Effects Of Cistanche On The Changes Of ATPase in Liver Mitochondria Of Septicemia Rats

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

Wang Zhiqiang Yin Gang

Abstract: Objective: To study the change of ATPase activity of liver mitochondria in sepsis and the effect of Cistanche on it. Methods: 40 SD rats were randomly divided into sham operation group, 12-hour control group, 12-hour administration group, 16-hour control group, and 16-hour administration group. The sepsis model was replicated by cecal ligation and perforation (C LP) and compared. The group survival rate, mean arterial pressure (MAP), liver mitochondrial ATPase activity changes. Results: After the rats replicated the sepsis model, the ATPase activity of liver mitochondria decreased, and the 24-hour survival rate decreased significantly. The ATPase activity of liver mitochondria after administration of Cistanche was significantly improved compared with the control group (P<0.05), which was significantly positively correlated with the reduction of rat mortality. Conclusion: Decreased mitochondrial ATPase activity in sepsis, cell calcium overload, and edema and necrosis are the direct causes of death in septic rats. Cistanche enhances ATPase activity is an important mechanism for the treatment of sepsis.

Keywords: Cistanche, cistanche sepsis mitochondria ATP enzyme

Sepsis is a clinically critical illness. Although antibiotics have been updated in the past 30 years, the mortality rate of sepsis has not decreased significantly and is still as high as 50% [1]. Western antibacterial "eliminating evil" treatment alone does not improve the prognosis of sepsis. In the pathogenesis of TCM, sepsis belongs to the "righteousness of the deficiency and the evil", the treatment must be based on the principle of "strengthening the body" and "eliminating the evil". Traditional Chinese medicine has shown great potential in the treatment of sepsis. Sepsis complicated by multiple organ failure is the root cause of the high mortality rate, and cell structure damage is the microscopic pathological basis of multiple organ failure [2]. Cistanche is a traditional Chinese medicine for tonic, modern pharmacological studies have shown that Cistanche has the effect of resisting membrane ester peroxidation and protecting cell function [3]. The function of mitochondrial energy metabolism is one of the basic functions of cells, and the activity of ATPase is an important indicator to measure the function of mitochondrial energy metabolism. In this experiment, we observed the changes of ATPase activity of liver cells in septic rats and the relationship between the effects of Cistanche on it and the mortality of rats and aimed to provide a theoretical basis and effective exploration for the clinical "strengthening" treatment of sepsis.

Cistanche

1. Materials and methods

1.1 Reagents

Cistanche was purchased from Xinjiang (identified by Professor Tan Defu of the Three Gorges University School of Medicine). 200g of dried Cistanche was taken and decocted twice in distilled water. The filtrate was evaporated and concentrated to a concentration of 1g/ml. Sodium Pentobarbital (001104), China National Pharmaceutical Group Shanghai Chemical Reagent Company; ATPase Reagent (20030108), Nanjing Jiancheng Bioengineering Research Institute.

1.2 Experimental animal model and grouping

40 male SD rats (purchased from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology), were randomly divided into 5 groups: sham operation group (n=8), 12-hour control group (n=8), 12-hour administration group (n=8), 16-hour control group (n=8), 16-hour administration group (n=8). The sham operation group and the control group were given normal saline by gavage, and the administration group was gavage with 1 g/ml Cistanche, twice a day, for up to 15 days. The sepsis model was replicated by cecal ligation and perforation [4]. Before the operation, the animals were free to drink water and fasted for 12 hours.

1.3 Determination of mean arterial pressure

The jugular vein was inserted with a scalp needle, heparin was used for anticoagulation, the left common carotid artery was cannulated to the aortic arch, and the self-made catheter was connected to the biologic multichannel instrument, and the average arterial pressure (MAP) was recorded. Experiment after the MAP stabilizes.

1.4 Determination of mitochondrial ATPase activity

Take 6g of rat liver and quickly place it in 0℃ separation medium [250mmol of sucrose, 2mmol of ethoxy-ethyl piperazine 2-ethyl sulfonic acid, 0.1mmol of ethylenediaminetetraacetic acid, pH7.4], open it and wash away impurities, Separate the medium suspension (1: 10), homogenize with a Teflon electric homogenizer (1200r/min), centrifuge at 2000r/min for 10 minutes, retain the supernatant, centrifuge at 10000r/min for 15 minutes, discard the supernatant, Take the precipitate and suspend it in 2ml of separation medium to obtain a mitochondrial suspension. The whole process is carried out at 0 to 4 ℃. The phosphorus determination method [5] was used to measure ATPase activity (the kit was purchased from Nanjing Jiancheng Institute of Biological Engineering).

1.5 Statistical processing

The experimental data are all expressed as mean±standard deviation (X±S), and SPSS software performs one-way analysis of variance and correlation analysis.

