| Objective:Parkinson’s disease(PD)is a neurodegenerative disease that is characterized by a significant loss of dopaminergic(DA)neurons in the substantia nigra(SN)of the midbrain and aggregation of Lewy bodies.The etiology of PD is related to various factors,such as genetic factors,environmental toxins,mitochondrial dysfunction,oxidative stress,and inflammatory reactions.Selenium(Se)is an essential trace element for the human body,with a wide range of biological functions such as anti-inflammatory,antioxidant,and anti-tumor.Se deficiency can decrease the activity of Se-dependent antioxidant enzymes,increase oxidative stress,and promote neurodegeneration in the brain.Se exists in nature in two forms: inorganic and organic selenium.Due to the extremely narrow range between the effective and toxic doses,it is easy to cause Se poisoning,which limits its application.Selenium nanocomposites(Se NPs)are nano-sized particles of elemental selenium that are prepared using nanotechnology.These particles have high biocompatibility,low toxicity,and remarkable biological activity.The Se NPs studied in this project is a cordyceps polysaccharide-functionalized selenium nanocomposites(Cs4-Se NPs),which were prepared by the research team of Dr.Wong Kahing of the Hong Kong Polytechnic University using patented technology.We utilized Drosophila PD models and SH-SY5 Y cell injury models induced by rotenone(ROT)to investigate the neuroprotective properties of Cs4-Se NPs and their underlying mechanisms.Methods:1.Rotenone was used to establish Drosophila melanogaster PD model.The climbing experiment was used to detect the climbing ability of Drosophila melanogaster,and quantitative real-time PCR(q PCR)was used to detect the m RNA expressions of tyrosine hydroxylase(TH)and dopamine decarboxylase(Ddc)in the head of the Drosophila melanogaster.The data was used to explore the protective effect of Cs4-Se NPs on DA neurons in the Drosophila melanogaster.2.Rotenone was used to establish the SH-SY5 Y cell injury model.MTT,q PCR and western blotting were used to detect the neuroprotective effect of Cs4-Se NPs on rotenone-induced cell viability,gene expression of selenoprotein and expressions of apoptosis,endoplasmic reticulum stress(ERs)and autophagy-related protein.Results:1.Protective effect of Cs4-Se NPs on rotenone-induced dopaminergic neuronal injury in Drosophila melanogaster.(1)Treatment of rotenone and different concentrations of Cs4-Se NPs(0.01,0.1,1 m M)did not affect the survival rate of Drosophila melanogaster during the 7 th day feeding period.The climbing ability of Drosophila melanogaster was significantly reduced on the 7 th day in the rotenone group(P < 0.001).0.1 m M and 1 m M Cs4-Se NPs could resist the reduced climbing ability of Drosophila melanogaster induced by rotenone(P < 0.001).(2)The results of q PCR showed that the gene expressions of Pale(TH)and Ddc of the Drosophila melanogaster heads in the rotenone groupwere significantly reduced compared with the control group(P < 0.05).0.1 m M Cs4-Se NPs treatment significantly attenuated the neurotoxicity of rotenone(P < 0.01).However,1 m M Cs4-Se NPs treatment antagonized the downregulation of Pale(TH)gene expression induced by rotenone(P < 0.05),but it had no effect to improve the gene expression of Ddc.2.Study on the protective effect and mechanism of Cs4-Se NPs on rotenone-induced injury in SH-SY5 Y cells(1)The MTT results showed that different concentrations of Cs4-Se NPs(0.01,0.1,1,10μM)and Se Met(1,10 μM)had no significant effect on cell viability.Rotenone could significantly reduce cell viability(P < 0.001),and different concentrations of Cs4-Se NPs and 1 μM Se Met pretreated could significantly attenuate the toxic effects of rotenone(P < 0.05,P < 0.001).(2)The protein expressions of TH and α-synuclein were detected using western blotting.The results showed that 1 μM Cs4-Se NPs could significantly antagonize the downregulation of TH protein and the upregulation of α-synuclein protein induced by rotenone(P < 0.05).0.1 and 10 μM Cs4-Se NPs could only reduce rotenone-induced upregulation of α-synuclein protein(P < 0.05,P < 0.01).Compared with Cs4-Se NPs,1 μM Se Met showed no improvement in the changes of TH and α-synuclein proteins induced by rotenone.(3)The expressions of apoptosis-related proteins were detected using western blotting.The results showed that rotenone significantly increased the phosphorylation level of p38 and the activation level of Caspase-3,downregulated the expression of antiapoptotic protein Bcl-2,and upregulated the expression of proapoptotic protein Bax(P < 0.05,P < 0.01,P < 0.001).Pretreatment with different concentrations of Cs4-Se NPs could attenuate the neurotoxic effects of rotenone(P < 0.05,P < 0.001).1 μM Se Met could only significantly reduce the protein expression of Bax(P < 0.001)induced by rotenone.There was no statistically significant improvement on Bcl-2protein expression induced by rotenone.(4)The gene expression of selenoprotein was detected by q PCR.The results showed that the expression of the Sel K gene in the rotenone group was significantly reduced compared with the control group(P < 0.05,P < 0.001),1 μM Cs4-Se NPs significantly increased the gene expression of Sel K(P < 0.001).1 μM Se Met pretreatment could also promote the gene expression of Sel K(P < 0.05),but the effect was weaker than that of Cs4-Se NPs treatment,and the difference was statistically significant(P < 0.01).GPx4 and Sel H gene expressions were not detected in the control group,rotenone group and Cs4-Se NPs treated group.(5)Western blotting was used to detect the expression of ERs-related proteins GRP78 and CHOP.The results showed that Cs4-Se NPs(0.1,1,10 μM)significantly reduced the upregulation of GRP78 and CHOP protein induced by rotenone(P < 0.001).(6)Western blotting was used to detect the expressions of autophagy-related proteins.The results showed that rotenone significantly upregulated the protein expression of LC3-II,downregulated theprotein expression of p62,and significantly reduced the phosphorylation levels of Akt and m TOR(P < 0.05,P < 0.01).1 μM Cs4-Se NPs could upregulate the expression of p62 protein and increase the phosphorylation levels of Akt and m TOR.0.1 μM Cs4-Se NPs could significantly inhibit rotenoneinduced LC3-II protein expression(P < 0.05).1 μM Cs4-Se NPs showed a trendency to inhibit the protein expression of LC3-II,but there was no statistical significance.Conclusions:1.Cs4-Se NPs can protect against rotenone-induced dopaminergic neuronal injury in Drosophila melanogaster.2.Cs4-Se NPs can protect against rotenone-induced apoptosis and excessive autophagy in SH-SY5 Y cells.Sel K is involved in the neuroprotective effect of Cs4-Se NPs. |
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