Does cistanche total glycoside have effects on the olfactory?

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The effect of cistanche total glycosides on rat olfactory ensheathing cells and their secretion of neurotrophic factors

Li Chuangfeng, Guo Jinyu, Zhang Xu, Liu Yunnan

Spinal cord injury (SCI) is a serious injury of the central nervous system, accounting for 0.2%—0.5% of total trauma. In spinal fractures, 16%—40% are complicated by spinal cord injury, often leaving severe disability, leading to Difficulties in daily life affecting the quality of life of patients, and causing a great burden to the family and society. Olfactory ensheathing cells (OECs) are a special type of nerve glue. Plasma cells can migrate into the brain with olfactory nerve axons and can secrete many growth factors such as NGF, GDNF, BDNF, NT-3, neuropeptide-Y, and S-100. Due to these characteristics of olfactory ensheathing cells, OECs are considered to be one of the promising seed cells for cell transplantation to repair SCI. At present, the treatment of SCI in the field of rehabilitation medicine is mainly based on comprehensive rehabilitation. In order to find a more effective method, the following research has been carried out on the effect of cistanche total glycosides on OECs:

Cistanche total glycoside for Olfactory ensheathing cells

1 Material and methods

1.1 Experimental materials

Wistar rat (provided by the Animal Center of Inner Mongolia University); cistanche total glycosides, DMEM/F12 medium, fetal calf serum (FCS), poly-D-lysine, arabinoside (Ara-C), trypsin, D -Hanks solution, rabbit anti-p75NGFR (Santa Cruz), ELISA kit (Promega), cell incubator (Thermo-HEPA Class 100), inverted phase-contrast microscope (Olympus CKX41, Japan), ultra-clean workbench (Suzhou Antai Air Technology Co., Ltd.) The company, SW-CJ-2FD), enzyme label analyzer (Wellscan MKH), flow cytometer, etc.

1.2 Research methods

1.2.1 OECs culture, purification, and identification: Wistar rats were sacrificed by decapitation and soaked in wine for 5 minutes. The anterior skull was opened to expose the olfactory bulb. The two olfactory bulbs were completely removed under a dissecting microscope. Wash in D-Hanks solution; cut the olfactory bulb with microsurgical scissors, add 2ml of 0.25% pancreatin and incubate at 37°C for 20 minutes; add D MEM/F12 culture medium containing 10% calf serum to a 2ml centrifuge tube Digestion was terminated in the middle; the bus pipette aspirated the incompletely digested tissue at 800 rpm and centrifuged for 5 min, discarded the supernatant; finally used the conditioned medium (20% FCS + 20|i, mol/LForskolin + 20|ig/mlBPE + DMEM /F12) Make it into lx 107ml single-cell suspension, inoculate it in a polylysine-coated cell culture flask, place it in a 37°C, 5% CO2 incubator, and then add Ara- c Purification was carried out at 36h. Use rabbit anti-p75E and FITC-labeled goat anti-rabbit IgG secondary antibody for immunohistochemical cell identification and fluorescence microscope observation.

1.2.2 Use MTT and flow cytometry to determine the effect of cistanche total glycosides on olfactory ensheathing cells: inoculate the cultured and purified olfactory ensheathing cells in a 96-well plate at 1X106, and add cistanche total glycosides (action concentrations are Ijig/ml, 10mg/ml, 100mg/ml), measure the OD value and measure the cell flow cytometry after 24h of action. Set up Control group and Cistanche total glycosides different concentration groups: Group A (Control group): Serum-containing culture medium is used as a negative control; Group B (cistanche total glycosides low concentration group: llig/ml); Group C (cistanche total glycosides medium concentration Group: L10jxg/MK); Group D (cistanche total glycosides high concentration group: 100|xg/mlo): 15 rats in each group.

1.2.3 Determination of neurotrophic factor content in the supernatant by ELISA: inoculate the cultured and purified olfactory ensheathing cells in a 6-well plate at 1x106, add cistanche total glycosides (the same concentration as above) after 24 hours and act for 24 hours, and collect the supernatant separately, Saved at -80 Long. The content of nerve growth factors in the fluid was determined by the ELISA method.

1.3 Statistical analysis

The data in this study are treated as measurement data, and the obtained data are expressed as mean ± standard deviation, and SPSS 11.0 statistical software is used for statistical analysis. The data obtained are analyzed by variance analysis, and P<0.05 indicates that there is a significant difference.

2 Results

2.1 The effect of cistanche total glycosides on olfactory ensheathing cells

2.1.1 Methyl oxazolyl tetrazole method (MT) determination results: Group A: 0.617±0.149; Group B: 0.589±0.147; Group C: 0.761±0.138; Group D: 0.732±0.234. Comparison between Cistanche total glycosides group (B) and Control group (A), P>0.05, the difference was not significant; Cistanche total glycosides 10mg/ml, 100mg/ml concentration group (C, D) compared with Control group (A) P<0.05, The difference is significant.

