Baicalein Protects Against Nerve Damage By Induction Of Autophagy In PD Mice

Backgrouds:(1)Parkinson's disease(PD),a most common neurodegenerative disorder with progressive extrapyramidal dysfunction in middle-aged and elderly people,is characterized by static tremor,rigidity,bradykinesia and postural instability.It seriously affect the patients' life quality and increase burden of the society and patients' family.Until now,the disease is mainly treated with medication and can only be released by medication or surgery.There is no therapeutic method to prove could exactly retard or prevent the progression of PD.It is urged to find more effective treatment for this disease and thus it need as deeply as possible explore the etiology,pathogenesis and therapeutic method.(2)Autophagy,exits in nearly all eukaryotic cell,is a highly conserved degradation process of damaged organelles and misfolded proteins.The bilayer membrane dropped from the rough endoplasmic reticulum wrapped the supplement that need to be degraded and formed the autophagosome.The autophagosome and lysosome grown together to form the autolysosome which would degraded the enfolded content to produce amino acid and some other micromolecule compound to be reused or afford energy in order to satisfy the need of cell metabolism,organelle update and cellular basic vital movement.Basic autophagy is essential for neuronic steady state and ensuring the nerve cell's function normal.The deficiency or defect of autophagy will lead the collection of metamorphic a-Syn and some other harmful things.The abnormal autophagy will effect on eliminating dysfunctional mitochondria and leading the PD.Above all,autophagy play a key role in pathogenesis of PD and the regulation of aotuphagy may be a new strategy for PD's treatment.(3)Baicalein is a flavonoid derived from the root of the traditional Chinese herb Scutellariabaicalensis Georgi.Recent studies have shown that baicalein could exert neuroprotective effects in PD model through anti-inflammatory,anti-apoptosis,and anti-oxidative actions.It has been found that baicalein plays a role in anti-tumor,reduces liver ischemia/reperfusion injury by inducing protective autophagy.However,the exact mechanism of baicalein remains to be elucidated and whether baicalein could be used as protective autophagy inducer in the treatment of PD need further study.Objective:(1)The purpose of this study was to observe the activation of baicalein on autophagy in neural cells and to explore a new mechanism for its neuroprotective effect.(2)Through investigate the effects of regulated autophagy in apoptosis and mitochondrial function to find the relationship between the autophagy protection for PD's nerve injury via baicalein and the regulation of apoptosis and mitochondrial function.(3)The study aimed to explore the mechanism of autophagy protection for PD's nerve injury via baicalein and provide a theoretical basis for the research and development of autophagy modulators as novel drugs for the treatment of Parkinson's disease.Methods:(1)The preparation of mice models of PD,animal grouping and drug administration:Male inbred C57BL/6 mice(one-year old)were randomly divided into 6 groups-----control group,vehicle group,simple baicalein group,PD model group,baicalein + PD model group and 3-Methyladenine(3-MA)+ baicalein + PD model group.Mice in PD model group,baicalein + PD model group and 3-MA + baicalein + PD model group were given rotenone solution(5mg/kg,O.Olml/g)for 5 days a week,and the mice were continuously gavaged for 12 weeks.Baicalein was administered(100 mg/kg,intraperitoneally)daily from 7 week to 12 week.Mice were treated with 3-MA(30 mg/kg,intraperitoneally)from 10 week to 12 week.The mice in simple baicalein group baicalin only were given the same dose of baicalein in seventh to 12 weeks.Mice in vehicle group were received vehicle(4%carboxymethylc-ellulose and 1.25%chloroform)orally daily for 12 weeks.Mice in control group were not given any treatment.To measure autophagic flux,chloroquine(60 mg/kg,intraperitoneally)was administered 24 h before sacrifice.(2)Behavioural tests and assessment of striatal dopamine content:All behavioral tests were carried out during the day,and there was training 3 days before,to avoid anxiety and panic in mice,affecting the results of the experiment.Rotsarod test and grid test are used to behavioural test.The content of DA in the striatum of mice was determined by high performance liquid chromatography with electrochemical detection.(3)Cell culture,drug administration and experimental cell grouping:SH-SY5Y cells were cultured in vitro.Appropriate amount of SH-SY5Y cells were inoculated into 6 or 96-well black plate,cultured at 37 ? under a gas phase of air/C02(95:5)for 24 hours.SH-SY5Y cells were added with rotenone solution(2?mol/L).In order to observe whether baicalein can alleviate cell injury,baicalein(10?mol/L)was added into the culture medium to co-treated with rotenone.After cultured for 24 hours,we detect damage cell viability,apoptosis and mitochondrial.To measure autophagic flux,chloroquine(10 ?mol/L)was administered 6h before cells were collected.To investigate whether baicalein can alleviate cellular injury by upregulating autophagy,cells were treated with autophagy inhibitor 3-MA(10 mmol/L)1 hours before baicalein treatment.To observe whether autophagy could alleviate cellular injury,the cells were treated with rapamycin(100 nmol/L),which was administered by rotenone.Experimental cell were divided into 7 groups-----control group,vehicle group,simple baicalein group,rotenone group,baicalein + Rotenone group,3-MA +baicalein + Rotenone group and rapamycin + Rotenone group.