Why Does Cistanche Have Effection On The Apoptosis Of PC12 Cells?

Effects of Cistanche on apoptosis of PC12 cells

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

Tian Jiyu¹, Chen Jianzong¹, Chen Xiaoli², Yang Hao³

¹Department of Traditional Chinese Medicine, ²Department of Pharmacy, Xijing Hospital, ³PLA Institute of Neurobiology, Fourth Military Medical University, Xi'an 710033, China

Abstract

AIM: To investigate the effects of Cistanche on 1- methyl-4-phenylpyridinium (MPP+)-induced PC12 cells apoptosis.

METHODS: PC12 cells were exposed to 200μmol/ L MPP+ alone or MPP+ combined with different concentrations of Cistanche from New Zealand White rabbit serum. Effects of Cistanche on apoptosis of PC12 Cells were detected by flow cytometry, electron microscopy, and fluorescence microscopy.

RESULTS: The sera collected from rabbits fed on an equivalent dosage of Cistanche or on a twofold equivalent dosage of Cistanche had obvious protective effects on PC12 cells and the apoptosis rates of PC12 cells incubated with these two sera were 2.8 % and 10 .2%, respectively, which were obviously lower than those of PC12 cells with MPP+ treatment only(18 . 6 %, P<0 .05).

CONCLUSION: Cistanche can protect PC12 cells from apoptosis induced by MPP+.

Keywords: Cistanche Deserticola, 1-methyl-4-phenylpyri- dinium, PC12 cells, apoptosis

Introduction

The pathogenesis of Parkinson's disease (Parkinson's disease, PD) is the damage and loss of substantia nigra dopaminergic neurons, and apoptosis may be an important reason. We use flow cytometry (FACS), transmission electron microscopy, and fluorescence staining of nuclear Shape analysis and other methods to detect the Chinese medicine Cistanche medicated serum and neurotoxin 1-methyl-4-phenylpyridinium (1-methyl-4-phenylpyridinium, M PP +) treated PC12 cells, to explore the effect of Cistanche on neurons The protective mechanism of action.

Effects of Cistanche on apoptosis of PC12 cells

1. Materials and methods

1.1 Materials

PC12 cells were purchased from the Japan Life Science Foundation; RP-MI1640 culture medium, calf serum, and rat tail collagen were purchased from Gibco; MPP+, Annexin V-FITC, PI, PBS, cell culture flasks and culture plates were purchased from Sigma; Chinese medicine Cistanche was purchased from Xi'an Decoction Piece Factory; New Zealand big-eared white rabbit (male, body weight 2.5 ~ 3 kg) was provided by the school animal center; Hoechst33342 was purchased from Biyuntian Company; Paraformaldehyde was domestically pure; LD4- Type 2 centrifuge (Beijing Medical Centrifuge Factory) Instruments Fluorescence microscope (Laika company), inverted microscope (Olympus company), JEM-2000EX transmission electron microscope (Japan), CO2 constant temperature incubator (Here-cus HERAcell, Germany), Coulter EPICS flow cytometer (Beck-man Coulter, USA).

