Preventive Effect Of Cistanche Salsa Phenylethanoid Glycosides On Rats With High-altitude Cerebral Edema

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Abstract

Objective To observe the effect and mechanism of phenylethanoid glycosides from Cistamnche Salsa on rats with high-altitude cerebral edema. Methods Phenylethanoid glycosides were given prophylactically to establish the rat model of high-altitude cerebral edema by using a hypobaric chamber in a simulated environment of 5000 m altitude. Pathologic changes in the rat lung tissue, the content of water, IL-6, TNF-a, MDA, and enzymatic activity of SOD and GSH-Px in the lung homogenate were measured. Results Compared with the control group, the high-altitude cerebral edema rats showed the remarkable character of cerebral edema and high content of water in the lung. The content of IL-6, TNF-a, and MDA in the lung homogenate was increased while the enzymatic activity of SOD and GSH-Px were significantly decreased. The phenylethanoid glycosides could improve the pathological changes in the lung tissue, decrease the content of water, IL-6, TNF-a, and MDA, while increasing the enzymatic activity of SOD and GSH-Px in the lung homogenate, Concision Phenylethanoid glycosides can prevent high-altitude cerebral edema. whose mechanism may be related to the anti-inflammation and anti-oxidation stress in the lung tissue.

Keywords: Cistanche Salsa; phenylethanoid glycoside; high-altitude cerebral edema

Cistanche phenylethanoid glycosides

High-altitude cerebral edema (HACE) generally occurs when the altitude is greater than 3000m for the first time. It is a serious life-threatening disease caused by the body's inadaptability to the altitude low-pressure and low-oxygen environment. It is related to altitude sickness. , High altitude pulmonary edema is the three manifestations of acute altitude sickness. Although there are fewer studies on HACE compared to high altitude pulmonary edema, related studies have shown that oxidative stress and inflammation play an important role in the pathogenesis of HACE (P-3).

Cistanche phenylethanoid glycosides (phenylethanoid glycosides from Cistanche Salsa, PhGCs) are extracted from Xinjiang Cistanche cistanche, and related studies have successfully isolated a variety of phenylethanoid glycosides, mainly including echinacoside and verbascoside 1. Research on PhGCs' pharmacological effects over the years has shown that it has similar pharmacological effects to salidroside, the active ingredient of anti-altitude sickness. It has anti-hypoxia, anti-radiation, scavenging free radicals, etc. P-7]. In this experiment, a rat HACE model network 91 was established in a special environment artificial experimental chamber in the northwest region, and PhGCs were administered preventively to observe its preventive effect on HACE and its possible mechanism of action.

Cistanche herb: Cistanche phenylethanoid glycosides

1 material

1.1 Animals

Experimental animal Wistar rat, SPF grade, half male and half male, weight 180～220g [Experimental Animal Center of Xinjiang Medical University, animal production license number SCXK (new) 2011-0004, animal use license number SYXK (new) 2011- 0004].

1.2 Apparatus

Northwest special environment artificial experiment cabin (Guizhou Fenglei Aviation Ordnance Co., Ltd., DY-2); optical microscope (Japan Nikon company, E 200); tissue slicer (German Mecang company, HM340E); electronic balance (Mettler- Toledo Instrument Co., Ltd., AL204, accuracy; 0.1 mg); microplate reader (Bio-RAD, XMark 7M, USA): UV spectrophotometer (Shanghai Lens Optical Technology Co., Ltd., SpectrumLab 22); electric heating constant temperature drying oven (Huangshi, Hubei) City Medical Equipment Co., Ltd., SKHG-01).

1.3 Test drug

PhGCs (Professor Tu Pengfei from Peking University, content: 90.70%, prepared with distilled water to make a solution of corresponding concentration); Rhodiola Rosea Oral Liquid (Tibet Tibetan Medicine Group Co., Ltd., batch number: 120503, specification: 10 mL); tumor necrosis Factor-α (tumor necrosis factor-α, TNF-α) kit (batch number: ZJAGBZAB01), interleukin-6 (interleukin-6, IL-6) kit (batch number: ZIBIBZAB 02) (Shanghai Yikesai Biological Products Co., Ltd. Company); Sodium Pentobarbital (American Amresco Company, Lot Number: 20110612); Paraformaldehyde (Chengdu Kelon Chemical Reagent Factory, Lot Number: 20110901); Superoxide Dismutase (SOD) Kit (Lot Number:20130812), malondialdehyde (MDA) kit (batch number: 20130812), glutathione peroxidase (GSH-Px) kit (batch number: 20130814) (Nanjing Jiancheng Institute of Bioengineering ); PBS phosphate buffer powder (Fuzhou Maixin Biotechnology Development Co., Ltd., batch number: 13061716).

