Effects Of Cistanche Deserticola Total Glycosides On Oxidative Stress Injury And Cell Apoptosis in Rats With Cerebral Ischemia-reperfusion Ⅰ

Abstract

Objective: To explore the effects and possible mechanisms of Cistanche deserticola total glycosides (GCs) on oxidative stress damage and nerve cell apoptosis in rats with cerebral ischemia reperfusion injury (CIRI). 

Methods: The middle cerebral artery occlusion (MCAO) model was constructed in model rats using the suture embolization method. After the model was successfully established, the rats in the drug group (GCs) were given GCs 50 mg/(kg·d) by gavage for 14 consecutive days; the rats in the sham operation group (Sham) and the model group (Model) were given the same volume of normal saline. Oral administration. After the administration, the Longa score method was used to evaluate the neurological deficit; rats were then sacrificed, and TTC staining was performed to calculate the percentage of cerebral infarct area; ELISA method was used to detect serum malondialdehyde (MDA) content, superoxide dismutase (SOD) and Glutathione peroxidase (GSH-Px) activity; Western blot to detect nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and B in brain tissue Cellular lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), and caspase-3 (Caspase-3) expression.

Results: Compared with the Sham group, the Model group had an increased neurological deficit score, increased cerebral infarction area percentage, increased MDA content, decreased SOD and GSH-Px activities, and decreased Nrf 2, HO -1 and Bc l-2 protein expression level The protein expression levels of Bax and Caspase-3 increased; compared with the Model group, the neurological deficit score of rats in the GCs group decreased, the percentage of cerebral infarction area decreased, the MDA content decreased, SOD and GS H-Px activity increases, The protein expression levels of Nrf 2, HO -1 and Bcl - 2 increased, and the protein expression levels of Bax and Caspase - 3 decreased. Conclusion: GCs can effectively reduce cerebral ischemia-reperfusion injury, and its mechanism may be related to activating the Nrf 2 / HO - 1 pathway, thereby reducing oxidative stress levels and inhibiting nerve cell apoptosis.

[Key words] Cistanche deserticola total glycosides; cerebral ischemia-reperfusion injury; oxidative stress; cell apoptosis

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Cerebral ischemia-reperfusion,

CIS is a disease with extremely high morbidity, disability and mortality rates. Brain cells are extremely sensitive to ischemia and hypoxia, so recanalizing cerebral blood vessels as soon as possible is the most effective way to deal with acute ischemic stroke. Intravenous thrombolysis and mechanical thrombectomy are currently widely used methods, but due to their relatively

Due to the narrow time window and other uncontrollable factors, only <10% of stroke patients can achieve vascular recanalization within an effective time. Cerebral ischemia reperfusion, CIRI is a type of cerebral blood vessel recovery from stenosis A superimposed pathological damage that occurs during blood flow. It is currently believed that its pathological mechanism may be related to excitatory amino acid toxicity, calcium overload, release of inflammatory factors, and accumulation of oxygen free radicals caused by mitochondrial damage [1], ultimately leading to blood-brain barrier destruction, microcirculation disorders, nerve cell apoptosis, and necrosis [1] 2] and a series of reactions, thereby further aggravating neurological deficits.

Cistanche deserticola total glycosides (glycosides of cistanche, GCs) are derived from the traditional Chinese medicine Herba Cistanche The main active ingredient is extracted from the stems and roots of inches). Previous studies have shown that GCs can effectively inhibit lipid peroxidation, improve mitochondrial function, scavenge excess oxygen free radicals, inhibit the release of inflammatory cytokines, reduce cytotoxicity, anti-platelet aggregation, inhibit cell proliferation, inhibit neuronal apoptosis, and Reduce hormone levels, thereby having anti-aging, anti-tumor, anti-depression, enhancing immunity, improving ischemic cardiovascular and cerebrovascular diseases, improving learning and memory abilities and cognitive functions, and improving reproduction [3].

Studies have shown that GCs can reduce oxidative damage to cells and maintain the integrity of the blood-brain barrier.

[4]; GCs can effectively improve cell viability and block inflammation and apoptosis induced after ischemia-reperfusion [5]; Nrf 2 plays an important role in the anti-inflammatory and antioxidant processes [6]. Based on the above background, this article aims to further study the effects and possible pathways of GCs on oxidative stress and apoptosis after CIRI by replicating the CIRI model.

1 Materials and methods

1. 1 Drugs and reagents GCs (SN-5692, purity 98%, Western

Anshenong Biotechnology Co., Ltd.), 2% triphenyltetrazolium chloride (TTC) dye solution (Beijing Solebao Technology Co., Ltd.), ELISA kit (Beijing Boosen Co., Ltd.), BCA protein quantitative analysis kit (U.S.A. Cell Signaling Technology Company), rabbit anti-mouse antibody Bcl-2, Bax, Caspas Antibodies such as e-3, Nrf 2, HO-1, and goat anti-rabbit secondary antibodies were all purchased from Proteintech Company of the United States.

