Experimental Study On The Protective Effect Of Cistanche Total Glycosides(CTG) On Renal Ischemic Reperfusion Injury
Mar 09, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
ZHU Jun, ZHAO Xi
Abstract: Objective To establish the rat model of hemorrhagic shock and resuscitation so as to observe the protective effect of cistanche total glycosides( CTG) on the renal ischemic-reperfusion injury. Meth ods SD rats were randomized into a sham － operation group, a shock, and resuscitation group( experiment group), and a CTG group, with 10 rats in each one. After modeling, the rats in the CTG group received the intraperitoneal injection of CTG 400 mg/kg. In the experimental group and the sham-operation group, physical saline 3 ml was injected. Cr, BUN, and Cys － C levels were determined to detect the kidney function in each group. The flow cytometry was used to detect renal cell apoptosis rate. ELISA was used to detect XIAP expression in kidney tissue. Results Compared with the experiment group, Cr, BUN, and Cys － C levels were apparently reduced in the CTG group( P ＜ 0.01), and renal cell apoptosis was reduced( P ＜ 0.01). CTG improved significantly XIAP expression( P <0.05). Conclusion CTG brings the protective effect on ischemic reperfusion kidney injury in the rats, which is possibly related to the promotion of XIAP expression.
【Key words】 Kidney; Ischemic Reperfusion Injury; Cistanche Total Glycosides; XIAP; Cell Apoptosis
Ischemia-reperfusion (I/R) injury is a common pathophysiological phenomenon of renal damage in the process of hemorrhagic shock, severe trauma, kidney transplantation, and renal tumor surgery. Studies have shown that apoptosis is an important link in renal function damage caused by I/R injury [1]. X-chromosome-linked inhibitor of apoptosis protein (XIAP) is one of the strongest endogenous inhibitors of apoptosis found so far [2]. There are few studies on the anti-apoptotic effect of XIAP in renal tissue after I/R injury. Cistanche total glycosides (CTG) are from the list Phenylethanol glycoside compounds extracted from Cistanche have diverse biological functions. There are few studies on its renal protection after I/R injury. In this experiment, a rat model of hemorrhagic shock resuscitation was established, and a systemic I/R injury model was established to explore the protective effect of CTG pretreatment on the kidney from I/R injury and explore its possible mechanism.
Effects of cistanche: prevent Ischemia-reperfusion (I/R) injury
1 Material and methods
1.1 Experimental materials
1.1.1 Laboratory animals 30 clean-grade healthy male SD rats, provided by the Experimental Center of Shanghai University of Traditional Chinese Medicine, animal certificate number: SYXK (Shanghai) 2009-0069, bodyweight (320 ± 30) g.
1.1.2 Experimental reagent CTG (purchased from Zhejiang Hangzhou Xinghui Tianli Pharmaceutical Co., Ltd., batch number: 20081516); sodium pentobarbital (batch number: P3761), propidium iodide (PI), Rhodamin123 (both purchased (From Sigma, USA); Rat XIAP determination kit ELISA Kit (purchased from Shanghai Kemin Biotechnology Co., Ltd., batch number CSB-ELO26172MO).
1.1.3 Laboratory instrument Microplate reader (American Thermo Company); Multi-parameter physiological detector (purchased from NIHON KOHDEN Company, Japan); Flow Cytometer (purchased from Becton-Dickinson Company, USA); 24 G trocar ( Shandong Jierui Medical Equipment Company).
1.2 Experimental methods
1.2.1 Preparation of animal model SD rats were fasted 12 hours before the experiment and anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The rats were fixed in the supine position, and a 3 cm incision was made in the left lower abdomen and separated. In the femoral artery, a 24 G trocar is inserted, a multi-parameter physiological tester is connected, a 20 ml syringe is heparinized, and the blood pressure drops to 40 mm Hg. The blood pressure drops to 40 mm Hg. It is considered that the shock has started, and the blood pressure is maintained for 90 minutes. It is (13 ± 3) ml, and then the autologous blood in the syringe is slowly returned. It takes about 30 minutes to restore the blood pressure to more than 90% of the basic value. It is considered that the resuscitation is successful [3]. Pull out the trocar needle, ligate the femoral artery, and sew the incision. After the rats were awake, they were returned to the cage for observation, and they had free access to food and water. 24 hours after resuscitation, the rats were anesthetized with pentobarbital sodium (50 mg/kg) by intraperitoneal injection, laparotomy, and 5 ml blood was collected from the inferior vena cava. The rats were killed immediately, and the left and right kidneys were taken out for use.
1.2.2 Animal grouping and administration 30 rats were randomly divided into sham surgery group, shock resuscitation group (experimental group), and CTG group, with 10 rats in each group.