cistanche herb

2 method

2.1 Grouping and medication

Wistar rats were randomly divided into 6 groups: normal control group, model group, Rhodiola Rosea oral solution group (1.78 mL·kg-'), PhGCs low-dose group (75 mg·kg-), PhGCs medium-dose group (150mg ·Kg-'), PhGCs high-dose group (300mg·kg-), 12 rats in each group. Each group was bred in an SPF environment. The normal control group and the model group were given intragastrically with distilled water (1.0 mL/100g). The other groups were given intragastrically according to the corresponding dose for 10 consecutive days. On the 8th day, the rest except the normal control group Each group was kept for 72 hours in an artificial experimental cabin that simulated a plateau environment at an altitude of 5 000 m. The height in the cabin rises at a constant speed of 10 m·s-' to the altitude of 5000 m (atmospheric pressure is 54.1 kPa, oxygen partial pressure is 11.52 kPa), during which the animals freely enter the water and eat, and the cabin is opened for 0.5 h every 24 h. Medicine, add feed and drinking water.

2.2 Animal handling

Rats in each group were anesthetized by intraperitoneal injection of sodium pentobarbital (2%, 0.2 mL/100g) immediately after leaving the cabin, blood was taken from the abdominal aorta, the cranial cavity was opened, the brain tissue was taken, and the upper half of the left brain was taken out and weighed. The package is used to determine the water content, and the lower half of the left brain is fixed with 4% paraformaldehyde for HE staining. Weigh the right brain, add PBS solution to prepare a 10% homogenate, centrifuge at 3 000 r·min-' for 10 min, aliquot the supernatant, and store it in a refrigerator at 80°C for the determination of TNF-a, IIL-6, SOD, MDA, GSH-Px.

2.3 Brain tissue pathology observation

After the brain tissue is fully fixed in 4% paraformaldehyde, dehydrated, embedded, sliced, and HE stained, the pathological changes are observed under an optical microscope under different magnifications, and pictures are taken.

2.4 Determination of brain water content and determination of related indexes of brain tissue homogenate inflammation and oxidative stress

Bake the brain tissue wrapped in tin foil in an oven (80 ℃, 72 h) to a constant weight, weigh the dry weight, and calculate the water content: water content = (tissue weight-tissue dry weight) / tissue weight × 100%. Use enzyme-linked immunoassay kit to measure TNF-α, IL-6 content, UV spectrophotometer to measure MDA, SOD, GSH-Px, the specific steps follow the instructions.

2.5 Statistical analysis

SPSS 16.0 software was used for analysis, the measurement data were all represented by the lower +s, the data were first tested for normality, the independent sample 1 test was used for the comparison between the conforming groups, and the logarithmic transformation was performed for the non-conforming group. The inspection level is α=0.05.

cistanche extract

3 Results

3.1 The effect of PhGCs on the pathological changes of brain tissue in HACE rats

Observing the pathological slices of the brain tissues of rats in each group under a light microscope showed that the molecular layer, outer granular cell layer, vertebral body layer cell layer, inner granular cell layer, and multi-row cell layer structure of the brain tissue of the normal control group were clear, and no lesions were found. In the model group, the brain tissues of the rats were hyperemia and edema of the capillaries, and the edema of the cells in the molecular layer and the vertebral body layer was obvious. PhGCs low-dose group of rats brain tissue molecular layer part of the vasodilation and congestion edema, mild cell edema, edema less than the model group: PhGCs middle-dose group of rats brain tissue part of the submeningeal vasodilation and congestion, occasionally cell edema, edema compared with the model group The group was significantly reduced; the molecular layer of the PhGCs high-dose group had no obvious edema, and there was mild perivascular edema, which was significantly reduced compared with the model group. The brain tissue molecular layer of rats in the Rhodiola Rosea group had scattered edema, and the scattered blood vessels had very mild edema. The edema was significantly reduced compared with the model group. It can be seen that the brain tissue of the rats in the model group is obviously edema, and the model is established. PhGCs and Rhodiola Rosea can reduce the degree of brain edema in HACE model rats. The results are shown in Figure 1.

Fig 1 Histopathological observation of the rat brain in different groups（HE ×400）

3.2 The effect of PhGCs on the brain water content of HACE rats

Compared with the normal control group, the brain tissue water content of the model group increased, and the difference was statistically significant (P<0.01), indicating that the model was established. The brain tissue water content of rats in the low, medium, and high doses of PhGCs and the Rhodiola Rosea group was lower than that of the model group, and the difference was statistically significant (P<0.01). There was no statistically significant difference in tissue water content.

3.3 The effect of PhGCs on TNF-a and IL-6 in the brain tissue of HACE rats

Compared with the normal control group, the contents of TNF-a and IIL-6 in the brain tissue homogenate of the model group increased significantly (P<0.01). The contents of TNF-α and IIL-6 in the brain tissue homogenate of PhGCs low, medium, and high dose groups and Rhodiola Rosea group were lower than those of the model group (P<0.05), and the difference was statistically significant. There was no statistically significant difference in brain tissue TNF-a and IIL-6 levels between the PhGCs dose groups and the Rhodiola Rosea group.

