Salidroside Protects Dopaminergic Neurons Via Regulation Of MEF2D-ND6 Pathway

Parkinson’s disease(PD) is a common neurodegenerative disease. Its main pathological characteristic is the degeneration, death and missing of dopamine(DA) neurons in substantia nigra pars compacta(SNpc). Oxidative stress and mitochondrial dysfunction has been widely believed to be the central pathological mechanism of PD. At present, without clear the etiology and pathogenesis of PD caused the clinical treatment is mainly symptomatic treatment, which cannot prevent or delay the process of PD, and also lack of the effective protection drugs for the DA neurons.Myocyte enhancer factor 2(MEF2) was firstly found in the study on the differentiation of muscle cells. The sub-types MEF2(including MEF2 A, MEF2 B, MEF2 C and MEF2D) play a central role in the control of cell proliferation, differentiation, morphogenesis, survival and apoptosis. MEF2 D has been proved to be closely related to the differentiation and survival of neurons, and importantly play a key role in the survival of DA neurons. Further, it has been found that MEF2 D expresses in the mitochondria. Rreducing mitochondrial MEF2 D expression level could significantly inhibit the function of mitochondrial respiratory chain complex I(referred to as the mitochondrial complex I) and leaded to neuronal death, whereas overexpression of mitochondrial targeting MEF2 D could resist neurotoxins induced DA neuronal death.Mitochondrial DNA(mt DNA) is the genetic substance in the mitochondria, and also the only substance with a genetic effect outside the nucleus. The mitochondrial complex I contain 46 subunits, 7 subunits of them are encoded by mt DNA, and the rest of the 39 subunits are encoded by the nuclear gene which import into the mitochondria and then assembled with the mt DNA to encoding the subunit. NADH dehydrogenase(ND) is a necessary encoding component of the mitochondrial complex I, and ND6 is the only protein encoded by the L strand, while the remaining 6 subunits are encoded by the H strand. Mitochondrial MEF2 D can directly bind to the MEF2 site in ND6 to regulate its transcription, and then regulate the activity of mitochondrial complex I and the function of mitochondrial. However, the site mutations will interfere with the formation of mitochondrial complex I. The studies have been found that the level of MEF2 D and ND6 in the brain of PD patients was significantly decreased. Thus, the MEF2D-ND6 pathway may be a new potential therapeutic target for the treatment of PD.Salidroside(Sal), also known as the 2-(4-Hydroxyphenyl)ethyl β-D-glucopyranoside, is one of the main active ingredient of Rhodiola rosea L. in Traditional Chinese Medicine. Pharmacological studies show that Sal has powerful antioxidant properties, and also play important roles in anti-inflammatory, anti-proliferation and anti-apoptosis. In our preivous study, we have found that Sal could improve the behavior coordination ability, increase the number of TH positive neurons and the content of DA in MPTP-PD mice. Moreover, Sal could also inhibit the activation of mitochondrial apoptotic pathway, and regulate the apoptosis factor, ROS-NO pathway and PI3K/Akt pathway, and activate the Nrf2 to reduce MPP~+ induced apoptosis in neuronal cells.In this study, the aim is investigate whether Sal can through regulating MEF2D-ND6 pathway to prevent the damage of mitochondrial complex I to protect DA neurons in MPP~+/MPTP-PD model. It will provide experimental basis evidents for Sal as a potential new drug in the treatment of PD.Part I Protective effect of Sal on MPP~+-PD model in SN4741 cells.Objective: This study aim to investigate whether Sal reduces cell apoptosis in the MPP~+-PD model in SN4741 cells. Methods: After treated by groups, cell activity was measured by MTT assay, cell toxicity was measured by LDH cytotoxicity assay, DHE was detected the level of superoxide anion(O2—), DCFH-DA was detected the level of ROS, Mito Tracker was detected the length of mitochondrial, Tetramethylrhodamine, ethyl ester(TMRE) was detected the mitochondrial