The experimental group was intraperitoneally injected with 3 ml of normal saline 2 hours before bloodletting. In the CTG group, CTG was given 2 hours before the bloodletting of the rats, and the dose was injected into the abdominal cavity at a dose of 400 mg/kg. The sham operation group only underwent anesthesia, laparotomy and separation of femoral artery trocar placement and suture incisions, etc., bloodletting and resuscitation were not performed, and the rest were the same as the experimental group.
1.3 Testing indicators and methods
1.3.1 Determination of blood creatinine (Cr), urea nitrogen (BUN), cystatin C Cr, BUN, and cystatin C are determined by the laboratory of our hospital. Cystatin C is an endogenous marker of changes in glomerular filtration rate. When renal function is impaired, the glomerular filtration rate decreases and the concentration of cystatin C in the blood can increase more than 10 times. The diagnostic accuracy of cystatin C is significantly better than that of serum creatinine.
1.3.2 Determination of cell apoptosis rate Flow cytometry was used to detect the apoptosis rate of kidney cells. Take the kidney and chop it with a blade, add a little PBS and transfer it into a test tube. Add 0.3% trypsin for digestion in a 37 ℃ water bath, and incubate for 20 min. When the substance in the test tube becomes turbid, add twice the PBS to stop the digestion, filter with a 300 mesh nylon mesh, discard impurities, and use a low-temperature centrifuge Centrifuge at 1500 g / s for 5 min, discard the supernatant, adjust the cell concentration to 1 × 106 ~ 1.5 × 106 / ml, take 0.4 ml of the above cell suspension, and add 10 μl / ml Rhodamin123 0 ．4 ml, incubate in a 37 ℃ water bath protected from light, cap the test tube, rinse with twice PBS after 30 min, then centrifuge at 1500 g/s for 5 min, discard the supernatant, and rinse again in the same way. Add PI 0.4 ml, stain for 15 min, and check the percentage of normal, necrotic, and apoptotic cells in the kidney. According to the affinity of these two dyes in various types of cells, it is known that only PI-colored cells are dead cells, only Rhodamin123 colored cells are living cells, and both colored cells are apoptotic cells.
1.3.3 Enzyme-linked immunoassay (ELISA) was used to detect the expression of XI-AP in rat kidney tissues. Kidneys of each group were taken, and fresh kidney tissues were treated with tissue lysate. Centrifuge at 1500 g/s in a low-temperature centrifuge for 5 min. Take the supernatant and operate according to the instructions of the XI-AP ELISA Kit. Shanghai Jiaoda Fenghe Technology Co., Ltd. is responsible for testing.
1.4 Statistical processing
All data were statistically analyzed using SPSS17.0 software, and the data were expressed as mean ± standard deviation (x ± s), and P<0.05 indicated that the difference was statistically significant.
cistanche total glycosides
2 results
2.1 Kidney function indexes Cr, BUN, Cystatin C Comparison of Cr, BUN, Cystatin C in the experimental group were significantly higher than those in the sham operation group (P<0.01), indicating that the rat kidney I/R model was prepared success.
Compared with the experimental group, the values of Cr, BUN, and Cystatin C in the CTG group were significantly reduced, and the difference was statistically significant (P<0.01). The results are shown in Table 1.
Table 1 Comparison of the renal function indexes Cr, BUN, and Cystatin C of rats in each group (x ± s)
Note: Compared with the sham operation group, a P＜0.01; compared with the experimental group, bP＜0.01
2.2 The rate of renal cell apoptosis and the expression of XIAP in the renal tissue. Only a few cells in the renal tissue of the sham operation group undergo apoptosis.
The number of apoptosis in the experimental group was significantly increased, and the difference was statistically significant compared with the sham operation group (P <0.01); the number of apoptosis in the CTG group was decreased compared with the experimental group, and the difference was statistically significant (P <0.01) ).
The sham operation group had only weak XIAP expression, while the experimental group had a significant increase in XIAP expression (P <0.01). Compared with the experimental group, the CTG group can significantly increase the expression of XIAP (P<0.05). The results are shown in Table 2.
Table 2 Comparison of the apoptosis rate and XIAP expression of rat kidney cells in each group (x± s)
Note: Compared with the sham operation group, aP＜0. 01; Compared with the experimental group, b P＜0. 05, cP<0. 01
3 Discussion
The mechanism of I/R kidney injury is very complicated. It is a pathological process that occurs through many pathways under the action of various factors, such as calcium ion overload, lipid peroxidation damage, apoptosis, and necrosis, among which endogenous caspases Caspase-dependent apoptosis is one of the main forms of renal tubular epithelial cell damage induced by I/R kidney injury [4]. The inhibitor of apoptosis protein (IAP) family is the only caspase inhibitor found in mammalian cells so far, which can effectively prevent the occurrence of apoptosis. XIAP is the strongest caspase inhibitory member of the IAP family, which can inhibit cell apoptosis caused by a variety of pathological stimuli and play a protective effect on damaged organs [5].