3.4 The effect of PhGCs on oxidative stress in the brain tissue of HACE rats

Compared with the normal control group, the SOD and GSH-Px enzyme activities in the brain tissue of the model group were significantly reduced, and the MDA content was significantly increased (P<0.01). The SOD and GSH-Px enzyme activities in the brain tissue of the PhGCs low, medium, and high dose groups and Rhodiola Rosea group were higher than the model group, and the MDA content was lower than that of the model group. The difference was statistically significant (P<0.05). There was no significant difference in the content of SOD, GSH-Px, and MDA in the brain tissue between the PhGCs groups and the Rhodiola Rosea group.

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4 Discussion

In recent years, domestic research on the prevention and treatment of altitude sickness with traditional Chinese medicine has achieved major innovative results, and traditional Chinese medicines with independent intellectual property rights for the prevention and treatment of altitude sickness have been successfully developed. For example, the Rhodiola Capsules developed by the Tibet Military Region have obtained the approval number of the military special-needed medicines and are used in the plateau border defense forces as special medicines for the prevention and treatment of plateau diseases. The Rhodiola Rosea oral liquid launched by Qinghai and Tibet local pharmaceutical companies has obtained the official approval number of national Chinese medicine varieties and is clinically suitable for the prevention and treatment of acute and chronic altitude sickness10. The pharmacological effects of PhGCs are similar to salidroside, so we chose Rhodiola Rosea oral liquid, which is sold and used in the market and has a drug standard, as the positive control drug.

A large number of studies [1H12 proved that the levels of TNF-α and IL-6 in HACE patients are elevated, Zhou Qiquan et al. [3 Studies have shown that TNF-α plays an important role in changing the permeability of the blood-brain barrier. An important factor for increased barrier permeability. TNF-α secretion increases during hypoxemia. When TNF-α increases abnormally in the blood, it can damage vascular endothelial cells, causing vascular endothelial cell morphology changes, cell membrane and organelle damage, etc., and TNF-a can also be used as a The intracellular signal of apoptosis induces the apoptosis of vascular endothelial cells, promotes the production of endothelin (ET-1) and aggravates the damage of the blood vessel wall, and has the effect of promoting the secretion of IIL-614.

Hypoxia is considered to be a key factor leading to the occurrence of acute altitude sickness. The oxidative stress response induced by hypoxia plays an important role in cerebral arterial hypertension and vascular leakage. Local free radicals are involved in causing vascular damage. Cerebrovascular damage and increased fluid leakage can cause brain edema 13i4. Zhou Qiquan et al. [7 Studies have shown that oxygen free radicals play an important role in the formation of HACE and are an important factor in increasing the permeability of the blood-brain barrier in high altitude environments. In a hypoxic environment, free radicals and MDA will be significantly increased, while the function of antioxidant enzymes is down-regulated. Therefore, the low function of antioxidant enzymes and the increase of lipid peroxidation damage may have important significance in the pathogenesis of acute altitude sickness. MDA is produced by the breakdown of fatty acid peroxides and is the most commonly measured lipid peroxidation product in biological systems. Antioxidant enzymes in the body include SOD, GSH-Px, etc., which either directly scavenges superoxide free radicals and hydrogen peroxide, or convert them into less active substances to play a defensive role. So we detected SOD, MDA, GSH-Px in brain tissue homogenate.

In this experiment, compared with the normal control group, the amount of TNF-a and IIL-6 in the brain tissue homogenate of the HACE model group increased significantly, the activity of SOD, GSH-Px enzymes decreased, and the content of MDA increased significantly It shows that hypoxia-induced oxidative stress dysfunction and inflammation in rats are involved in the formation of HACE. PhGCs preventive administration can reduce the water content of brain tissue in rats with cerebral edema, and significantly improve the pathological changes of brain tissue edema, indicating that it has the effect of preventing the occurrence of HACE. PhGCs can increase SOD and GSH-Px in rat brain tissue during hypoxia. Enzyme activity reduces the content of MDA in brain tissue caused by hypoxia. The content of TNF-α and IL-6 in brain tissue of rats in the PhGCs preventive administration group is significantly lower than that in the model group, suggesting that our anti-oxidative stress and anti-inflammatory may be the prevention and treatment of PhGCs One of the mechanisms of altitude sickness. Based on the results of this experiment, the effect of PhGCs in the middle dose group is slightly better than the high dose, but the difference is not statistically significant. No related literature reports PhGCs are liver drug enzyme inducers, which can rule out the possibility that drugs induce the increase of liver drug enzyme activity and accelerate self-metabolism. It is speculated that the possible reason is that as the concentration of the drug increases, the concentration of the drug in the body exceeds the range of the dose-effect relationship. At the middle dose, the receptors in the body are all occupied and the receptors are saturated, so there is no continued increase in the effect of high doses, and the drug It is a glycoside, which may hydrolyze sugar in the stomach, and the increase in viscosity will limit the effective absorption of the drug when the concentration is too high.

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