Cistanche extract

2. results

2.1 The effect of Cistanche on mean arterial pressure and 24-hour rat mortality

The blood pressure of the control group decreased significantly at 12 hours after cecal ligation and perforation, and it became more obvious at 16 hours. Compared with the sham operation group, there was a significant difference. The blood pressure of the Cistanche treatment group was improved. The death rate of the control group was significantly higher than that of the sham operation group, and the death rate of the administration group was significantly lower than that of the corresponding control group.

2.2 The effect of Cistanche on mitochondrial ATPase activity

The activities of sodium-potassium ATPase, calcium ATPase, magnesium ATPase, and calcium-magnesium ATPase in the administration group were significantly higher than those of the corresponding control group, reaching the level of the sham operation group, which was significantly positively correlated with the reduction of rat mortality (r=0.834) ).

Cistanche

3. Discussion

Na +-K+ -ATPase, also known as sodium-potassium pump, plays an important physiological role in maintaining cell physiological activities, body temperature, and normal metabolism, and regulating the balance of ions inside and outside the cell. In sepsis, there is severe sodium pump dysfunction in the inner mitochondrial membrane, causing mitochondrial edema. The Na+ -K+-ATP enzyme is damaged, the imbalance of mitochondrial ion concentration increases, energy metabolism enzyme activity is inhibited, cell energy supply is depleted, and cytotoxic edema is caused. At the same time, the membrane permeability increases, which ultimately leads to cell death. In the experiment, we found that the mitochondrial Na+-K+-ATPase activity of septic hepatocytes was significantly reduced (P<0.01), indicating that the liver tissue is seriously damaged. After administration of Cistanche, the Na+-K+-ATPase activity of liver mitochondria was significantly increased (P<0.05), and the mortality of septic rats was significantly reduced. The experimental results suggest that the decrease of Na+ -K+ -ATPase activity is significantly positively correlated with the development of sepsis in rats. The inhibition of Na+ -K + -ATPase activity, cell edema, and death leading to multiple organ failure may be the rapid development of sepsis. One of the important mechanisms. In recent years, there have been many reports about Na + -K + -ATPase and many diseases, which have attracted the attention of clinical and basic workers. It is of great significance to study the law of changes in the activity of Na + -K + -ATPase and its regulation [6]. Little is known about the specific mechanism of Na + -K+-ATPase in the development and outcome of sepsis, and further research is needed.

The stability of intracellular calcium and magnesium is the necessary material basis for maintaining intracellular release response and information transmission [7]. In the experiment, the decrease of Ca2 + -ATPase activity in the control group directly affects the activity of the calcium pump, and the decrease of calcium pump activity weakens the role of mitochondrial calcium pump to excrete Ca2 + out of the cell, and Ca2 + overload in mitochondria. Calcium overload in mitochondria is an important cause of mitochondrial oxidative phosphorylation disorder, structural damage, and cell necrosis [8, 9]. Ca2 + overload reduces the mitochondrial membrane potential and the tissue ATP content [10], and the energy utilization of tissue cells is impaired. Ca2+ can activate the phospholipase on the mitochondria, cause damage to the mitochondrial membrane, destroy the normal ATP synthesis function of the mitochondria, and hinder mitochondrial energy production [11]. Disorders of mitochondrial calcium metabolism can directly affect mitochondrial respiratory function, causing mitochondrial oxygen uptake and proton oxidation disorders in the electron transport chain [12]. At the same time, the increase of Ca2+ can aggravate the damage of ester peroxidation to mitochondria [13]. These all directly or indirectly lead to the failure of cell energy supply and the death of tissues and organs and accelerate the development of pathological changes in sepsis. In the experiment, we also found that the activities of M g2 + -ATPase and Ca2 +-M g2 + -ATPase in septic rats were also significantly decreased. Mg2 + -ATPase and Ca2 + -M g2 + -ATPase are important substances to maintain the intracellular calcium and magnesium balance. Widner reported that the decrease of intracellular magnesium ions is the inducing factor of irreversible cell damage, and the ratio of Mg 2 +/Ca2 + is An important indicator reflecting the irreversible damage of cells [14]. The Mg2 + -ATPase and Ca2 + -M g2 +-ATPase activities of the Cistanche-administered group were enhanced, A TPase is a sulfhydrylase, and Cistanche has an anti-membrane ester peroxidation effect, which may be due to the enhancement of ATPase activity by Cistanche Basis of Chemistry and Basis of Pharmacology [15].

In sepsis, mitochondrial energy metabolism disorder, decreased ATP enzyme activity, mitochondrial edema, intracellular calcium overload, and calcium-magnesium balance disorder are the direct causes of sepsis cell death and multiple organ failure. Cistanche antagonizes membrane ester peroxidation, enhances ATPase activity, maintains intracellular calcium and magnesium homeostasis, and protects the normal operation of cell functions. Cistanche is an important mechanism for reducing the mortality of septic rats. It demonstrates the good prospects of Cistanche in the treatment of sepsis