2.1.2 The result of flow cytometry determination is shown in Figure 1. Compared with the Control group, the cistanche total glycosides group was higher than the Control group, and the percentage of apoptotic cells was lower than the Control group.

2.2 Enzyme-linked immunosorbent assay (ELISA) results

The test results showed that the content of glial-cell-line-derived neurotrophic factor (GDNF): before dosing: 302.1±16.6 pg/ml, group A: 340.5±13.2pg/ml; group B: 333.2±23.6pg/ml; Group C: 355.2±17.6pg/ml; Group D; 379.2±15.3pg/ml. Compared with the pre-administration, the GDNF content was significantly increased; compared with the Control group (A), the GDNF content of the Cistanche total glycosides 100mg/ml concentration group (D) was significantly increased.

3 Discussion

Difficulty in the regeneration of axons is one of the main reasons why it is difficult to recover after spinal cord injury. OECs have a role in the regeneration of central neuron axons that other types of cells are difficult to replace. In recent years, the research of olfactory ensheathing cells has become a hot spot. Olfactory ensheathing cells are a special type of glial cells that have the characteristics of Schwann cells and astrocytes. They originate from the olfactory basement membrane and are widely distributed on the surface of the olfactory mucosa, olfactory nerve, and olfactory bulb. It has the ability to divide and regenerate for life and can accompany the olfactory nerve axons to migrate into the brain. The axons of the olfactory nerve can pass through the junction between the peripheral nervous system and the central nervous system every 30-60 days, re-grow into the olfactory sheath and establish synaptic connections4. Olfactory ensheathing cells can secrete many growth factors such as NGF, BDNF, GDNF, NT-3, neuropeptide-Y, and S-100. Studies have shown that nerve growth factor (NGF) can prevent spinal red nucleus neurons from atrophy and strengthen the corticospinal tract Sprout and promote the regeneration of the red nucleus spinal cord nerve pathway; both the brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) can prevent the red nucleus spinal cord cell apoptosis in adult rats with axotomized injury and rescue Dying neurons and promote axonal regeneration P. In addition, olfactory ensheathing cells also express other factors on their cell membranes, such as nerve cell adhesion factor (N-CAM), which are involved in cell adhesion and axon growth. Due to these characteristics of olfactory ensheathing cells, OECs are considered to be one of the promising seed cells for cell transplantation to repair SCI.

The chemical components of Cistanche mainly include phenylethanoid glycosides, iridoids, volatile components, lignans, polysaccharides, and alkaloids. The study found that Cistanche and its chemical components have neuroprotective effects. Deng et al. studied the effect of tubulosa B on the apoptosis of SH-SY5Y neurons induced by tumor necrosis factor α (TNFα). The results showed that after 100μg/ The SH-SY5Y neurons after 36 hours of TNFα treatment by L showed typical apoptosis characteristics, while the treatment of tubulin B for 2 hours could significantly reduce the apoptosis induced by TNFα. In addition, studies have also shown that phenylethanoid glycosides can inhibit the apoptosis of rat cerebellar granule cells by inhibiting caspase-3 and caspase-8, and against the C57 mouse black caused by methyl phenyl tetrahydropyridine (MPTP). The damage of quality dopaminergic neurons has a protective effect. Not only that, but Cistanche can also alleviate various damages such as ischemia-reperfusion of the nervous system. The mouse brain ischemia injury experiment shows that the infarct range of 24h and 48h after ischemia in the cerebral ischemia-reperfusion group is 72.98% and 60.45% on average. In contrast, the cistanche total glycosides large, medium, and low dose groups (250.0mg·kg/d; 125.0mg·kg/d; 62.5mg·kg/d) respectively can make the brain of mice after cerebral ischemia-reperfusion 24h The infarct size was reduced to 25.94%, 25.81%, and 31.13%, and the cerebral infarction size was reduced to 22.14%, 20.06%, and 22.27% after 48 hours. There was a significant difference between the two, showing a significant inhibitory effect on neuronal apoptosis.

In this study, different concentrations of cistanche total glycosides were applied to OECs. The MTT method and flow cytometry measurement confirmed that 10mg/ml100mg/ml cistanche total glycosides can promote the proliferation of olfactory ensheathing cells. The results of flow cytometry showed that cistanche total glycosides can promote the proliferation of olfactory ensheathing cells It mainly promotes the proliferation of the G1 cycle of olfactory sperm cells and significantly reduces apoptotic cells. Therefore, Cistanche total glycosides can not only promote the proliferation of olfactory ensheathing cells but also increase cell activity and inhibit their apoptosis. Enzyme-linked immunosorbent assay proves that Cistanche total glycosides can significantly promote the secretion of GDNF by olfactory ensheathing cells. The above results provide an experimental basis for the clinical application of Cistanche total glycosides.

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