Objective

2

To investigate the effects of Paning Ⅰ Formula on oxidative stress-mediated ferroptosis pathway in dopaminergic neurons of Parkinson's disease（PD） mice，and to explore the mechanism of this formula in treating PD.

Methods

2

Sixty SPF-grade C57/BL6 mice were injected intraperitoneally with 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine （MPTP） to induce a PD model. Fifty successfully modeled mice were randomly divided into model group （9 g/L sodium chloride solution）， madopar group （97.5 mg/kg levodopa and benserazide hydrochloride solution）， and TCM low， medium and high dose groups （9.05 g/kg， 18.10 g/kg， 36.20 g/kg

Paning Ⅰ Formula）， with 10 mice in each group； an additional 10 mice were set as the normal group. The mice were administered the corresponding interventions via gavage for 14 consecutive days. The motor function of mice was evaluated by climbing pole， gait and open field analysis. The activity of mice substantia nigra neurons was detected by Nissl staining. The contents of dopamin （DA）， 3，4-dihydroxyphenylacetic acid （DOPAC） and 3-hydroxy-4-methoxyphenylacetic acid （HVA） in striatum of mice were detected by enzyme-linked immunosorbent assay （ELISA）. The expression of tyrosine hydroxylase （TH） in substantia nigra of mice was detected by immunohistochemistry. The deposition of Fe

3+

in substantia nigra of mice was detected by Prussian bl

ue staining. The contents of serum Fe

2+

， and the level of catalase （CAT）， glutathione （GSH）， glutathione peroxidase （GSH-PX）， malonic dialdehyde （MDA） and superoxide dismutase （SOD） were detected by colorimetry. Flow cytometry was used to detect reactive oxygen species （ROS） levels in mouse dopaminergic neurons. The protein expressions of acyl-CoA synthetase long chain family member 4 （ACSL4）， glutathione peroxidase 4 （GPX4） and solute carrier family 7 member 11 （SLC7A11） in the substantia nigra were detected by Western blot.

Results

2

①Motor function results showed that compared with the normal group， the step length and total moving distance of model group were shortened， and the pole climbing time was prolonged in the model group （

P

<

0.01）； Compared with the model group， dyskinesia of the each drug group was gradually decreased with the increased of gavage number of times， the improvement in the motor function was the most obvious at the day 12 after gavage （

P

<

0.05）. ②Nissl staining showed that compared with the normal group， the expression of average optical density in mice substantia nigra neurons was decreased in the model group （

P

<

0.01）； compared with the model group， the expression of average optical density in mice substantia nigra neurons was increased in medium， high dose groups and madopar group （

P

<

0.01，

P

<

0.001）. ③ELISA results showed that compared with the normal group， the content of DA， DOPAC and HVA were increased in the mice striatum of the model group （

P

<

0.001）； Compared with the model group， the content of DA was increased in high dose group and madopar group （

P

<

0.001）； the content of DOPAC and HVA were increased in medium， high dose groups and madopar group （

P

<

0.05，

P

<

0.001）. ④The immunohistochemical method results showed that， compared with the normal group， the expression of

TH in mice substantia nigra neuron was decreased （

P

<

0.001）； Compared with the model group， the expression of TH in high dose group and madopar group were increased （

P

<

0.01，

P

<

0.001）. ⑤Prussian blue staining showed that compared with the normal group， iron deposition and the iron death in the nigra of mice were increased in the model group； Compared with the model group， iron deposition and the iron death in the nigra of mice were decreased in each drug group. ⑥The colorimetric results showed that compared with the normal group， in the model group， the content of Fe

2+

was increased （

P

<

0.001）， the activity of CAT， GSH-PX and SOD were decreased （

P

<

0.001）， the level of GSH was decreased （

P

<

0.001）， the level of MDA was increased （

P

<

0.01）； Compared with the model group， in the medium， high dose groups and madopar group， the content of Fe

2+

decreased （

P

<

0.05，

P

<

0.001）， the activity of GSH-PX and GSH were increased （

P

<

0.05，

P

<

0.001）， in madopar group， the activity of CAT was increased （

P

<

0.05）， the level of MDA was decreased （

P

<

0.05）， in high dose group and madopar group， the activity of SOD was increased （

P

<

0.05）. ⑦Flow cytometry results showed that， compared with the normal group， the level of ROS in the nigra of mice was increased in the model group （

P

<

0.001）； Compared with the model group， the level of ROS was decreased in high dose group and madopar group （

P

<

0.01）； ⑧Results of Western blot showed that， compared with the normal group， in model group， the expression of ACSL4 protein in the nigra of mice was increased （

P

<

0.001）， the expression of GPX4 and SLC711 protein were decreased （

P

<

0.001）； Compared with the model group， in each drug group

， the expression of ACSL4 protein was increased （

P

<

0.05，

P

<

0.001）， the expression of GPX4 and SLC711 protein were increased （

P

<

0.01，

P

<

0.001）.

Conclusions

2

Paning Ⅰ Formula can alleviate motor dysfunction in MPTP-induced PD mice and has a neuroprotective effect. Its mechanism may be related to regulating iron metabolism， improving oxidative stress and inhibiting ferroptosis of dopaminergic neurons.