Effect of Reynoutria Japonic and Resveratrol on Sirt1/SOD/MDA of Adriamycin-Induced Renal Injury: A Randomised Trial

Background: Mechanisms of drug-induced kidney injury include mitochondrial dysfunction and oxidative stress. Resveratrol is a natural activator of sirt1 that is related to oxidative stress. Objectives: To explore the mechanism of treating drug-induced kidney injury with Reynoutria japonica and its extract (Resveratrol). Methods: Fifty adult male SD rats were randomly divided into ve groups: blank group, model group, Reynoutria japonica group, resveratrol group and Benazepril group. Except the blank group, each group used a one-time tail vein injection of 7.5mg/kg adriamycin to make the rat model of drug-induced renal injury. After three days, the proteinuria test strip showed green, which was positive for proteinuria. Each group was given the corresponding drug. ACR was measured on the seventh day every week. All rats were anaesthetized death on the fourth weekend to obtain blood and kidneys. Results: At the fourth week, the MDA levels of blank group and Reynoutria japonica group were signicantly lower than those of benazepril hydrochloride group (P<0.05), and the MDA levels of resveratrol group and model group were signicantly higher than those of benazepril hydrochloride group (P<0.05).The Sirt1 mRNA levels of the blank group and the Reynoutria japonica group were signicantly higher than those in the benazepril hydrochloride group (P<0.05), and the Sirt1 mRNA levels of resveratrol group and model group were signicantly lower than those of the benazepril hydrochloride group (P<0.05). The comparison results between groups of the Sirt1 protein expression were the same as those of the Sirt1 mRNA expression (P<0.05). Conclusions: The therapeutic effect of the Reynoutria japonica group was better than that of the Benazepril group and resveratrol group.


Introduction
In recent years, the number of patients with renal damage caused by antineoplastic drugs, targeted drugs, antipsychotics and painkillers has increased year by year. In the future, drug-induced renal injury will become an increasingly serious economic and major public health problem. As early as the 1960s, people had a clear understanding of the renal damage caused by drugs. All renal structural and functional damage caused by drugs, chemicals or biotoxins, with clinical manifestations, can be called drug-induced kidney injury or toxic nephropathy. [1][2] Adriamycin is a widely used anticancer drug. Dose-related nephrotoxicity greatly limits its clinical application. It was previously thought that the mechanism of drug-induced kidney injury induced by adriamycin may include in ammatory mediators, oxidative stress, necrosis, apoptosis, autophagy, etc. [3][4][5][6] Sirtuin1 (abbreviated as sirt1) is an anti-ageing gene. It is mainly expressed in renal tubular epithelial cells. Recent studies suggest that the development of sirt1 may serve it as a new marker for the diagnosis and prognosis of drug-induced kidney injury. [7][8][9] These studies suggest that sirt1 may be an effective target for the prevention of drug-induced kidney injury.
Benazepril can reduce renal injury by dilating blood vessels, reducing renal load and reducing renal hyper ltration as a common kidney disease drug. [10] Traditional Chinese medicine believes that blood stasis, damp heat and toxin are closely related to the occurrence and development of drug-induced renal injury. Therefore, the active use of methods such as promoting blood circulation and removing blood stasis, promoting dampness and resolving phlegm and detoxi cation is of great positive signi cance for the prevention and treatment of drug-induced renal injury. Reynoutria japonica is a traditional Chinese medicine, it is the root of Reynoutria japonica Houtt., it is a genus of Polygonaceae plants. In China, it is taken directly by boiling water with its roots. Reynoutria japonica has the effects of clearing heat and detoxi cation, diuresis, promoting blood circulation and removing blood stasis, it has a good effect on chronic renal insu ciency. According to the above TCM theories, we believe that the role of Reynoutria japonica in promoting blood circulation and removing blood stasis may play an important role in the prevention and treatment of drug-induced renal injury.
Resveratrol, an extract of Reynoutria japonica (Reynoutria japonica Houtt.), has antiaging and antifungal effects [11] and inhibits the proliferation of vascular endothelial cells. [12] Resveratrol is a natural sirt1 activator. To date, there is little study on the pathogenesis and prevention mechanism of drug-induced kidney injury by Reynoutria japonica and resveratrol through the sirt1 pathway.
Recent studies [13] have increased the understanding of the subcellular mechanisms of drug-induced kidney injury, including mitochondrial dysfunction, oxidative stress, direct toxicity to renal tubules and cell apoptosis. At present, there is no speci c drug to prevent drug-induced kidney injury, and thus it is necessary to explore prevention strategies for drug-induced kidney injury. National Institute of Health guidelines. All animals were housed in a standard feeding environment (room temperature 21-23℃, 12 hours of day/night cycle) and were free to eat standard feed and drinking water.