1. 2 instruments

Centrifuge (Model: TDL-40B, Shanghai Anheng Scientific Instrument Factory), Microplate reader (Model: Max-M5, American Molecular Devices Company), SDS- PAGE electrophoresis instrument (model: DYY-7C, Beijing Liuyi Biotechnology Co., Ltd. Company), gel scanner (Model: 4466611, US BIO-RAD Company).

1. 3 animals

  Male Wistar rats [SCXK (Beijing) 2019-0010], clean grade, 8 weeks old, body weight 250-280 g.

1. 3 methods

1. 3. 1 Grouping and modeling

Rats were divided into sham group (Sham), model group (Model), and drug group (GCs) using a simple randomization method. There were 15 rats in each group. They were adaptively raised for 1 week and then modeled. Strictly fasting and fasting 12 hours before surgery, the Model group and the GCs group used the suture embolization method [7] to create middle cerebral artery occlusion (middle cerebral artery occlusion). occlusion, MCAO) rat model, embolus removal was performed after 2 hours of ischemia to achieve reperfusion. , record the start time of reperfusion. The wound was sutured, the skin was disinfected with iodophor, and the animals were returned to the cage and reared, and then perfused for 24 h. The operation for the rats in the Sham group was the same as that for the model rats, but no plugging was performed. Finally, 10 rats in each group were retained for subsequent experiments.

1. 3. 2. Administration method

Take GCs powder and add pure water to prepare a 15 mg/mL solution. Preliminary studies have shown that the dose of 50 mg/(kg·d) is better than other dose groups [8], so the dose selected for this experiment is the same as before. Rats in the GCs group were given GCs by gavage after 24 hours of ischemia and reperfusion, and rats in the Sham group and Model group were given an equal volume of normal saline by gavage for 14 days.

1. 3. 3 Neurological deficit score

  The Longa scoring method [7] was used to score 24 hours after surgery, and model rats with scores of 1 to 3 were retained for subsequent experiments. Score after the last dose. 0 points: None

Neurological symptoms; 1 point: slight neurological deficit, the contralateral forelimb flexes and elevates when the tail is lifted, and the shoulder adducts; 2 points: moderate neurological deficit, the contralateral forelimb flexes and elevates when the tail is lifted , shoulder adduction, turning in circles to the opposite side of the lesion when walking; 3 points: severe neurological deficit, leaning to the opposite side of the lesion when walking; 4 points: unconscious and autonomous activities.

1. 3. 4 Determination of percentage of cerebral infarction area

After the last administration, 4 rats from each group were randomly selected, deeply anesthetized, decapitated, and their brains were removed quickly.

Quickly freeze the brain tissue in a -20°C refrigerator for 20 minutes. After the brain tissue is relatively fixed, take it out. Starting from the intersection of the anterior pole of the brain to the optic chiasm, and from the forehead to the occiput, cut the rat brain along the coronal plane. Cut into 5 brain slices with a thickness of 2 mm, immerse them in 2% TTC solution, place them in a 37°C water bath for dyeing for 15 minutes, turn them over, and dye them for another 15 minutes. Finally, the brain slices were immersed in 4% paraformaldehyde solution and fixed for 12 hours before being photographed. Normal brain tissue was shown in red and infarcted brain tissue was shown in white. Use images software to analyze and calculate the infarction rate of each brain slice. Infarction rate = infarct area/total area of the brain slice × 100%.

1. 3. 5 Detection of oxidative stress related indicators

Anesthetize the rat, take 4 mL of abdominal aorta blood, aliquot it into clean EP tubes and place it in a 4°C refrigerator for 0. Centrifuge at 3 500 r/min for 5 h at 4°C for 15 min, take the supernatant, and finally retain the serum of 3 rats in each group. The content of malondialdehyde (MDA) and superoxide dismutase (superoxide dismutase) were determined according to the ELISA kit operating method. e dismutase,

SOD), glutathione peroxidase (glutathione peroxidase, GSH-Px) activity.

1. 3. 6 Western blot detection

Oxidative stress and apoptosis-related proteins were measured to measure the expression of Nrf 2, HO -1, Bcl -2, Bax, and Caspase -3 proteins in brain tissue. Three rats were selected from each group, and an appropriate amount of cerebral cortex from the diseased area was taken, lysis solution was added to it, and the protein concentration was calculated after protein extraction. After protein gel electrophoresis, transfer to membrane, blocking with blocking solution for 2 h, rinsing, and then incubated with primary antibody at 4°C overnight, the next day, the TBST was removed, rinsed, and secondary antibody was added for incubation for 2 h. Place the target band on the imaging analyzer to analyze the gray value. Calculate the relative strip gray value, relative strip gray value = target strip gray value / internal reference strip gray value.

1. 4 Statistical analysis methods

  Data application SPSS 25. 0 software for data analysis, and the statistical graphs in this article were drawn with GraphPad Prism 8 software. Data results consistent with normal distribution are expressed as mean ± standard deviation. One-factor analysis of variance was used for comparisons between multiple samples, and LSD-t was used for comparisons between two independent samples. Test, with P < 0. 05 indicates that the difference is statistically significant.