At present, the method of establishing renal I/R injury animal models is mostly to remove one side of the kidney and clamp the other side of the renal pedicle or renal artery [6] to simulate the state of kidney transplantation. The author used the hemorrhagic shock/resuscitation model in this study, which is closer to the pathophysiological process of systemic ischemia-reperfusion that occurs in clinical traumatic shock. In this experiment, the blood BUN, Cr, and cystatin of SD rats The amount of C is significantly increased and renal function is severely impaired. It is an ideal animal model for studying I/R renal injury.
CTG is an important medicinal component of the traditional Chinese medicine Cistanche. Cistanche, which is warm in nature, sweet and salty, belongs to the kidney and large intestine meridian and was originally contained in Shennong's Materia Medica. "Ben Jing," says: "Maintains five labors and seven injuries, nourishes the middle, nourishes the five internal organs, strengthens yin, and nourishes essence and qi." Also." Cistanche has been used in my country for two thousand years. It ranks first in strength-increasing formulas in the past, and second only to ginseng in nourishing formulas. Cistanche can improve sexual function, regulate neuroendocrine, and improve the immune function of the body. Antioxidants, promote material metabolism, protect ischemic myocardium and other functions. As the main medicinal ingredient of Cistanche, CTG has many functions. Research on cerebral ischemia-reperfusion model
Studies have found that CTG can reduce the excitatory amino acids in the brain tissue after ischemia-reperfusion and protect the brain tissue of SD rats [7]. CTG has an antioxidant effect on mouse tissues and has a certain protective effect on acute cerebral hypoxia in mice and cardiac muscle damage in mice [8]. CTG can significantly improve the neurobehavior of Parkinson's model mice and inhibit the decrease in the number of dopamine neurons in the substantia nigra [9]. By enhancing the activity of free radical scavenging enzymes, preventing lipid peroxidation, inhibiting the deposition of β-amyloid peptide in the brain, and inhibiting brain cell apoptosis, CTG significantly increased β-amyloid peptide-induced Alzheimer’s disease mice Learning and memory ability [10]. This study showed that after the application of CTG pretreatment, the blood BUN, Cr, and cystatin C contents of SD rats decreased significantly, and the renal function damage was reduced. CTG increased the apoptosis-inhibiting protein XIAP in the renal tissues of I/R injury and blocked the cells. The occurrence of apoptosis plays a protective role in damaging the kidney.
references
[1] Cassis P, Azzollini N, Solini S, et al. Both darbepoetin alfa and carbamylated erythropoietin prevent kidney graft dysfunction due to ischemia/reperfusion in rats ［J］． Transplantation, 2011, 92 (3): 271-279.
[2] West T, Stump M, Lodygensky G, et al. Lack of X-linked inhibitor of apoptosis protein leads to increased apoptosis and tissue loss following neonatal brain injury［J］． ASN Neuro, 2009, 1 (1). PII: e00004. doi:10. 10421AN20090005.
[3] Fu X, Tian H, Wang D, et al. Multiple organ injuries and failures caused by shock and reperfusion after gunshot wounds［J］． J Trauma, 1996, 40 (3 Suppl): S135-139.
[4] Padanilam BJ. Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis［J］． Am J Physiol Renal Physiol, 2003, 284 (4): F608-F627.
[5] Galbán S, Hwang C, Rumble JM, et al. Cytoprotective effects of IAPs revealed by a small molecule antagonist［J］． Biochem J, 2009, 417 (3): 765-771.
[6] Wu Jianguo, Zhang Dan, Wu Xinsheng, etc., Establishment and experience of acute ischemia-reperfusion kidney injury model in mice [J]. Medical Information: Handbook of Surgery, 2007, 20 (12): 1103-1105.
[7] Zhu Zheng. Effects of Cistanche Glycosides on Excitatory Amino Acids in Brain Tissue of Rats after Cerebral Ischemia and Reperfusion [J]. Anhui Medicine, 2010, 14 (12): 1393 -1394.
[8] Julia Y, Wang XW, Wubuli AS, et al. Protection effect of total glycosides of cistanche deserticola on lacking oxygen ［J］． Rep Chin Pharm, 2003, 20 (1): 73-76.
[9] Li Wenwei, Yang Ru, Cai Dongfang, et al. The protective effect of the total glycosides of cistanche on dopaminergic neurons in the substantia nigra of MPTP Parkinson's disease model mice
[10] Liu Fengxia, Wang Xiaowen, Luo Lan, etc. The effect and mechanism of the total glycosides of cistanche on the learning and memory of Alzheimer's disease mouse model induced by β-amyloid peptide [J]. Chinese Pharmacology Bulletin, 2006, 22 (5): 595-599.
