Experimental Study Of Protective Effect Of Cistanche Glycosides Pretreatment On Renal Ischemia-reperfusion Injury

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Zhu Jun, Zhao Ximin

(Surgery Department, Baoshan District Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai 201900)

【Abstract】Objective To establish a resuscitation model of hemorrhagic shock in rats and observe the protective effect of Cistanche total glycosides (CTG) on renal ischemia-reperfusion injury. Methods SD rats were randomly divided into 3 groups: sham operation group, experimental group (shock resuscitation group), CTG treatment group, 10 rats in each group. The experimental group and the CTG treatment group established renal ischemia-reperfusion models. The rats in the treatment group were injected with CTG 400 mg/kg intraperitoneally, and the experimental group and the sham operation group were injected with saline. The blood myocardial content, urea nitrogen content and cystatin C of each group were detected to determine renal function, flow cytometry analysis was used to detect renal cell apoptosis rate; enzyme-linked immunoassay (ELISA) was used to detect X-chromosome linkage in rat kidney tissue Expression of X-linked inhibitor of apoptosis protein (XIAP). Results Comparing the CTG group with the experimental group, blood Cr, BUN, and Cystatin C were significantly reduced (P<0.01), renal cell apoptosis was reduced (P<0.05), CTG can significantly increase the expression of XIAP (P<0.05). Conclusion The total glycosides of cistanche have a protective effect on renal ischemia-reperfusion injury in rats, which may be related to the promotion of the expression of XIAP.

[Keywords] kidney; ischemia-reperfusion injury; total glycosides of cistanche; chromosome-linked inhibitor of apoptosis protein; apoptosis; rat

[Chinese Library Classification Number] R692 [Document Marking Code] A [Article Number] 1008 -0392 (2015) 02-0037 -04

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Ischemia-reperfusion (I/R) injury is a common pathophysiological phenomenon of renal damage in the process of hemorrhagic shock, severe trauma, kidney transplantation, and kidney tumor surgery. Studies have shown that apoptosis is an important part of renal function damage caused by I/R injury (1). X-linked inhibitor of apoptosis protein (XIAP) is one of the strongest endogenous apoptosis inhibitors 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) is a phenylethanoid total glycosides compound extracted from Cistanche, which has diverse biological functions. There are few studies on its renal protection after I/R injury. . This study established a model of hemorrhagic shock resuscitation in rats and established a model of systemic I/R injury to explore the protective effect of total glycosides of meat hernia on the kidney from I/R injury and explore its possible mechanism.

1Materials and methods

1.1 Animals and grouping

There are 30 clean and healthy male rats provided by the Experimental Center of Shanghai University of Traditional Chinese Medicine, weighing (320 ± 30) g. Randomly divided into 3 groups: sham operation group, experimental group (shock resuscitation group), CTG group, each with 10 rats.

1.2 Main reagents and instruments

The total glycosides of glutinous rice (batch number: 20081516) were purchased from Zhejiang Hangzhou Xinghui Tianli Pharmaceutical Co., Ltd.; sodium pentobarbital, Hoechst 33258, and propionyl iodide were purchased from Sigma, USA; mouse X chromosome-linked apoptosis inhibitor Protein Assay Kit (XIAP) and ELISA Kit were purchased from Shanghai Kemin Biotechnology Co., Ltd.; microplate reader was purchased from Thermo Company, USA; multi-parameter physiological detector was purchased from Nihon Kohden Company, Japan; flow cytometer BDFACSAria was purchased from Becton, USA -Dickinson Company; 24G trocar was purchased from Shandong Jierui Medical Devices Company.

1.3 Experimental method

1.3.1 Preparation of animal model SD rats were fasted 12h before the experiment and anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg).

The rat was fixed in the supine position, a 3 cm incision was made in the left lower abdomen, the femoral artery was separated, a 24G trocar was inserted, and a multi-parameter physiological detector was connected. The average blood pressure of the rat was measured as (113 ± 15) mmHg, 20 ml syringe heparin The blood pressure was dropped to 40 mmHg (l mmHg = 0. 133 kPa) with a chemical connection three-way device. The shock was considered to have started, and the blood pressure was maintained for 90 min. The bloodletting volume was (13 ± 3) ml, and then the autologous blood in the syringe Slow reinfusion takes about 30 minutes, and the blood pressure returns to more than 90% of the basic value, and the resuscitation is considered successful (3). The trocar was pulled out, the femoral artery was ligated, and the incision was sutured. After the rats were awake, they were returned to the cage for observation, and they were free to eat and drink. 24 h after resuscitation, intraperitoneal injection of sodium pentobarbital (50 mg/kg) was used for anesthesia, laparotomy, 5 ml blood was collected from the inferior vena cava, the rats were immediately sacrificed, and the left and right kidneys were taken out for use.

1.3.2 Medication Methods CTG treatment group was given CTG 2 hours before the bloodletting of the rats and injected into the abdominal cavity at a dose of 400 mg/kg. The experimental group was intraperitoneally injected with 3 ml of normal saline 2 hours before bloodletting; the sham operation group only underwent anesthesia, laparotomy and separation of the femoral artery, placement of a trocar, and suture of the incision, but no bleeding or resuscitation. The rest were the same as the experimental group.