membrane potential(ΔΨm), mitochondrial complex I activity assay kit was detected the mitochondrial complex I activity. Results: MTT tests showed that Sal cannot change the cell activity in differenct dose(P>0.05), the MPP~+(50 μM) Group could incduced the cell activity decreased into IC50 level(P<0.01), Sal +MPP~+ Group could protect the cell activity maintaining at 86.7±9.5%(P<0.01). TUNEL and Annexin V-FITC/PI tests showed that Sal could not increase the rate of apotosis(P>0.05), MPP~+ Group increased the rate of apotosis(P<0.01), however pre-treatment with Sal decreased the rate of apotosis in Sal+MPP~+ Group(P<0.01). The LDH tests showed that Sal could not increase the cytotoxicity in Sal Group(P>0.05), it increased the cytotoxicity in MPP~+ Group(P<0.01), however pre-treatment with Sal decreased the cytotoxicity in Sal+MPP~+ Group(P<0.01). The level of O2— and ROS had no changes in Sal Group(P>0.05), it increased their level in MPP~+ Group(P<0.01), however pre-treatment with Sal decreased their level in Sal+MPP~+ Group(P<0.01). Mito Tracker tests showed that Sal could not change the length of the mitochondrial(P>0.05), it reduced the length of mitochondrial which occurred fragmentation in MPP~+ Group(P<0.01), however pre-treatment with Sal increased the length of mitochondrial in Sal+MPP~+ Group(P<0.01). ΔΨm assay showed that Sal could not change the level of ΔΨm in Sal Group(P>0.05), it decreased the level of ΔΨm in MPP~+ group(P<0.01), however pre-treatment with Sal increased the level of ΔΨm in Sal+MPP~+ Group(P<0.01). The ratio of mitochondrial complex I activity had no change in Sal Group(P>0.05), it decreased its level in MPP~+ group(P<0.01), however pre-treatment with Sal increased its level in Sal+MPP~+ Group(P<0.01). Conclusion: Sal can improve the cell activity, inhibit the apoptosis, and also stabilize mitochondrial morphology and function. It also has antioxidant effect in the MPP~+-PD model in SN4741 cells.Part II Protective effect of Sal for MEF2D-ND6 pathway in MPP~+-PD model in SN4741 cells.Objective: This study aims to investigate whether Sal protects the MEF2D-ND6 pathway in MPP~+-PD model in SN4741 cells. Methods: Luciferase reporter gene was used to detect the transcription level of MEF2 D. Western blot were used to detect the protein level and the protein level in cytoplasm, mitochondria and nucleus of MEF2 D and ND6, q PCR was detected the m RNA level of MEF2 D and ND6, immunofluorescence was detected the co-localization level of MEF2 D and mitochondria. Results: Luciferase reporter gene detection showed that Sal could not increase the transcription level of MEF2D(P>0.05). It significantly decreased its level in MPP~+ Group than that of in WT MEF2 D Group(P<0.01), however its level was increased in Sal+MPP~+ Group(P<0.01). Western blot assay showed that the protein level of MEF2 D had no changes in Sal Group(P>0.05), it decreased in MPP~+ group(P<0.01), however pre-treatment with Sal increased its level in Sal+MPP~+ Group(P<0.01). The q PCR assay showed that the m RNA level of MEF2 D had no changes in Sal Group(P>0.05), it decreased in MPP~+ group(P<0.01), however pre-treatment with Sal increased its level in Sal+MPP~+ Group(P<0.01). The results of immunofluorescence and Western blot assay showed that the proteins level of MEF2 D in cytoplasm, mitochondria and nucleus had no changes in Sal Group(P>0.05), they were obviously decreased in MPP~+ group(P<0.01), however pre-treatment with Sal were obviously increased their level in Sal+MPP~+ Group(P<0.01). Western blot assay showed that the protein level of ND6 had no changes in Sal Group(P>0.05), it decreased in MPP~+ group(P<0.01). Pre-treatment with Sal increased the protein level of ND6 in Sal+MPP~+ Group(P<0.01), however it still lower than that of in Normal Control Group(P<0.01). The q PCR assay showed that the m RNA level of ND6 had no changes in Sal Group(P>0.05), it decreased in MPP~+ group(P<0.01). Pre-treatment with Sal increased the m RNA level of ND6 in Sal+MPP~+ Group(P<0.01), however it still higher than that of in Normal Control Group(P<0.01). Conclusion: