Neuroprotective Effects of Green Tea Seed Isolated Saponin Due to the Amelioration of Tauopathy and Alleviation of Neuroinflammation: A Therapeutic Approach to Alzheimer's Disease

Abstract

Tauopathy is one of the major causes of neurodegenerative disorders and diseases such as Alzheimer's disease (AD). Hyperphosphorylation of tau proteins by various kinases leads to the formation of PHF and NFT and eventually results in tauopathy and AD; similarly, neuroinflammation also exaggerates and accelerates neuropathy and neurodegeneration. Natural products with anti-tauopathy and anti-neuroinflammatory effects are highly recommended as safe and feasible ways of preventing and /or treating neurodegenerative diseases, including AD. In the present study, we isolated theasaponin E1 from ethanol extract of green tea seed and evaluated its therapeutic inhibitory effects on tau hyper-phosphorylation and neuroinflammation in neuroblastoma (SHY-5Y) and glioblastoma (HTB2) cells, respectively, to elucidate the mechanism of the inhibitory effects. The expression of tau-generating and phosphorylation-promoting genes under the effects of theasaponin E1 were determined and assessed by RT- PCR, ELISA, and western blotting. It was found that theasaponin E1 reduced hyperphosphorylation of tau and Aβ concentrations significantly, and dose-dependently, by suppressing the expression of GSK3 β, CDK5, CAMII, MAPK, EPOE4(E4), and PICALM, and enhanced the expression of PP1, PP2A, and TREM2. According to the ELISA and western blotting results, the levels of APP, Aβ, and p-tau were reduced by treatment with theasaponin E1. Moreover, theasaponin E1 reduced inflammation by suppressing the Nf-kB pathway and dose-dependently reducing the levels of inflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha etc.

Keywords: SHY-5Y cells; kinases; neurodegeneration; neurofibrillary tangles; neuroinflammation; taupathy.

Figure 1

LC-MS-TOF analysis of the green tea seed extracted saponin. (a). LC-MS chromatogram of various saponins extracted from green tea seeds via HPLC (b) and the purified fraction containing the pure, isolated theasaponin E1 (c) The base peak intensity mass spectrum of green tea seed extracted saponins.

Figure 2

Determination of non-toxic concentration level of theasaponin E1 to SHY-5Y neuroblastoma and glial cells (a,b), respectively via MTT assay. Cells were cultured in their respective media in a 96-well microtiter plate. After attachment and treatment with saponin, cell viability was determined for each dose by reading the absorbance of the wells after addition of the MTT reagent and calculation of the results. Cells without treatment were employed as the control. Data are shown as mean ± standard error of means (SEM) (n = 3).

Figure 3

In vitro activities of various kinases and phosphatases involved in tau phosphorylation and dephosphorylation under the effects of theasaponin E1, quantified by ELISA. Percent activity for each enzyme was measured. (a) Changes in GSK3beta activity before and after treatment with saponins. (b) Changes of CDK5 activity by theasaponin E1. (c) Effect of theasaponin E1 on CAMII activity. (d) Effect of theasaponin E1 on JNK activity. (e) Effects of theasaponin E1 on ERK activities. (f) Effects of theasaponin E1 on PP1 activities. (g) Effects of theasaponin E1 on PP2B activities. Data are the values of mean ± SEM and were analyzed using one-way ANOVA (n = 3) (* p < 0.05; ** p < 0.01).

Figure 4

mRNA expression levels of various kinases and phosphatases involved in tau phosphorylation and dephosphorylation, determined by RT-PCR. (A). PCR products of mRNA expression of various genes of the kinases and phosphatases presented in gel band form after gel electrophoresis. (B). Relative mRNA expression levels of genes of various kinases and phosphatases after treatment with theasaponin E1. (a) Relative mRNA expression levels of GSK3 beta. (b) Relative mRNA expression levels of CDK5. (c) Relative mRNA expression levels of JNK. (d) Relative mRNA expression levels of ERK. (e) Relative mRNA expression levels of CAMII. (f) Relative mRNA expression levels of PP1. (g) Relative mRNA expression levels of PP2A. mRNA expression levels of genes of various kinases and phosphatases that were visualized as PCR products on agarose gel. Data are the mean values ± SEM and were analyzed using one-way ANOVA (n = 3). Data are statistically significant at p < 0.05 (* p < 0.05; ** p < 0.01 to the control group).

Figure 5

The gene expression of APP, PESN1, PESN2, EPOE4, IDE, VPS35, PCALM, NF-kB, and IL-1B, as under the influence of theasaponin E1, determined by RT-qPCR. (A) Expression of APP, PESN1, PESN2, EPOE4, IDE, VPS35, PCALM, NF-kB, and IL-1B, as determined by reverse transcription quantitative PCR, visualized by PCR products bands on membrane after gel electrophoresis. (B) Relative mRNA expression levels of various genes. (a) Relative protein expression levels of APP. (b) Relative mRNA expression level of PESN1. (c) Relative mRNA expression level of PESN2. (d) Relative mRNA expression level of EPOE4. (e) Relative mRNA expression level of IDE. (f) Relative mRNA expression levels of VPS35. (g) Relative mRNA expression levels of PCALM. (h) Relative mRNA expression levels of NF-kB. (i) Relative mRNA expression levels of IL-1B. Data are shown as mean ± SEM analyzed with one-way ANOVA (n = 3). Data considered statistically significant at p < 0.05 (* p < 0.05; ** p < 0.01).

Figure 6

Inhibitory effects of theasaponin E1 on the inflammation-promoting cytokines IL-1B, IL-6, and TNF alpha in glial cells determined by specific ELISA kits. (a). Inhibitory effects of theasaponin E1 on IL-1B. (b). Inhibitory effects of theasaponin E1 on TNF alpha. (c). Inhibitory effects of theasaponin E1 on IL-6. Data shown here are the mean values ± SEM. Data were analyzed using one-way ANOVA (n = 3) and were statistically considered significant at p < 0.05 (* p < 0.05; ** p < 0.01 to the control group).

Figure 7

Reduction in the levels of Aβ peptides and p-tau after treatment with theasaponin (due to enhancement or reduction in the activities of various kinases and phosphatases), determined by ELISA. (a) Aβ quantification with ELISA after the treatment of cells with different concentrations of theasaponin E1. (b) p-tau quantification with ELISA after the treatment of cells with different concentrations of theasaponin E1. Data were analyzed using one-way ANOVA (n = 3) and expressed as mean ± SEM. Data are statistically significant at p < 0.05 (* p < 0.05; ** p < 0.01 to the control group).

Figure 8

Effects of theasaponin E1 on tau phosphorylation levels determined by WB. Protein expression as fold change relative to that of the control (actin) after normalization. (A) Western blotting results after treatment of cells and expression of tau, p-tau, and GAPDH on PAGE. (B) Phosphorylated-tau relative protein expression levels determined by western blotting. Data are shown as mean (n = 3) ± SEM (* p < 0.01 and ** p < 0.001 compared to the control).