1.2 Method

PC12 cells were grown in RPMI1640 cell culture medium, which contained 100 mL/L calf serum. The cell culture flask and culture plate were covered with 0.2 g/L rat tail collagen before use. The cultured cells were placed at 37 ℃, In a 50 mL/L CO2 incubator, change the medium for 3 days. When the monolayer culture cells are about to fuse, digest with 1.25 g/L trypsin and subculture. When the subculture cells enter the logarithmic growth phase, you can add drugs. According to the bodyweight of the rabbit, the equivalent dose of the animal is converted according to the body surface area. The traditional Chinese medicine Cistanche is prepared as a freeze-dried powder, which is diluted with distilled water when used, and the animal is administered twice a day for 3 days (Chinese Experimental Formula Chinese Journal of Science, 1998; 4:13-15), 1 h after the last ig, carotid bloodletting, overnight at 4 ℃, centrifugation at 2500 r/min for 25 min, serum separation, inactivation at 56 ℃, 30 min, and 0. Sterilization with 22 μm filter membrane and storage at -20 ℃. Under the same conditions, blank rabbit control serum, 1/2, 1, and 2 times equivalent dose of serum were prepared. The experiment was divided into 5 groups: Group A was a blank rabbit Serum group, group B is 2 times the equivalent dose of serum + MPP + group (the gavage dose is cistanche crude drug 6.4 g/kg), group C is the equivalent dose of serum + MPP + group (the gavage dose is cistanche crude drug 3.2 g/kg), group D is a half-equivalent dose of serum + MPP + group (in dose is Cistanche crude drug 1.6 g/kg), group E is a blank rabbit serum + MPP + group. Subculture for 12 h In the future, observe the cells under an inverted microscope and add the drug before the confluence. The serum content of each group is 10%. After culturing for 3 days, MPP + is added to the final concentration of 200 μmol/L. Placed at 37 ℃, 50 mL/L CO2 culture and Check it after 24 hours incubation.

1.2.1 PC12 cell fluorescence staining observation

Add Hoechst33342 to the cultured PC12 cells at a final concentration of 10 mg/L, incubate at 37 ℃ for 30 min, mix 40 g/L paraformaldehyde and the culture medium at 13: to make the final concentration of paraformaldehyde 10 g/L After fixation for 5-10 min, discard the supernatant, wash with PBS, and observe under a fluorescence microscope.

1.2.2 Observation by transmission electron microscope

After 24 hours of drug treatment, the cells were collected and transferred to a centrifuge tube, centrifuged at 1500 r/min, 4 ℃ for 5 min, and washed twice with 10 mmol/L PBS. Centrifuge at 1500 r/min, 4 ℃ for 5 min, discarded Supernatant, slowly add 25 mL/L glutaraldehyde along the wall of the tube and fix at 4 ℃ for 2 h, peel off the cell clumps with a straight pipette, wash 3 times with PBS, 5 min each time, fix for 1 h after osmium acid at 4 ℃ Post-gradient acetone dehydration, epoxy resin soaking, and embedding, sliced with LKB-2188 ultra-thin microtome, stained with uranyl acetate-lead citrate, observed by transmission electron microscope and photographed.

1.2.3 Flow cytometry detection

M PP + treatment for 24 h, digest 106 adherent PC12 cells with 1.25 g/L trypsin, wash twice with PBS, add 490 μL binding buffer, mix the cells thoroughly, and add 5 μL Annexin V-FI TC Mix 10 μL of the dissolved PI in the cell suspension, mix gently, place the test tube at 4 ℃, incubate for 10 min in the dark, and perform detection on a flow cytometer. Each group has 4 replicate wells.

Statistical processing: The data is represented by x ±s, the comparison between multiple groups is performed by the Kruskal-Wallis test, and the pairwise comparison is performed by the Mann-Whitney test, and P<0.05 is considered as a significant difference.

Effects of Cistanche on apoptosis of PC12 cells

2. Results

2.1 Fluorescence microscope observation of PC12 cells

Normally grown PC12 cells can emit uniform fluorescence after being stained by Hoechst33342, and densely stained granular mass fluorescence and dotted white fluorescence can be seen in the apoptotic cell nucleus. The nucleus of most cells in the normal group is relatively uniform. Blue fluorescence, faintly visible dot-shaped condensed dot-shaped fluorescence, its condensed dot-shaped fluorescence is weaker than MPP + control cells. Normally grown cells show uniform blue fluorescence after Hoechst staining, and the nucleus morphology is regular (Fig. 1A); After MPP+, the nuclear chromatin appears concentrated, or highly condensed, showing irregular dot fluorescence, or marginalized; late cell nuclei split into fragments (Fig 1B). Equivalent dose and 2 times the equivalent dose The cells in the Cistanche medicated serum + M PP + group were close to normal (Fig 1C, D).

