Are Cistanche extracts useful for treating PD(Parkinson's disease)?

Contact: joanna.jia@wecistanche.com

Introduction: DJ-1 mutation is a causative reason for familial Parkinson's disease (PD). Leucine166Proline (L166P) and C106S are two important DJ-1 mutations. In this study, we established hydrogen peroxide(H, O,)induced L166P and C106S DJ-1-transfected neuroblastoma (SH-SY5Y) cellular models of PD(Parkinson's disease) and investigated the effects of Cistanche extracts and key bioactive compounds, including acteoside, echinacoside,caffeic acid, and Cistanche total glycosides on these two models. Methods: After expressing FLAG-tagged L166P and C106S DJ-1 plasmids in Escherichia coli, the expressed plasmids were collected, treated with a restriction enzyme, and identified using DNA electrophoresis. After purification, the L166P DJ-1 and C106S DJ-1 plasmids were separately transfected into SH-SY5Y cells using liposomes. Transfected SH-SY5Y cells were detected by western blotting and immunocytochemistry. Cell viability was determined using an MTT assay.

Results: Both western blotting and immunocytochemistry showed that L166P and C106S DJ-1 were highly expressed in the transfected SH-SY5Y cells. MTT assays showed that transfection withL166P or C106S DJ-1 reduced the viability of SH-SY5Y cells exposed to H, O, as compared to untransfected SH-SY5Y cells. In addition, Cistanche extracts and key bioactive compounds, including acteoside, echinacoside, caffeic acid, and Cistanche total glycosides, significantly inhibited the decreases of cell viability caused by H, O,inL166P, and C106S DJ-1-transfected SH-SY5Y cells. Conclusions: These findings suggest that we successfully established sensitive and stable H, O, induced L166P DJ-1-and C106S DJ-1-transfected SH-SY5Y cell models of PD(Parkinson's disease) and Cistanche extracts may thus be useful for treating PD(Parkinson's disease).

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DISCUSSION

Mutations of DJ‐1 are of being close association with autosomal recessive early‐occurrence PD(Parkinson's disease) (Bonifati et al., 2003; Park et al., 2015). In one Italian family, the cause is a missense mutation in DJ‐1 (L166P) (Bonifati et al., 2003). Leu‐166 localizes in the middle of the C‐terminal helix (Bonifati et al., 2003). The presence of Pro, a strong helix breaker, likely destabilizes the terminal helix in the DJ‐1 mutant (Bonifati et al., 2003), leading to the unfolding of the C‐terminal portion of the protein, which would impair its homo-oligomerization (Bonifati et al., 2003). In transfected cells, L166P DJ‐1 is unstable due to excessive degradation (Bonifati et al., 2003; Gorner et al., 2004; Moore, Zhang, Dawson, & Dawson, 2003). Bonifati et al. (2003)suggested that L166P mutation impairs the homodimerization and normal function of DJ-1, and so leads to the early onset of PD(Parkinson's disease).

In this study, we found that L166P DJ-1 is present within a polymer/complex with a molecular mass of 70 kDa in transfected SH-SY5Y cells. The structure of DJ-1 was assumed to adopt the same a/βsandwich structure as protease PH1704(Bonifati et al.,2003). Due to the similar structures of DJ-1 and PH1704, we assumed that, like PH1704, DJ-1 forms higher aggregates (trimers of dimmers). Gorner et al. (2004)showed that within transfected cells and the lymphocytes of PD(Parkinson's disease) patients, monomeric L166P DJ-1 is present within the higher structures in transfected cells and lymphocytes of PD(Parkinson's disease) patients. This may be a direct result of L166P DJ-1 protein misfolding or the formation of complexes between monomeric L166P DJ-1 and other proteins. Moreover, in addition to the structure of the protein, L166P DJ-1 mutation also changed the antioxidative function of DJ-1(Macedo et al.,2003; Taira et al,2004). This is noteworthy, as oxidative stress is an important factor in PD(Parkinson's disease), and SH-SY5Y cells transfected with L166P DJ-1 were more sensitive to H, O, than untransfected cells. Combined with the previous research that wild-type DJ-1 transfected SH-SY5Y cells were successfully established in our laboratory (Zhang, Wang,& Pu,2007), we suggest that H, O, induced L166P DJ-1-transfected SH-SY5Ycells are a useful new cellular model of PD(Parkinson's disease).

Cistanche extract can prevent Parkinson's disease

DJ-1 has been reported to reduce oxidative stress(Taira et al, 2004; Zhou & Freed,2005). One of the mechanisms is the oxidation of the protein itself(Taira et al.,2004). Among the three Cys residues of DJ-1, Cys-106 is most sensitive to oxidative stress. Moreover, the

antioxidative function of DJ-1is regulated through oxidation of Cys-106 (Freed &Zhou,2006), and mutation of Cys-106leads to loss of the antioxidative function of DJ-1 (Blackinton et al,2009; Kinumi, Kimata, Taira, Ariga,& Niki,2004).DJ-1 also acts as a molecular chaperone that inhibits the formation of α-synuclein aggregates(Shendelman et al.,2004)when Cys-106 is oxidized to sulfinic acid (Zhou & Freed,2005). These findings indicate that Cys-106 is a key contributor to proper DJ-1 function. As shown in Table 1, SH-SY5Y cells transfected with C106S DJ-1 were more sensitive to H, O, than untransfected cells. Based on the previous research(Zhang et al.,2007), this also suggests H, O, induced C106S DJ-1-transfected SH-SY5Y cells may also be a useful cellular model of PD(Parkinson's disease).

Using SH-SY5Y cells transfected with L166P DJ-1 or C106S DJ-1, we detected the effect of Cistanche extracts and key bioactive compounds, including acteoside, echinacoside,caffeic acid, and Cistanche total glycosides on HO, induced reductions in cell viability. The present results show that acteoside, echinacoside, caffeic acid, and Cistanche total glycosides all increased cell viability in a concentration-dependent manner, indicating a stable and linear relation between L166P DJ-1and C106S DJ-1 levels and SH-SY5Y cell viability.

In sum, we successfully established sensitive and stable H, O2 induced L166P DJ-1-and C106S DJ-1-transfected SH-SY5Y cell models of PD(Parkinson's disease) and confirmed that Cistanche extracts ameliorated the neurotoxicity induced by H, O, in these two models. We anticipate that these two cellular models will be effectively used for PD(Parkinson's disease) research and Cistanche extracts may thus be useful for treating PD(Parkinson's disease) in the future.

Benefits of Cistanche extracts: anti-Parkinson's disease

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Benefits of Cistanche extracts: treating Parkinson's disease