Modelling
After three days of adaptive feeding, fty adult male SD rats were randomly divided into ve groups: blank group, model group, Reynoutria japonic group, resveratrol group and benazepril hydrochloride group, with 10 rats in each group. The model group, Reynoutria japonic group, resveratrol group and benazepril hydrochloride group used a one-time tail vein injection of 7.5mg/kg adriamycin to make the rat model of drug-induced renal injury. After three days, the proteinuria test strip showed green, which was positive for proteinuria. For the rats that failed to make the model, the above methods can be repeated. In this experiment, the rats were successfully made.

Gastric perfusion method and collection of specimens
The Reynoutria japonica group was given 10g/kg/d, which converted from human dose, resveratrol group was given 40 mg/kg/d.respectively, while the Benazepril group was given 0.90 mg/kg/d. The blank and model groups were given double distilled water at the same time. All the drugs were gavaged twice daily for four weeks. Doses of doxorubicin [14] and resveratrol [15] were based on previous literature. The rats were weighed on the rst morning of each week to adjust the dosage. On the morning of the seventh day of each week, 24-hour urine was collected with a metabolic cage for urine volume and urinary protein excretion measurement. All rats were anaesthetized death with 3% sodium pentobarbital (0.1 0.2 ml/100g) on the fourth weekend to obtain blood and kidneys, the blood was taken from the heart to measure serum creatinine and urea nitrogen. The kidneys of both sides were divided into two halves along the sagittal line on ice and preserved in three parts: the kidneys were frozen in liquid nitrogen after being in ltrated with RNA protective solution and stored in -80℃ for real-time PCR experiment; for detection of oxidative stress index after tissue homogenization saved as above; the kidneys were xed in paraformaldehyde for immunohistochemistry and PAS staining.

Immunohistochemistry and pathological section observation
Part of the right kidney was para n-embedded, dewaxed, dehydrated, soaked and washed. The negative control used PBS instead of primary and secondary antibodies. For observation, DAB was used for coloration, as described previously. [16] Pathological section observation The kidneys were observed under a light microscope as described previously. [16] Western blot The total pulmonary protein was extracted from 100 mg of frozen lung tissue. Then the total proteins either from cells or tissues by RIPA lysis buffer were applied for the western blot analysis, which was processed following our previous methods. [17] Detection of urinary albumin/urinary creatinine Urinary albumin/urinary creatinine (mg/g) was detected by BECKMAN COULTER iRICELL3000 Automatic urinalysis line on November 12, 2019.

Detection of serum creatinine and urea nitrogen
Serum creatinine (µmol/L) and urea nitrogen (mmol/L) were detected by HITACHI 7600 automatic biochemical analyzer on November 28, 2019.

Detection of oxidative stress index
The levels of malonic dialdehyde (nmol/L) and superoxide dismutase (U.ml-1) in the kidney tissue were measured by mature commercial kits by colorimetry on November 18, 2019.

Quantitative real-time PCR
Real-time polymerase chain reaction analysis was performed as described previously. [16] Oligonucleotide PCR primers for rat genes were as Table 1.

General conditions
After injection of doxorubicin, the rats gradually appeared laziness, drowsiness and other phenomena. Compared with the blank group, the hair had no luster, and was accompanied by severe ascites, oliguria and weight loss in the later stage. The rats in resveratrol group and benazepril hydrochloride group were relatively poor, and the mortality was higher than that in Reynoutria japonica group.

