Injury Of Exercise——induced Renal Ischemia——reperfusion And The Protective Efect Of Herba Cistanches Combined With Vitamin C in Rats
Mar 07, 2022
Injury of exercise-induced renal ischemia-reperfusion and the protective effect of herba Cistanches combined with vitamin C in rats
for more information:ali.ma@wecistanche.com
Abstract：Objective： To study the Herba Cistanches combined with vitamin C’s protective effect and mechanism of movement in rats renal ischemia-reperfusion injury．Methods： Randomly divide 96 7一week—old male Wistar rats into 6 groups： control group(group C，12)， general training group (group M，1 2)， overtraining group (group OM，24)，cistanche + overtraining group (group COM，16)， vitamin C+ overtraining group(group VCOM，16)， and cistanche + vitamin C+overtraining group(group CVCOM，1 6)， take a 56一day of exhaustive swimming training． The COM group， CVCOM group with professional gavage for gastric lavage(ig)， once a day， the dose of 6．0 l g．kg～， Ig volume 5 mL．kg～； group VCOM， group CVCOM IP take an intraperitoneal injection， 100 mg． kg～， once a day， the injection volume is 0．5 mL； other groups ig normal saline． Detect biochemical parameters of serum urea nitrogen and serum creatinine 24 hours later after the last training．Results：1)In the 8 weeks of overtraining， rats resulted in the movement of“renal ischemia-reperfusion"， the ultrastructure of the kidney was severely damaged， the histopathological changes obviously， renal function was seriously damaged．2)Serum creatinine and blood urea nitrogen rise．OM group (P<0．01)， COM group(P<0．O1)， VCOM group(P<0．01)， CVCOM group(P<0．05) higher than in group C； COM group(P<0．05)， VCOM group(P<0．05)， CVCOM group(P<0．01)lower than the OM group； COM group(P<0．05)， CVCOM groups(P<0．01)lower than the VCOM group．3)SOD activity decreased．OM group(P<0．01)， COM group(P<0．01)， of VCOM group(P<0．01)， CVCOM group(P<0．05)lower than in group C； M group(P<0．05)， COM group(P<0．05)， VCOM group(P<0．05)， CVCOM group(P<0．01)higher than the OM group； COM group(P<0．05)， CVCOM group(P<0．01)higher than the VCOM group．4)The content of MDA increased， OM group(P<0．01)， COM group(P<0．01)， VCOM group(P< 0．O1)， CVCOM group(P<0．05)higher than in group C； COM group(P<0．05)， VCOM group (P<0．05)， CVCOM group(P<0．01)lower than the OM group； COM group(P<0．05)， CV— COM groups(P<0．01)lower than the VCOM group．Conclusion：1)Overtraining can induce renal ischemia-reperfusion in rats．2)Cistanche deserticola Y．C．Ma and vitamin C can enhance the activity of SOD， scavenging oxygen free radicals， reduce lipid peroxidation， they have a protective effect on exercise-induced ischemia-reperfusion kidney．3)Cistanche is better than vitamin C， but they worked even better when taken both．
Keywords：herba Cistanches； vitamin C； exercise-induced ischemia-reperfusion
Click to Cistanche herba products
With the development of modern Chinese medicine theory, modern pharmacology theory, and modern biotechnology, more single agents or formulas are applied to sports practice. Traditional Chinese medicine shows its unique advantages because of its multi-target, multi-channel effect, and almost no prohibited components. It has made some achievements in improving athletes' physical function and alleviating fatigue. When the body is in a stable state, renal blood flow can be maintained relatively constant through its regulation mechanism. During strenuous exercise, renal blood flow changes under the influence of nerve and humoral factors. Due to the redistribution of blood flow of various organs, the blood flow of active organs, especially muscles, increases, and the renal blood flow decreases sharply, which is more obvious with the continuous and increasing intensity of the exercise process. This incomplete ischemic state of the kidney forms "exercise-induced renal ischemia". After exercise stops, the renal blood supply recovers, forming "reperfusion" after exercise-induced renal ischemia. The abnormal increase of free radical production plays an important role in the process of ischemia-reperfusion injury. The results show that Cistanche deserticola has the pharmacological effects of increasing the activity of peroxide dismutase and reducing lipid peroxide, and can eliminate the damage of excessive free radicals to body cells; Vitamin C, as an important antioxidant in the body, can also effectively scavenge free radicals. This paper studies the effects of Cistanche deserticola combined with vitamin C on rat kidney ischemia-reperfusion injury, and discusses their effects and mechanisms in ischemia-reperfusion injury, to provide a theoretical basis for their clinical application.
1 material
1.1 Animals
100 clean grades male Wistar rats, 42 days old, average weight (201.8 ± 14.1) g, provided by the Department of experimental animal science, Department of Medicine, Peking University, license No. scxk2006-0008. During the whole test, the temperature in the laboratory is maintained at 22 ± 2 ℃, the relative humidity is 55% ~ 75%, and the illumination time changes with nature. All experimental rats were fed with basic feed (provided by the Department of experimental animal science, Department of Medicine, Peking University) and distilled water. The experimental time was 63 days and the formal training time was 56 days.