1. 4 detection indicators and methods

1.4.1 Determination of blood BUN, Cr, and cystatin C content Cystatin C is an endogenous marker of changes in glomerular filtration rate. When renal function is impaired, glomerular filtration rate Decreases, the concentration of cystatin C in the blood can increase 10 times, and the diagnostic accuracy of cystatin C is significantly better than that of serum muscle intoxication. The blood BUN, Cr, and Cystatin C content were submitted to the laboratory of our hospital for determination.

1.4.2 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 to digest in a 37 P water bath, incubate for 20 min, when the contents in the test tube become turbid, add 2 times PBS to stop the digestion, filter with a 300 mesh nylon mesh, discard impurities, and centrifuge in a low-temperature centrifuge. Centrifuge radius 8 cm, 12,000 r/min, centrifuge for 5 min, discard the supernatant, adjust the cell concentration to 1 ~ 1.5 x 106/ml, take 0.4 ml of the above cell suspension, and add 1.2 ml Hoechst 33258 (10 pil/ml), dark 37 t: incubate in a water bath, cap the test tube, rinse with PBS after 5 min, centrifuge radius 8 cm, 12 000 r/min, centrifuge for 5 min, discard the supernatant, Rinse again in the same way. Add 0.4 ml of PI, stained for 5 minutes, and detect the percentage of normal, necrotic, and apoptotic cells in the kidney. According to the affinity of the two dyes of various types of cells, it is known that only PI-colored cells are dead cells, only Hoechst 33258 colored cells are apoptotic cells, and neither of them is colored normal living cells.

1.4.3 Enzyme-linked immunoassay (ELISA) to detect XIAP expression in rat kidney tissues Take each group of kidneys and treat fresh kidney tissues with tissue lysate. Centrifuge in a low-temperature centrifuge at 12 000 r/min for 5 min. Take the supernatant and operate according to the instructions of the XIAP ELISA Kit kit. Shanghai Jiaotong University Fenghe Technology Co., Ltd. is responsible for testing.

1.5 Statistical processing

The experimental data of each group is represented by Yuan soil s, and SPSS 17. 0 software is used for statistical analysis. P <0.05 indicates that the difference is statistically significant.

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2 results

Kidney function indexes Cr, BUN, and Cystatin C values were statistically significant (F <0.01); the experimental group was significantly higher than the sham operation group (P <0.01), indicating that the rat The kidney I/R model was successfully prepared; compared with the experimental group, the values of Cr, BUN and Cystatin C in the CTG group were significantly reduced (P<0.01). Renal cell apoptosis rate: In the sham-operated group, only a few cells in the kidney tissue experienced apoptosis. Apoptotic cells accounted for 0.42% ± 0.08%, and the experimental group had a significant increase in apoptosis, accounting for 18. 22% ± 5. 32% (P <0.01). Compared with the experimental group, the CTG group showed apoptosis The number decreased to 12.71% ± 3.81% (P< 0.01). The effect of CTG on the expression of XIAP in I/R injured renal tissue: The sham operation group only had weak XIAP expression, while the experimental group XIAP expression increased significantly, (F< 0.01). Compared with the experimental group, the CTG group can significantly increase the expression of XIAP (F <0.05), see Table 1.

Table 1 Kidney function indexes Cr, BUN, and Cystatin-C values of rats in each group Tab. 1 Renal function indexes in three groups

Compared with the sham operation group, *P<0.01; compared with the experimental group, △P<0.05/P <0.01

         The mechanism of ischemia-reperfusion kidney injury is very complicated. It is a pathological process that occurs in many ways under the action of various factors, such as calcium ion overload, lipid peroxidation damage, cell apoptosis and necrosis, among which caspase-dependent cell 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 endogenous caspase inhibitor found in mammalian cells so far, which can effectively prevent the occurrence of cell apoptosis. XIAP is the member with the strongest inhibitory effect on caspase in the IAP family. It can inhibit cell apoptosis caused by a variety of pathological stimuli and play a protective effect on damaged organs (5).

At present, most of the methods for establishing animal models of renal I/R injury are to remove one kidney and clamp the other side of the renal pedicle or renal artery ⑹ to simulate the state of kidney transplantation. This study uses a hemorrhagic shock/resuscitation model, 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 C levels of SD rats Significant improvement, severely impaired renal function, is an ideal animal model for studying I/R renal injury.

CTG is an important medicinal ingredient of traditional Chinese medicine meat girl paste. Eggplant paste, which is warm in nature, sweet and salty, returns to the kidney and large intestine meridian and was originally contained in Shennong's Materia Medica. "Ben Sutra": Main five labors and seven injuries, nourish the middle, nourish the five internal organs, strengthen yin, nourish essence and qi; Cistanche has been used in my country for two thousand years. It ranks No. 1 in strength-increasing formulas in the past and ranks No. 2 in nourishing formulas after ginseng. Studies have shown that Cistanche can improve sexual function, regulate neuroendocrine, and improve immune function. , Antioxidants, promote material metabolism, protect ischemic myocardium and other functions. As the main medicinal ingredient of meat family puree, CTG has many functions. Research on cerebral ischemia-reperfusion models 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 (9). 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 B-amyloid peptide in the brain, and inhibiting brain cell apoptosis, CTG significantly improves the learning of Alzheimer's disease mice caused by B-amyloid peptide Memory ability w. This study showed that after the application of CTG pretreatment, the blood BUN, Cr, and cystatin C contents of rats decreased significantly, and the renal damage was reduced. CTG increased the apoptosis-inhibiting protein XIAP in I/R injured renal tissue and blocked cell apoptosis. The occurrence of death has a protective effect on the damage to the kidney.