Comparison of weight changes in each group of rats within four weeks
At the rst week, the body weight levels in the benazepril hydrochloride group and the blank group were signi cantly higher than those of the model group (P<0.05), there were no signi cant differences between the Reynoutria japonica group, the resveratrol group and the model group (P>0.05). The body weight levels in the model group was signi cantly lower than that in the benazepril hydrochloride group (P<0.05), There were no signi cant differences in the blank group, the Reynoutria japonica group, the resveratrol group and the benazepril hydrochloride group (P>0.05). At the second week, the body weight levels in the benazepril hydrochloride group, the Reynoutria japonica group and the blank group were signi cantly higher than those of the model group (P<0.05), there was no signi cant difference between the resveratrol group and the model group (P>0.05). The body weight levels in the model group and the resveratrol group were signi cantly lower than those in the benazepril hydrochloride group (P<0.05), There were no signi cant differences between the blank group, the Reynoutria japonica group and the benazepril hydrochloride group (P>0.05). At the third week, the body weight levels in the benazepril hydrochloride group and the blank group were signi cantly higher than those of the model group (P<0.05), There were no signi cant differences between the model group, the Reynoutria japonica group and the resveratrol group (P>0.05). The body weight level in the blank group was signi cantly higher than that of the benazepril hydrochloride group (P<0.05), the body weight level in the model group was signi cantly lower than that of the benazepril hydrochloride group (P<0.05), there were no signi cant differences between the benazepril hydrochloride group, the Reynoutria japonica group and the resveratrol group (P>0.05). At the fourth week, the body weight levels of the benazepril hydrochloride group, the Reynoutria japonica group and the blank group was signi cantly higher than those of the model group (P<0.05), there was no signi cant difference between the resveratrol group and the model group (P>0.05). The body weight level of the blank group was signi cantly higher than that in the benazepril hydrochloride group (P<0.05), the body weight levels of the resveratrol group and the model group were signi cantly lower than the benazepril hydrochloride group (P<0.05). there was no signi cant difference between the benazepril hydrochloride group and the Reynoutria japonica group (P>0.05) (  Comparison of Urine volume changes in each group of rats within four weeks At the rst week, the urine volume values in the blank group and the benazepril hydrochloride group were signi cantly higher than that of the model group (P<0.05), and there were no signi cant differences between the resveratrol group, the Reynoutria japonica group and the model group (P>0.05). The urine volume value in the blank group was signi cantly higher than that of the benazepril hydrochloride group (P<0.05), the urine volume values in the resveratrol group, the Reynoutria japonica group and the model group were signi cantly lower than that of the benazepril hydrochloride group (P<0.05). At the second week, there were no signi cant differences between the model group and the other groups (P>0.05). The urine volume value of blank group was signi cantly higher than that of the benazepril hydrochloride group (P<0.05). There were no signi cant differences between the resveratrol group, the Reynoutria japonica group, the model group and the benazepril hydrochloride group (P>0.05). At the third week, the urine volume values of the blank group, the Reynoutria japonica group and the benazepril hydrochloride group were signi cantly higher than those of the model group (P<0.05), there was no signi cant difference between the resveratrol and the model group (P>0.05). The urine volume value of the blank group was signi cantly higher than that of the benazepril hydrochloride group (P<0.05), the urine volume value in the model group was signi cantly lower than that of the benazepril hydrochloride group (P<0.05).
There was no signi cant difference between the resveratrol the Reynoutria japonica group and the benazepril hydrochloride group (P>0.05). At the fourth week, the urine volume values of the blank group, the Reynoutria japonica group and the benazepril hydrochloride group were signi cantly higher than those of the model group (P<0.05), there was no signi cant difference between the resveratrol group and the model group (P>0.05). The urine volume value of the blank group was signi cantly higher than that of the benazepril hydrochloride group (P<0.05), the urine volume value in the model group was signi cantly lower than that of the benazepril hydrochloride group (P<0.05). There was no signi cant difference between the resveratrol, the Reynoutria japonica group and the benazepril hydrochloride group (P>0.05) ( Table 3, Figure legend 2). Comparison of urinary albumin to creatinine ratio (ACR) in the urine of rats in each group At the rst week, the ACR levels in the blank group, the Reynoutria japonica group and the benazepril hydrochloride group were signi cantly lower than those of model group (P<0.05), and there was no signi cant difference between the resveratrol group and the model group (P>0.05). The ACR level in the blank group was signi cantly lower than that in the benazepril hydrochloride group (P<0.05), the ACR levels in the Reynoutria japonica group, the resveratrol group and the model group were signi cantly higher than those of benazepril hydrochloride group (P<0.05).At the second week, the ACR levels in the blank group, the Reynoutria japonica group and the benazepril hydrochloride group were signi cantly lower than those of model group (P<0.05), and there was no signi cant difference between the resveratrol group and the model group (P>0.05). The ACR level in the blank group was signi cantly lower than that in the benazepril hydrochloride group (P<0.05), the ACR levels in the Reynoutria japonica group, the resveratrol group and the model group were signi cantly higher than those of benazepril hydrochloride group (P<0.05).At the third week, the ACR level in each group was signi cantly lower than those of the model group (P<0.05), The ACR level in the blank group was signi cantly lower than that in the benazepril hydrochloride group (P<0.05), the ACR levels in the resveratrol group and the model group were signi cantly higher than those of the benazepril hydrochloride group (P<0.05), and there was no signi cant difference between the Reynoutria japonica group and the benazepril hydrochloride group (P>0.05). At the fourth week, the ACR levels in each group was signi cantly lower than those of the model group (P<0.05), The ACR level in the blank group was signi cantly lower than that in the benazepril hydrochloride group (P<0.05), the ACR levels in the resveratrol group and the model group were signi cantly higher than those of the benazepril hydrochloride group (P<0.05), and there was no signi cant difference between the Reynoutria japonica group and the benazepril hydrochloride group (P>0.05) (Table 4, Figure legend 3).