1.2 Experimental medication
Cistanche deserticola, produced in Inner Mongolia, was purchased from Tongrentang drugstore in Beijing, batch No.: 120224536
Vitamin C injection (batch No.: 20120406), provided by Beijing Shuanghe Pharmaceutical Co., Ltd.
1.3 Reagents
Serum creatinine (SCR) was determined by Jaffe picric acid method, blood urea nitrogen (BUN) by diacetyl MONOOXIME method, and malondialdehyde (MDA) by colorimetry; Superoxide dismutase (SOD) was determined by xanthine oxidase method, using the kit provided by Nanjing Jiancheng Bioengineering Institute, kit No. 2012051
1.4 Instruments
Bs224s electronic analytical balance (Sartorius, Germany), optical microscope (Olympus, Japan), alcyon300 automatic biochemical analyzer (Abbott, USA), 756mc ultraviolet-visible spectrophotometer (Shanghai Precision Instrument Factory), and gl-20g high-speed freezing centrifuge.
2 Methods
2.1 Animal grouping
After 4 days of adaptive feeding, the experimental rats were screened for 3 days with 20 minutes of daily exercise, and individual unsuitable swimmers were eliminated. The remaining rats were randomly divided into 6 groups: 12 in the control group (Group C), 12 in the general training group (group M), 24 in the overtraining group (OM group), 16 in the Cistanche deserticola + overtraining group (COM group) and 16 in the vitamin C + overtraining group (VCOM group) 16 rats in Cistanche deserticola + vitamin C + overtraining group (cv com group) were trained for 56 days. During the training period, the com group and cv com group were gavaged with a professional gavage device once a day, with a dose of 6.01 g / kg and a volume of 5 ml/kg; VCOM group and cv com group were injected intraperitoneally at a dose of 100 mg/kg once a day, with an injection volume of 0.5 ml, and the same amount of normal saline in other groups.
2.2 experimental method
2.2.1 training and test scheme
Group C was fed routinely without any intervention and did not exercise at ordinary times. Group M received moderate-intensity swimming training and formal swimming training for 8 weeks. Train 6 days a week, once a day, swim 20 laps for the first time, and then gradually increase. Swim 60 minutes each time on the first weekend, 90 minutes a day on the second weekend, and 120 minutes a day on the third weekend. Maintain this amount of exercise for the next 5 weeks. The training arrangement of other groups in the first 3 weeks is the same as that of group M, and high-intensity training will be arranged from the fourth week. Rats were trained to exhaustion each time. The exhaustion standard was that the rats did not emerge from the water surface for 10 seconds after sinking. Increase the weight by 0.5% in the first three weeks, 1% in the fourth week, and 2% in the fifth week. Train once a day. In the 6th week, they trained once every day in the morning and afternoon, and in the 7th-8th week, they trained once every day in the morning, afternoon, and night, all of which increased the weight by 5%. By the end of the 8th week, the rats in groups C and M grew normally without accidental death. The mortality of rats in other groups was higher due to increased weight, failure to recover from fatigue, and accidental death after training. There were 14, 13, 11, and 13 rats in the OM group, com group, VCOM group, and CVCOM group respectively. 10 rats in each group were selected for the experiment, and the others were randomly excluded.
2.2.2 index determination
24 hours after the last swimming training, the rats in each group were moderately anesthetized with ether. Blood was taken from the common carotid artery, anticoagulated with sodium citrate solution, placed in 37 ℃ water for 30 minutes, centrifuged at 4 ℃ 3000 rpm for 10 minutes, separated and prepared serum, and stored in a refrigerator at minus 20 ℃ for future reference. Quickly remove both kidneys, remove fascia, put them in pre-cooled normal saline to wash bloodstains, and visually observe the size, color, and texture of kidneys. Separate the left kidney, take 0.5g of renal upper pole tissue, grind it with 1.5ml normal saline at 12000 rpm at 1 ℃ to make 10% renal tissue homogenate; Renal tissue specimens were fixed with 10% formaldehyde, wrapped in paraffin, made into 4 um thick sections, stained with HE, and the histopathological changes were observed.
3 Results
3.1 pathological changes of exercise, Cistanche deserticola and vitamin C on rat kidney
After the determination of SCR and bun, the renal tissues of each group were made into paraffin sections, stained with HE, and the histomorphological changes were observed under a light microscope. Under the light microscope, the renal tissue structure of rats in the static control group and general training group was normal, without congestion, degeneration, and edema, and there was no tubular type in the lumen of renal tubules. In the overtraining group, there was congestion in the glomerulus, edema, vacuolar degeneration and lumen expansion of tubular epithelial cells, and a small number of exfoliated villi and epithelial cells in the lumen. The histopathological changes in other groups were lighter than those in the overtraining group, with slight edema, vacuolar degeneration, lumen expansion of tubular epithelial cells, and no protein tube type and cell tube type. There was no significant difference in the Paller score of renal tubules between the static control group and the general training group (P > 0.05), the static control group was significantly lower than that of other groups (P < 0.01), and the treatment group was significantly lower than that of the overtraining group (P < 0.05).