Sal protects the MEF2D-ND6 pathway in MPP~+-PD model in SN4741 cells.Part III Effect of Sal in MPP~+-PD model in SN4741 cells after used sh MEF2 D and Mt2 Ddn.Objective: This study aims to investigate whether the protective effect of Sal in MPP~+-PD model after used sh MEF2 D and Mt2 Ddn in SN4741 cells. Methods: The cell activity was tested by MTT, MEF2 D and ND6 protein level was tested by Westerning Blot, Mito Tracker was dected the mitochondrial morphology, mitochondrial complex I activity assay was detected the mitochondrial complex I activity. Results: After transfected the sh MEF2 D, the cell activity had no significant difference compared with the Control Group(P>0.05). In sh MEF2D+MPP~+ Group, the transfected sh MEF2 D increase the susceptibility for MPP~+, which showed that the cell activity decreased, the protein level of MEF2 D and ND6 decreased, mitochondrial length reduced to 27.2±4.5%, the mitochondrial complex I activity reduced in MPP~+ treated only 12 h(all P<0.01). However, sh MEF2D+Sal+MPP~+ Group, it could not increase the cell activity, the protein level of MEF2 D and ND6, the mitochondrial length and the mitochondrial complex I activity, which had no statistical significance compared with sh MEF2D+MPP~+ Group(all P>0.05). After transfected the Mt2 Ddn, which was blocked the MEF2 D binding site in ND6, the cell activity was significantly decreased, the protein level of MEF2 D and ND6 were reduced, mitochondrial length also shortened and the mitochondrial complex I activity reduced(all P<0.01). In Mt2Ddn+Sal Group, Sal could not increase the cell activity, the protein level of MEF2 D and ND6, the mitochondrial length, the mitochondrial complex I activity, which had no significance differences compared with the Mt2 Ddn Group(P>0.05). Conclusion: The protective effect of Sal may be through the MEF2D-ND6 pathway in MPP~+-PD model in SN4741 cells.Part IV Neuroprotective effects of Salidroside via MEF2D-ND6 pathway in the MPTPPD model.Objective: This suty aims to investigate whether Sal through MEF2D-ND6 pathway plays a neuroprotective effect in the MPTP-PD model. Methods: All mice were randomly divided into 4 groups(n=10 per group). The pole test was used to detect the coordination of body movement in mice. The open field test was used to detect the changes in exercise capacity of mice. Immunofluorescence staining was used to observe the TH positive cell number in SNpc. Western blot was used to detect the protein level of MEF2 D and ND6. The mitochondrial complex I activity assay kit was detected the mitochondrial complex I activity. Results: In pole test, T-turn and T-LA time had no changes in Sal Group(P>0.05). They were significantly increased their time in MPTP Group(P<0.01). Pre-treatment with Sal significantly reduced the T-turn and T-LA time in Sal+MPTP Group(P<0.01). In open field test, the locomotion frequency and the immobility time had no changes in Sal Group(P>0.05). The locomotion frequencwere significantly reduced and immobility time was increased in MPTP Group(P<0.05). Pre-treatment with Sal significantly restored locomotion frequency and reduce the immobility time in Sal+MPTP Group(P<0.01). Immunofluorescence showed the number of TH positive neurons had no changes in Sal Group(P>0.05). It was significantly decreased in MPTP group, which had a statistical significant when compared with the Control Group(P<0.01). However, Pre-treatment with Sal could protect its number in Sal+MPTP Group(P<0.01). In addition, the protein level of MEF2 D and ND6 had no changes in Sal Group(P>0.05). They were significantly decreased in MPTP Group(P<0.01). Pre-treatment with Sal could recover their protein level in Sal+MPTP Group(P<0.01). Mitochondrial complex I activity detection showed that the activity of mitochondrial complex I had no changes in Sal Group(P>0.05). It significantly decreased its activity in MPTP Group(P<0.01). Pre-treatment with Sal could recover the mitochondrial complex I activity(P<0.01). Conclusion: Sal protects the MEF2D-ND6 pathway to promoting the survival of dopaminergic neurons in MPTP-PD model.