Comparison of Scr and BUN in the blood of rats in each group
At the fourth week, the Scr levels of the blank group, the Reynoutria japonica group, the resveratrol group and the benazepril hydrochloride group were signi cantly higher than that of model group (P<0.05). The Scr levels of the blank group and the Reynoutria japonica group were signi cantly higher than those of the benazepril hydrochloride group (P<0.05). The Scr levels of the resveratrol group and the model group were signi cantly lower than those of the benazepril hydrochloride group (P<0.05). The comparison results between groups of the BUN values were the same as those of the Scr values (Table 5, Figure  legend 4). Comparison of SOD activity and MDA level in renal tissue of rats in each group At the fourth week, the SOD activities of blank group, Reynoutria japonica group, resveratrol group and benazepril hydrochloride group were signi cantly higher than that of model group (P<0.05). The SOD activities of blank group and Reynoutria japonica group were signi cantly higher than that of benazepril hydrochloride group (P<0.05), and the SOD activities of resveratrol group and model group were signi cantly lower than that of benazepril hydrochloride group (P<0.05). The levels of MDA in blank group, Reynoutria japonica group, resveratrol group and benazepril hydrochloride group were signi cantly lower than those in the model group (P<0.05). The MDA levels of blank group and Reynoutria japonica group were signi cantly lower than those of benazepril hydrochloride group (P<0.05), and the MDA levels of resveratrol group and model group were signi cantly higher than those of benazepril hydrochloride group (P<0.05) ( Table 6, Figure legend 5).

Sirt1 mRNA and protein expression level on the fourth weekend
At the fourth week, the Sirt1 mRNA levels in the blank group, the Reynoutria japonica group, the resveratrol group and the benazepril hydrochloride group were signi cantly higher than those in the model group (P<0.05). The Sirt1 mRNA levels of the blank group and the Reynoutria japonica group were signi cantly higher than those in the benazepril hydrochloride group (P<0.05), and the Sirt1 mRNA levels of resveratrol group and model group were signi cantly lower than those of the benazepril hydrochloride group (P<0.05). The comparison results between groups of the Sirt1 protein expression were the same as those of the Sirt1 mRNA expression (P<0.05) ( Table 7, Figure legend 6 and Fig. 1 and Fig. 2).

Changes in each group of rat renal ultrastructures
The histological examination of the kidney under a light microscope showed that on the fourth weekend, the rats in the model group had the characteristics of brosis and hyperplasia, interstitial hyperaemia, glomerular haemorrhage, in ammatory cell in ltration, tubular protein, and tubular and glomerular atrophy. In the treatment group, there was slight glomerular haemorrhage and a small amount of in ammatory cell in ltration in interstitial tissue. The lesion degree of Reynoutria japonica group was better than that of Benazepril group, while the Benazepril group was better than that of Resveratrol group (Fig. 3).
Pearson linear correlation analysis was used to analyse the correlation between Sirt1, SOD and MDA According to the correlation results table, there was a signi cant correlation between Sirt1, SOD and MDA. The correlation coe cients were between 0.8-1, indicating that there was a strong positive correlation between Sirt1 and SOD, there was a strong negative correlation between Sirt1 and MDA, SOD and MDA (Table 8).