3.2 effects of exercise, Cistanche deserticola, and vitamin C on serum BUN and SCR in rats
Blood urea nitrogen and serum creatinine increased significantly in each group compared with the control group, and decreased significantly in com group, VCOM group and CVCOM group compared with OM group (P < 0.05, P < 0.05 and P < 0.01 respectively); Compared with VCOM group, COM group and CVCOM group decreased significantly (P < 0.05, P < 0.01, respectively).
3.3 effects of exercise, Cistanche deserticola, and vitamin C on SOD activity and MDA content in rat renal homogenate.
Compared with OM group, SOD activity in COM group, VCOM group and CVCOM group increased significantly (P < 0.05, P < 0.05 and P < 0.o1, respectively); Compared with VCOM group, COM group and CVCOM group increased significantly (P < O.05, P < 0.o1, respectively). The content of MDA in each group was significantly higher than that in the control group and decreased significantly in the COM group, VCOM group, and CVCOM group compared with OM group (P < 0.05, P < 0.05 and P < 0.01 respectively); Compared with VCOM group, COM group, and CVCOM group decreased significantly (P < 0.05 and P < 0.o1, respectively).
4 Discussion
Renal ischemia-reperfusion (R / R) injury is a complex pathophysiological process, and its specific mechanism has not been fully clarified. However, oxygen-free radicals (OFR) play an important role in the occurrence and development of I / R. Under normal circumstances, a small number of free radicals can be produced in the body, but due to the inactivation of the free radical enzyme system in the body, the free radicals are quickly removed without damage. During exercise, I / R increases the production of free radicals, which can lead to membrane lipid peroxidation, resulting in the changes of membrane fluidity, fluidity, and permeability, and then cause membrane dysfunction, especially the destruction of mitochondrial membrane, which will affect cell metabolism and function, and interfere with the physiological function of the whole organ. Antioxidant enzymes are the main substances in the body to eliminate free radicals and reduce their harm. Sod is the only enzyme with an oxygen-free radical as substrate among thousands of enzymes in aerobic organisms. It removes superoxide anion by catalyzing superoxide anion to form hydrogen peroxide and protects the body from damage. At the same time, in a certain range, SOD will increase compensably when free radical metabolism is enhanced. Therefore, the level of SOD activity is a sign of the strength of antioxidant capacity. MDA is the main product of cellular lipid peroxidation. The content of MDA in mitochondria is recognized as a sensitive index to measure free radical metabolism. Creatinine and urea nitrogen are catabolic products of muscle and protein respectively. They are mainly excluded from the kidney through blood circulation. Their concentration in the blood depends on the glomerular filtration capacity. When the renal essence is damaged and the glomerular filtration rate drops to the critical point, their concentration will rise significantly.
The results showed that overtraining for 8 weeks resulted in significant changes in renal histopathology. The experimental results show that Cistanche deserticola and vitamin C can effectively scavenge free radicals, reduce renal ischemia and hypoxia, inhibit lipid peroxidation and improve SOD activity. It can effectively reduce renal ischemia-reperfusion injury and play a protective role. The possible mechanisms are as follows: 1) Cistanche deserticola can improve the activity of antioxidant enzymes and accelerate the elimination of free radicals. At the same time, a variety of polyphenols, glycosides, and flavonoids play a positive role in scavenging free radicals, reducing the damage of free radicals to the mitochondrial membrane and sarcoplasmic reticulum membrane, and reducing the oxidative damage to enzyme proteins. 2) Vitamin C is an important antioxidant vitamin. As a strong antioxidant, it can effectively eliminate oxygen free radicals, inhibit the initiation of apoptosis cascade and reduce the ischemia-reperfusion injury of cells and organs. In addition, the results of three treatment groups suggest that Cistanche deserticola may be better than vitamin C in the treatment of exercise-induced renal ischemia-reperfusion injury; The combination of the two may be more conducive to the protection of kidney from ischemia-reperfusion injury.
In conclusion, heavy load swimming training caused renal ischemia-reperfusion injury in rats. Cistanche deserticola and vitamin C have obvious protective effects on renal ischemia-reperfusion injury, which may be by enhancing superoxide dismutase activity, scavenging free radicals, reducing lipid peroxidation, and improving renal function. In terms of curative effect, the combination of the two is better than the two alone, and Cistanche deserticola is better than vitamin C, but it may also be related to the dose of the two in the application. The relationship between dose and curative effect needs to be further studied.