(4)Cell viability assay:Cell viability was detected using the conventional MTT assay.The cell culture supernatant LDH was detected,and the cytotoxicity was analyzed.(5)Detection of apoptosis:The activity of Caspase-3 was determined by colorimetric assay,and the expression of Cleaved caspase-3 protein was detected by Western blot.(6)Assessment of mitochondrial function:Mitochondrial membrane potential was measured by JC-1 mitochondrial membrane potential analysis tool.(7)Autophagy detection:The expression of LC3B protein was detected by Western blot.The application of tool drugs(chloroquine,3-MA,rapamycin)artificially regulate autophagy and observe its changes in vivo and in vitro model.Results:(1)Behavioral detection in mice:Rotenone administration induced catalepsy in mice of PD model group.Compared with the control group or vehicle group,the time of descent latency of rotarod test and grid test were significantly shortened.However,baicalein treatment significantly ameliorated these behavioral deficits induced by rotenone toxicity.These results suggest that baicalein can reverse PD-related behavioral deficits caused by rotenone.(2)Assessment of striatal dopamine content in mice:Rotenone administration significantly reduced striatal dopamine content in PD model group compared to those in control group or vehicle group.However,baicalein significantly increased the striatal dopamine content in baicalein + PD model group when compared to those in PD model group.The above results show that baicalein partially restores striatal dopamine content in rotenone-treated mice.(3)Apoptosis detection of mice striatum:Compared to the control group or vehicle group animals,caspase-3 activity was significantly increased in the striatum of PD model group mice.Baicalein treatment significantly inhibited the increase of caspase-3 activity by rotenone.Furthermore,similar results were obtained by Western blot to detect the expression of cleaved-caspase3 protein,rotenone treatment increased cleaved-caspase3 protein expression levels,which were significantly prevented by baicalein treatment.Baicalein suppressed rotenone-induced apoptosis.(4)Detection of mitochondrial membrane potential in striatal cells of mice:Rotenone administration caused a decrease in mitochondrial membrane potential compared to the control group or vehicle group animals.However,baicalein treatment significantly prevented the rotenone-induced decrease in mitochondrial membrane potential.According to the above results,it is conjectured that baicalein prevented rotenone-induced mitochondrial dysfunction.(5)Modulation of baicalein on autophagy in striatal cells of mice:The expression levels of LC3B-II in PD model group mice were significantly decreased compared to the control group or vehicle group.However,baicalein significantly prevented the decrease in LC3B-II protein levels.LC3B-II expression levels were measured in the presence and absence of chloroquine.The expression levels of LC3B-II in brains from the chloroquine + baicalein +PD model was higher than those in the chloroquine + PD model,indicating baicalein could increase autophagic flux in the rotenone-treated mice.To determine whether the neuroprotective effect of baicalein was mediated via induction of autophagy,baicalein-induced autophagy was inhibited by a pharmacological autophagy inhibitor 3-MA.The protective role of baicalein on rotenone-induced apoptosis and mitochondrial dysfunction was blocked by 3-MA treatment.(6)The effect of baicalein on neurotoxicity induced by rotenone in SH-SY5Y cell:Compared with control group and vehicle group,Rotenone group significantly decreased in LC3B-? protein expression,the cell vitality and mitochondrial membrane potential,also increased significantly in the cell culture supernatant LDH,caspase 3 activity and Cleaved caspase 3 protein expression.Compared with rotenone group,baicalein + rotenone group significantly enhanced on LC3B-? expression,the cell vitality and mitochondrial membrane potential,also significantly reduced LDH activity,caspase-3 activity and Cleaved caspase 3 protein expression.Furthermore,a significant accumulation of LC3B-? by chloroquine was evident in baicalein-treated SH-SY5Y cells,indicating a robust autophagic flux with baicalein.Further studies have been conducted after the addition of autophagy inhibitor 3-MA or autophagic inducer rapamycin for autophagy intervention.3-MA + baicalein + rotenone group was compared with baicalein + rotenone group,significantly reduced the LC3B-? protein expression,the cell vitality and mitochondrial membrane potential,At the same time,significantly enhanced LDH activity,caspase-3 activity and Cleaved caspase 3 protein expression.There was no significant difference in rapamycin + rotenone group and baicalein+ rotenone group.Conclusions:(1)The PD model induced by environmental toxin rotenone can produce similar performance with human PD in behavioral and biochemical characteristics,which can be used to evaluate the neuroprotective effect of PD drugs.(2)In vivo and in vitro test results showed that baicalein could activate autophagy and promote autophagy flow.The protective effects of baicalein in inhibiting the neurotoxicity of rotenone,the function of anti-apoptosis,the protection of mitochondrial function and the enhancement of cell vitality are related to the induced autophagy.(3)Autophagy is a protective molecular pathway in PD.Autophagy regulation is a promising new approach to PD therapy.