Discussion
The results showed that the urinary albumin/urinary creatinine of the treatment group and the model group were higher than that of the blank group, indicating that the model was successful. At the rst week, the urinary albumin/urinary creatinine value of the Benazepril group was signi cantly lower than that of the model group (P<0.05), suggesting that the effect of Benazepril is rapid and obvious. At the third week, one rat died in the Benazepril group, which indicated that the therapeutic effect of Reynoutria japonica was slow, but the side effect was less than that of Benazepril.
It can be seen from the observation of the oxidation indices of each group that the level of superoxide dismutase in the kidney of each treatment group can be increased, and the level of malonic dialdehyde can be reduced.
MDA is an important product of lipid peroxidation in vivo, which can indirectly re ect the degree of lipid peroxidation, and is an important index to evaluate the level of oxidative stress. SOD can eliminate oxygen free radicals in vivo and alleviate the damage to tissue caused by free radicals, and superoxide can affect the activity of SOD. When tissue damage is serious, SOD function will decline or even become inactive. [18] Therefore, SOD plays an important role in antioxidation. The results of this study suggest that the level of MDA in the treatment group was lower than that in the model group, and the level of SOD in the serum was higher.Studies have shown that the sirt1 protein has an antioxidative stress effect. Whether sirt1 can improve drug-induced renal injury by regulating the synthesis and activity of SOD and MDA has not been reported. [19] The sirt1 gene and protein expression was higher than that of the control group, which indicated that oxidative stress played an important role in the model of drug-induced renal injury.
Some studies have shown that Sirt1-SOD-ROS is the signalling mechanism of antioxidant stress in the kidney. [20] In our experiment, the SOD value in the model group was lower than that of the normal group, and the MDA value was higher than that of the normal group at the same time, suggesting that the pathogenesis of DKI may be related to oxidative reactions. The better the treatment effect, the higher the content of sirt1 and the content of SOD, the lower of the MDA. The increase of SOD means the decrease of ROS level in vivo, the high correlation among SIRT1, SOD and MDA indicates that they can affect each other, sirt1 and SOD are positive regulatory relationships, suggesting that sirt1 could enhance SOD expression and inhibit oxidative stress. [21] The effect of traditional Chinese medicine on diseases is multi-target, there are many mechanisms for drug-induced renal injury, possible mechanisms include local tissue in ammation, tissue peroxidation, injury and other pathological changes, other components in Reynoutria japonica can also play a role in the treatment of nephropat-hy. In this experiment, resveratrol may only protect the kidney from oxidation and lipid peroxidation, therefore, the protection of kidney is not as comprehensive as Reynoutria japonica. This is also one of the reasons why the experimental results of resveratrol are not as good as those of Reynoutria japonica.

Conclusion
In our experiment, the content of MDA in DKI rats was signi cantly higher than that in normal group, which also suggested that oxidative stress played an important role in the pathogenesis of drug-induced renal injury. In the experiment, the tested drugs can up regulate the expression of SIRT1, and all indexes are better than the model group. It has a certain therapeutic effect, and it can improve the expression of SIRT1 gene, increase SOD activity and reduce MDA level, indicating that it may play a therapeutic role through oxidative stress. It also suggests that the tested drugs may regulate the anti lipid peroxidation of the body through mediating SIRT1, Increase the synthesis of SOD in cells, so as to reduce the synthesis of reactive oxygen species in cells, then inhibit the oxidative damage of rat kidney induced by adriamycin, and nally realize its antioxidant physiological function. The therapeutic effect of Reynoutria japonica group is signi cantly better than that of resveratrol group and benazepril hydrochloride group, indicating that the effect of Reynoutria japonica on kidney is multi-target and the protective effect on kidney disease is more comprehensive, including not only anti lipid peroxidation, but also other mechanisms, which need to be further studied.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing Interest
All the authors declare that they have no known competing nancial interests. Authors' Contributions S. T. and S. C. designed the study. S. T. performed experiments, acquired and analyzed data, drafted and edited the manuscript; X. L. performed molecule dynamic simulation and docking. S. C., X. S. and X. C.
assisted with experiments. S. C. and S. T.contributed to provide analytical instruments and review of the manuscript.
All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.