Protective Effects Of Proanthocyanidin On LPS-Induced Microglia Inflammation And Rotenone-Induced Dopaminergic Neuron Injury

Parkinson's disease(Parkinson's disease, PD) is the second largest neurodegenerative diseases, Which have a seriously impacts on the health of elderly patients, with the Pathological features of dopaminergic neuron degeneration and necrosis in substantia nigra pars compacta, the detail pathogenesis of which is not clear yet. There is no accurate reliable drug delay or blocking the onset of PD so far. Studies have shown that proanthocyanidin possess efficient neuroprotective effects in the brain, showing prosperous prospects in the prevention and treatment of PD. This study commences with establishing the cell model of Parkinson's disease, by using LPS to activate microglia BV2 cells for inflammatory damage and rotenone to induce oxidative-stress damage in dopaminergic neuronal SH-SY5Y cells, and then observe the influences of proanthocyanidin on them.To further explore the protective effects of proanthocyanidin(PC) on microglia inflammation and dopaminergic neurons oxidative damage based on the perspectives of inflammation, mitochondrial dysfunction, oxidative stress, and apoptosis.The experimental scheme is made up of two parts as follows,The first part:Protective effects of proanthocyanidin on LPS-induced microglia inflammatory injury in vitroPurposesBV2 cells were treated with LPS as oxidative stress damage cell model of Parkinson disease, and to explore the protective effects of proanthocyanidin on inflammatory mediators secreting in LPS-induced BV2 cells, and the effects of PC on activated BV2 cell induced inflammatory lesion in SH-SY5Y cells lines. And to investigate the possible mechanisms for suppression effect of PC on activated BV2 cells based on the transcription factor NF-?B pathway.Methods1. BV2 cells were stimulated with different concentrations of LPS(0.01,0.1,1.0,10.0 ?g/mL) for 24 h, Griess assay was used to measure the nitric oxide(NO) levels in supernatants of cultures, select the appropriate concentration of LPS to construct inflammatory injury model of BV2 cells in subsequent experiments.2. MTT assay was used to evaluate the effects of LPS as well as proanthocyanidin on BV2 cytotoxic. The experimental groups divided into the zero setting group, normal control group, LPS(1.0 ?g/mL) treated group, proanthocyanidin(0.1,0.5,1.0,5.0 ?g/mL) groups, and LPS(1.0 ?g/mL) plus proanthocyanidin (0.1,0.5,1.0,5.0 ?g/mL) groups, after incubation 24h and the absorbance was detected by the enzyme standard instrument, then the survival rate was calculated.3. The cell cultures of LPS(1.0 ?g/mL) activated BV2 as inflammatory cell cultures of conditioned medium(conditioned medium, CM) to incubate dopaminergic neurons SH-SY5Y cells,and divided into control group, LPS groups, resting BV2 supernatant(C-CM) groups, LPS-activated BV2 cells conditioned medium(LPS-CM) group and LPS-CM+proanthocyanidin(0.1,0.5,1.0,5.0 ?g/mL) group, respectively, incubated for 24h, MTT assay was used to detect cell viabilities in above groups.4. Groups were setting by LPS activated BV2 cells, along with a control group, LPS group and LPS+ proanthocyanidin(0.1,0.5,1.0,5.0 ?g/mL) group. The culture supernatant in each group were collected after 24h, the Griess method was used for nitric oxide (NO) levels detection; ELISA assay were used to measure the level of tumor necrosis factor-a(TNF-a), interleukin-1?(IL-1?) and interleukin-6(IL-6) secretion levels; and WST-1 assay was used for evaluate superoxide(superoxide) consentration.5. To investigate whether the inhibition effects of proanthocyanidin were associated with the activation of NF-?B, groups were seted as control group, proanthocyanidin(5.0 ?g/mL), LPS(1.0 ?g/mL) group and LPS+proanthocyanidin(0.1,0.5,1.0,5.0 ?g/mL) group, treated for 1h, Western blot was used to detect the expression of NF-?B p65 and phospho-NF-?B p65's.Rresults1. Compared with the control group, there was a significant increase in NO release in BV2 cells treatet with various concentrations of LPS, among them, the group of 10.0 ?g/mL LPS and 1 ?g/mL present no significantly difference in the increasing NO level, based on which, a lower concentration of 1 ?g/mL LPS was selected for model construction.2. Results of MTT assay, showed that, proanthocyanidin of 0.1,0.5,1.0,5.0 ?g/mL alone or with 1.0 ?g/mL LPS treated BV2 cells for 24 h, presented no significant statistically difference in survival rates compared with the control group(P>0.05), indicating