Fig 1 PC12 cells stained with Hoechst33342(Ho)

A :Control:The chromatin evenly distributed and stained regularly ;B : MPP+ treated:The condensation and margination of the chromatin on nuclear membrane;C :Equivalent Cistanche + MPP+ treat ed;D :Twofold equivalent Cistanche + MPP+ treated , the chromatin is normal.

2.2 Observation by projection electron microscope

Normally cultured PC12 cells have uniform nuclear chromatin, clear and complete nuclear membrane, abundant cytoplasmic mitochondria, and complete organelles (Fig 2A). After the MPP+ treatment, PC12 cells have changed significantly, the cell membrane is intact, the surface microvilli disappeared, and the cytoplasm is concentrated, Chromatin is condensed, broken, highly condensed, and gathered on the inner surface of the nuclear membrane, showing the characteristics of apoptosis, mainly in early and mid-stage apoptotic cells (Fig 2B, C). Apoptotic cells in each group containing Cistanche sera are obvious Decrease, the nuclear chromatin is evenly distributed, the nuclear membrane is clear and complete, and the organelles are intact, close to the cell shown in Fig 2A.

2.3 Flow cytometry test results

Phosphatidylserine (PS) located on the inner side of the cell membrane migrated to the outer side of the cell membrane in the early stage of apoptosis. The apoptotic rates of each group were (2.3 ± 0.11)%, (2.8 ± 0.21)%, (10.2 ± 0.60)%, (16.0 ± 0.87)% and (18.6 ± 0.67)%. The rank-sum test analysis of B, C, D group compared with E group P <0.05. There is a significant difference between serum-containing Chinese medicine and the blank serum group.

Fig 2 PC12 cells ultrastructure EM ×6000

A: Without MPP + uniformly dispersed chromatin lightly electron-dense cytoplasm, normal organelle structure and intact cell membrane; B, C: Exposed to 200 μmol/ L M PP +.Early apoptotic phase, peripheral chromatin condensation, nearly normal cytoplasm structure(B). Late post-ot- ic phase, further chromatin condensation, increased cytoplasmic electron density, absence of normal organelle structure, and disruption of cell membrane integrity(C).

3. Discussion

The neurotoxicity of M PP + is a complex process of multi-component action, including the effect of hindering the replacement of mitochondria and synaptic vesicles with dopamine to produce reactive oxygen species [1]. At present, most of them are believed to be related to mitochondrial cell apoptosis and endogenous The neurotoxic effect of dopamine leads to apoptosis-related [,2] In the process of observing the apoptosis of the substantia nigra dopamine cell line SN4741 by M PP +, there is the activation of Caspase-1 and Caspase-3 [3] .1 μmol/ L’s MPP+ acts on SH-SY5Y cells of adrenal neuroblastoma and can increase the expression of apoptosis-related Bcl-2 protein after a few days [4]. Cistanche is a dry, scaly leaf of the Cistanche genus (Cistanche) of the family Cistanche. The fleshy stem of cistanche has a good curative effect in the treatment of Alzheimer’s disease and PD. The total glycosides of cistanche can effectively scavenge various active oxygen free radicals and can protect the DNA oxidation caused by OH. [5]. Sheng et al. [6] research confirmed that Cistanche The extract tubulcoside B can attenuate MPP+-induced cytotoxicity, weaken the accumulation of reactive oxygen species in the cell, and have an antagonistic effect on MPP+-induced apoptosis and oxidative stress. We have observed that Cistanche has an antagonistic effect on MPP+-induced PC12 The effect of cell apoptosis, and the protective effect of PC12 cells increases with the increase of dose.

Effects of Cistanche on apoptosis of PC12 cells

Reference

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From: Journal of the Fourth Military Medical University (J Fourth Mil Med Univ) 2004; 25(10)

Article ID: 1000-2790 (2004) 10-0955-03