some extent. Therefore, it is plausible that the degradation pAkt and pPI3K may be regulated by UF and the UF treatment could activate PI3K–Akt pathway.

**Figure 3.** *Cont*.

**Figure 3.** UF effect on the expression of PI3K, Akt and its phosphorylation. (**a**) PI3K, Akt and its phosphorylation results by western blot; (**b**) Akt and its phosphorylation results by immunochemistry method; (**c**) PI3K and its phosphorylation results by immunochemistry method; # Vs NC *p* < 0.05; ## Vs NC *p* < 0.01; \* Vs MPP *p* < 0.05; \*\* Vs MPP *p* < 0.01; ˆ Vs MPPLY *p* < 0.05; ˆˆ Vs MPPLY *p* < 0.01.

#### *2.4. UF E*ff*ect on the Expression of NGF*

To determine whether UF treatment could promote the survival of CNS neurons and activation PI3K/AKT pathway, we tested the effectiveness of UF on NGF. The protein expression level of the NGF decreased significantly in the MPP group, however, NGF expression elevated in different doses of the UF treatment groups, especially in the dose of 100-μg/mL UF group (Figure 4a). Moreover, the treatment of cells with 100-μM MPP<sup>+</sup> and 20-μM LY294002 significantly decreased the mRNA level of NGF to 0.28 ± 0.03 of the NC group and the UF (800 μg/mL) combined with LY294002 treatment group increased that to 0.75 ± 0.06 of the NC group, respectively (Figure 4b).

**Figure 4.** Protective effects of UF on MPP<sup>+</sup>-induced SH-SY5Y cell of NGF. (**a**) NGF results by immunochemistry method; (**b**) NGF results by RT-PCR method; # Vs NC *p* < 0.05; ## Vs NC *p* < 0.01; ### Vs NC *p* < 0.001; \* Vs MPP *p* < 0.05; \*\* Vs MPP *p* < 0.01; ˆ Vs MPPLY *p* < 0.05; ˆˆ Vs MPPLY *p* < 0.01.

#### *2.5. UF E*ff*ect on the Expression of Apoptosis Related Proteins*

Figure 5 summarizes apoptosis-related proteins in the PI3K/AKT pathway. From our data we concluded that UF has a certain inhibitory effect on the expression of pro-apoptotic proteins Bax, P53 and GSK3β, and also has a certain promotion effect on the anti-apoptotic protein Bcl-2 (Figure 5a,b). The ratio of Bax/Bcl-2 was increased to 4.2 and 4.7-fold in MPP and MPPLY groups compared with NC group, respectively. UF treatment group decreased the ratio of Bax/Bcl-2 at a dose dependent manner. The ratio of Bax/Bcl-2 in UF3 group was lower than in the NC group. The ratio of Bax/Bcl-2 in UF3LY group was nearly the same as in the NC group. The level of mRNA expression following MPP<sup>+</sup> and UF treatment were examined by RT-PCR in order to confirm the protein changes in process of the cell apoptosis (Figure 5c,d). The PD model induced by MPP<sup>+</sup> increased the mRNA expression level of pro-apoptosis protein such as Bax, p53 and GSK3β, and decreased the mRNA expression level of anti-apoptosis protein Bcl-2. However, there was a marked decrease in the mRNA expression level of pro-apoptosis protein levels and increase in the mRNA expression level of anti-apoptosis protein levels in the UF-treated groups. We also found that the mRNA expression of the key apoptosis-related

proteins Bax, P53 and GSK3β was consistently upregulated to 5.97 ± 0.15, 6.24 ± 0.44 and 7.63 ± 0.52, respectively, in cells treated with 100-μM MPP<sup>+</sup> and LY294002, whereas that of Bcl-2 was downregulated to 0.44 ± 0.07 of the control values. However, UF together with LY294002 treatment inhibited the up- or downregulation of Bax, P53, GSK3β and Bcl-2. Compared to the UF treated group, the up- or downregulation ability was weaker.

**Figure 5.** *Cont*.

**Figure 5.** Protective effects of UF on MPP<sup>+</sup>-induced SH-SY5Y cell of apoptosis relative protein. (**a**) protein expression of Bcl-2 and Bax by immunochemistry method; (**b**) protein expression of p53 and GSK3β by immunochemistry method; (**c**) mRNA expression of Bcl-2 and Bax by RT-PCR method; (**d**) mRNA expression of Bcl-2 and Bax by RT-PCR method # Vs NC *p* < 0.05; ## Vs NC *p* <0.01; ### Vs NC *p* < 0.001; \* Vs MPP *p* < 0.05; \*\* Vs MPP *p* < 0.01; ˆ Vs MPPLY *p* < 0.05; ˆˆ Vs MPPLY *p* < 0.01; ˆˆˆ Vs MPPLY *p* < 0.001.

#### *2.6. Caspase-3, -8 and -9 Activity*

The treatment of cells with 100-μM MPP<sup>+</sup> significantly increased the relative activity of caspase-3 (cas3), caspase-8 (cas8) and caspase-9(cas9) to 0.73 ± 0.2, 2.90 ± 0.43 and 2.06 ± 0.18 of the NC group, respectively (Figure 6); however, this significantly decreased to 0.58 ± 0.13, 1.45 ± 0.32 and 1.16 ± 0.36 of the NC group, respectively, under UF treatment at 100 μg/mL. Moreover, the treatment of cells with 100-μM MPP<sup>+</sup> and 20-μM LY294002 significantly increased the relative activity of cas3, cas8 and cas9 to 3.21 ± 0.11 and 4.53 ± 0.56, 3.02 ± 0.27 of the NC group, respectively, which were much higher than MPP group; however, this significantly decreased to 0.94 ± 0.24, 0.84 ± 0.35 and 0.73 ± 0.18 of the control group, respectively, when treated with UF at 800-μg/mL and 20-μM LY294002. These results showed that UF treatment could promote the activity of caspase-3, caspase-8 and caspase-9 did not inhibited by LY294002.

**Figure 6.** Protective effects of UF on MPP<sup>+</sup>-induced SH-SY5Y cell of relative activity of caspase-3, caspase-8 and caspase-9. # Vs NC *p* < 0.1; ## Vs NC *p* < 0.01; \* Vs MPP *p* < 0.1; \*\* Vs MPP *p* < 0.01; ˆ Vs MPPLY *p* < 0.1; ˆˆ Vs MPPLY *p* < 0.01.