that among the dose range used in this experiment, proanthocyanidin exhibit no toxic effects on resting state or LPS activated BV2 cells.3. Compared with the control group, cell viabilities of LPS(1.0 ?g/mL) group, and resting BV2 cell supernatant of SH-SY5Y cell lines showed no significant difference. While the groups treated with LPS-activated BV2 cell conditioned medium caused a 55% decline in cell viability, and a co-incubation of proanthocyanidin significantly improved the survival rate of SH-SY5Y cells.4. Compared with the control group, LPS treatment significantly increased the secreting of inflammatory mediators NO, TNF-?, IL-1?, IL-6 and the levels of superoxide in BV2 cell lines. While different concentrations of proanthocyanidin inhibited the release of inflammatory mediators activated BV2 cells in a dose dependent manner.5. Compared with the control group, LPS significantly increased the NF-?B p65 phosphorylation levels in BV2 cell lines, each concentration of proanthocyanidin showed a reduction in LPS-induced expression of phosphorylated NF-?B p65, suggesting that proanthocyanidins inhibit NF-?B pathway activation.ConclusionLPS activated BV2 cells induce massive release of inflammatory mediators, which damage the SH-SY5Y cells; proanthocyanidin reduced the release of inflammatory mediators from BV2 cells by the down regulated transcription factor NF-?B activation, thus protected against microglia-mediated SH-SY5Y cell injury.The second part:The protective effect of proanthocyanidins on rotenone-induced SH-SY5Y cell injuryPurposesThe invitro model of Parkinson's disease cell model was constructed by rotenone acting dopaminergic neuronal SH-SY5Y cells to induced oxidative injury, and to investigate the protective effects of proanthocyanidin on rotenone-induced SH-SY5Y cells damage in the perspective of oxidative stress and apoptosis in cells.Research method1. Different concentrations of rotenone(0.04?0.2?1.0?5.0 ?M) were used to treat SH-SY5Y cells for 24h, MTT assay was used to detect the impact of rotenone on SH-SY5Y cell activity, select the appropriate concentration of rotenone to injury SH-SY5Y cells and construct cells model of Parkinson's disease for subsequent experiments.2. To clarify the injury mechanisms of rotenone, SH-SY5Y cells was exposed to rotenone(1.0 ?M) 24h.oxidative stress was evaluated by detecting SOD, MDA levels in endogenous cells, and mitochondrial function were assessed by JC-1 fluorescent probe for detecting mitochondrial membrane potential. At last Annexin V-FITC/PI double staining for apoptosis rate, and colorimetric detection of activation level of caspase-3 were carried out to analyze whether rotenone induces apoptosis of SH-SY5Y cells.3. Proanthocyanidin(0.1,0.5,1.0,5.0 ?g/mL)+rotenone(1.0 ?M) were used to treat SH-SY5Y cells after 24h, cell viability, the levels of SOD, MDA, mitochondrial membrane potential and apoptosis rate and caspase-3 activation level were assayed to analyze whether proanthocyanidin reduced SH-SY5Y cells apoptosis by enhancing the ability against rotenone-induced-oxidative stress and improving mitochondrial function.Results1. Viabilities of SH-S Y5 Y cells decreased sharply with the increase concentration of rotenone. There was no statistically significant difference between 1.0 ?M-rotenone group and 5.0 ?M group, with cell viability of 50% in rate of control groups, based on which, the lower concentration 1.0 ?M rotenone was chose in model construction.2. SH-SY5Y cells were damaged after rotenone-induced, with the level of intracellular SOD and mitochondrial membrane potential decline, while, MDA content increased, resulting in an increase in apoptosis rate, and caspase-3 activation level, indicating that rotenone caused apoptosis in SH-SY5Y by increasing the cellular oxidative stress and inducing mitochondrial dysfunction.3. Compared with rotenone group,cell viability in groups pretreated with different concentrations of proanthocyanidin, were increased significantly, as well as the SOD levels in SH-SY5Y cell lines, and simultaneously, and mitochondrial membrane potential were partial restored, while the level of MDA content decreased and finally the level of caspase-3 activation and the rate of apoptosis reduce.ConclusionAll the results suggest that the mechanisms of Proanthocyanidin protection on rotenone-induced dopaminergic neuronal SH-SY5Y cell death by adjusting the level of oxidative stress, recovoring mitochondrial membrane potential, and reducing apoptosis of SH-SY5Y cells.