To Explore The Protective Effect Of Dabuyin Pill And Xiezheng Powder On The Mitochondria Of Parkinson 's Disease Cell Model From The Perspective Of PINK1 Gene

BackgroundParkinson’s disease (PD) is a neurodegenerative disease, characterized by the death of dopaminergic neurons in the substantia nigra pars compacta and lead to the dopamine depletion in the striatum. Its major symptoms includes tremor at rest, bradykinesia, and cogwheel rigidity. Also, there are non-motor symptoms, such as constipation, sleep disorder, olfactory problems, depression, dementia, etc. Currently, most treatments towards Parkinson’s disease are focused on the enhancement of dopamine level in the striatum by using L-dopa. However, long-term use of L-dopa can cause dyskinetic and psychotic symtoms. Reports show Traditional Chinese Medicine (TCM) may relieve PD symptoms as well as improve dyskinetic.Etiology study reviews that many factors are related to the pathogenesis of PD, including environmental toxins, gene mutation and/or the interactions between these two factors. Recently, the role of mitochondria in PD catches the eyes of researchers. Environmental toxins, such as 1-Methyl-4-phenylpyridinium (MPP+),6-Hydroxydopamine (6-OHDA) and rotenone, can inhibit the functions of mitochondria complex I and cause Parkinsonism in relatively short period of time. These toxins are now used to establish PD animal and cell models. In addition, PD-related autosomal recessive gene PINK1 and Parkin (PARK2) are directly related to mitochondria quality control. Full length PINK1 accumulated on mitochondria outer membrane to call in Parkin and activate Parkin’s E3 ubiquitin ligase activity. This may lead to the result of mitophagy.Da-Bu-Yin-Wan and Qian-Zheng-San (DBYW&QZS) are commonly used clinical decoctions in treating PD in China. Former studies suggested that it can relieve mtDNA damage, regulate mtDNA expression, and have protective effects on mitochondrial function.ObjectiveTo further the understanding of DBYW&QZS’s mechanism in dealing with PD, here choose to establish a PINK1 overexpression model as well as a MPP+ induced PD cell model. Experiments are designed to investigate how DBYW&QZS modulates mitochondria in these cell models and whether DBYW&QZS has influences on PINK1/Parkin pathway. The answer to these questions may help to build more useful and specific clinical strategies.Method1. Establishing cell models: ① PD cell model:Apply 1mM MPP+ to SH-SY5Y cells for 24h. ② PINK1 overexpression cell model:Use Lipofectamine(?) 2000 to transfect SH-SY5Y cells with EGFP-PINK1 plasmid; optimize transfection condition by RT-PCR.③PINK1-PD cell model:Give PINK1 overexpression cells 1mM MPP+ treatment for 24h.2. DBYW&QZS treatment and groups:Use CCK-8 to select DBYW&QZS doses, cells are divided into①control group:treat cells with normal culture media only; ②BYW&QZS group:introduce 5 μg/ml or 25μg/ml DBYW&QZS to cells for 24h;③mdel group:1mM MPP+ treat cells for 24h; ④BYW&QZS treatment group:after 24h MPP+ treatment, change media to 5μg/ml or 25μg/ml DBYW&QZS for another 24h; ⑤negat ive control group:transfect cells with negative plasmid;⑥negative model group:transfect cells with negative plasmid, then apply lmM MPP+ to cells for 24h; ⑦egative DBYW&QZS treatment group:transfect cells with negative plasmid for 24h, give 1mM MPP+ treatment for 24h, then change media to 5μg/ml DBYW&QZS for another 24h;⑧PINK1 overexpression group:transfect cells with EGFP-PINK1 plasmid; ⑨ PINK1 overexpression model group:transfect cells with EGFP-PINK1 plasmid for 24h and change media to 1mM MPP+ for 24h;⑩PINK1 overexpression DBYW&QZS treatment group:transfect cells with EGFP-PINK1 plasmid for 24h, apply 1mM MPP+ to cells for 24h, then treat cells with 5 μg/ml DBYW&QZS for 24h.3. Apply CCK-8 to test cell viabilities in different groups.4. Stain live cells with MitoTracker(?) Red CMXRos, col lecting photos under confocal microscope to analyze mitochondria morphology and distribution; ImageJ is used to measure form factor (F-factor) and aspect ratio (AR) of mitochondria.5. Mitochondria function analyzing:include①mitochondria membrane potential (ΔΨm) analyzing by staining cells with TMRM; ②Use ATP assay kit to detect cell ATP level in different groups.6. Immunofluorescence test:Tom20 is used to represent mitochondria, which is a mitochondrial outer membrane protein commonly used to visualize mitochondria in immunofluorescence; observe whether Parkin and mitochondria are colocalized under different treatment.Result1. DBYW&QZS’s concentration selection:5 DBYW&QZS’s concentrations(3.906μg/ml、15.625μg/ml、62.5μg/ml、250μg/ml 1000μg/ml) were chosen, all of them show no inhibition on SH-SY5Y cell viability. Select 5μg/ml and 25μg/ml to observe DBYW&QZS’s effects on cells, after discover ing that 25μg/ml treatment tended to have more fragmented mitochondria, thus abort 25μg/ml and choose 5μg/ml as DBYW&QZS’s final treatment concentration for the rest experiments.3. Cell viability:treat cells with 1mM MPP+ for 24h can largely reduce cell viability by 50% (P< 0.001). Overexpress PINK1 or DBYW&QZS has little influence on cell viability.4. Mitochondria morphology:DBYW&QZS treatment alone would significantly increase F-factor (P<0.01); MPP+ would lead to more fragmented mitochondria (P< 0.001). Compared to model cells, apply DBYW&QZS would improve mitochondria length (P< 0.05). Also, up-regulated PINK1 would decrease both F-factor and AR of mitochondria (P< 0.001), but PINK1-PD cells have longer mitochondria than negative-PD cells. In addition, treat PINK-PD cells with DBYW&QZS would also increase mitochondria length (P< 0.001).5. Mitochondria membrane potential (ΔΨm):MPP+ treatment leads to significant ΔΨm drop in both negative model cells and PINK1 overexpression model cells (P< 0.01). While apply DBYW&QZS to model cells may have the inclination to improve ΔΨm, but statistically showed no different.6. Cell ATP level:Overexpress PINK1 would dramatically increase cellular ATP level (P< 0.05).24h MPP+ treatment will decrease cell ATP by 15%, while DBYW&QZS does not seem to improve model cells’ATP level.7. Colocalization of Parkin and mitochondria:Parkin is more likely to translocate to mitochondria after PINK1 being up-regulated (P< 0.05). MPP+ treatment also increase Parkin’s mitochondrial localization. In addition, treat PD cells with DBYW&QZS inclined to decrease Parkin and mitochondria colocalization.Conclusion1. DBYW-QZS shows no inhibition on SH-SY5Y cells viability.2. DBYW-QZS displays protective effects on mitochondria by increasing mitochondrial network in normal SH-SY5Y cells and improving PD model cells’mitochondria morphology. It also shows potential on maintaining PD cells’ATP level.3. Overexpress PINK1 leads to more fragmented mitochondria, increased cellular ATP level and more Parkin being recruited to mitochondria; also, PINK1 up-regulation shows resilient to MPP* induced mi tochondrial damage.4. DBYW-QZS increases PINK1-PD cells’mitochondral length, displays potential on maintaining ATP level and ΔΨ.