Effect Of Cistanche Deserticola Bushen Huoxue Prescription On Oxidative Stress，PI3K/Akt， Bcl-2/Bax in Rats With Chronic Renal Failure

LIN Guan-Hao， HUANG Mei， WU Mei 

（Dept. of Pharmacy， Haikou Hospital of Traditional Chinese Medicine， Haikou 570216 Hainan， China） 

Abstract： Objective To investigate the therapeutic effect and mechanism of the Kidney Tonifying and Blood Activating Formula Composed of Cistanche deserticola and Other Prescriptions （BHP， a herbal compound mainly with the actions of replenishing kidneys and activating blood）in rats with chronic renal failure. Methods Forty SD rats were divided into a normal group（10 rats）， a model group（9 rats）， BHP low-dose group（10 rats）, and a high-dose group（10 rats）. Except for the normal group， the rats in all groups were replicated with the chronic renal failure model by adenine gavage. After successful replication， the rats in the BHP low- and high-dose groups were given 0.52 and 2.08 g/kg of BHP by gavage， respectively， once daily for 28 consecutive days. After the end of the administration， serum levels of renal function indicators blood urea nitrogen （BUN）， blood creatinine（SCr）and uric acid（UA）， and levels of oxidative stress indexes malondialdehyde （MDA）and superoxide dismutase（SOD）in renal tissue were measured by enzyme-linked immunosorbent assay （ELISA）， the histopathological changes of rat kidney tissue were observed by hematoxylin-eosin（HE）staining， and the protein expressions of phosphatidylinositol-3-kinase（PI3K）， protein kinase B（Akt）， B cell lymphoma 2 （Bcl-2）and Bcl-2 associated X protein（Bax）in renal tissue were detected by Western Blot. Results（1）Compared with the normal group，the levels of BUN，SCr and UA were increased in the model group（P＜0.05）；compared with the model group，the levels of BUN，SCr and UA were decreased significantly in the BHP low- and highdose groups（P＜0.05）.（2）Compared with the normal group， MAD level was increased and SOD level was decreased in the model group（P＜0.05）；compared with the model group，MAD level was decreased and SOD level increased in the BHP low- and high- dose groups（P＜0.05）；（3）Compared with the normal group， pathological changes such as mild necrosis and inflammatory cell infiltration，tubular dilatation and glomerular deformation appeared in the kidney tissue of the model group；compared with the model group，the pathological conditions in both the BHP low- and high- dose groups were improved.（4）Compared with the normal group，the protein expression levels of PI3K and Akt in the kidney tissue in the model group were significantly increased（P＜ 0.05）；compared with the model group，the protein expression levels of PI3K and Akt in the kidney tissue in the BHP low- and high- dose groups were significantly decreased（P＜0.05）.（5）Compared with the normal group， the protein expression level of Bcl- 2 in the kidney tissue in the model group was decreased and the protein expression level of Bax was increased（P＜0.05）；compared with the model group，the expression level of Bcl-2 protein in the kidney tissue in the BHP low- and high- dose groups was increased and the protein expression level of Bax was decreased（P＜0.05）. Conclusion BHP has an effect in improving kidney injury in rats with chronic renal failure by a mechanism related to the inhibition of PI3K/Akt signaling pathway and regulation of related apoptotic protein expression. 

Keywords：Kidney Tonifying and Blood Activating Formula Composed of Cistanche deserticola and Other Prescriptions；chronic renal failure；oxidative stress；phosphatidylinositol-3-kinase （PI3K）；protein kinase B（Akt）；B cell lymphoma 2（Bcl-2）；Bcl-2 associated X protein（Bax）； rats

Chronic renal failure (CRF) is a kind of clinical syndrome that damages the kidney due to autoimmune reaction, heredity, diet, infection, and other factors, leading to weakened renal function, or even complete loss of function. The incidence rate is increasing year by year, and it is difficult to cure [1-3]. In recent years, the therapeutic effect of traditional Chinese medicine on chronic renal failure has increasingly received widespread attention. Chronic renal failure belongs to the category of "Guange" and "edema" in traditional Chinese medicine. The disease mainly occurs in the kidney, which is a syndrome of deficiency and excess. The main pathogenesis is kidney deficiency and blood stasis, and the main treatment is to replenish the kidney and promote blood circulation [4]. Previous studies have shown that the method of tonifying the kidney and activating blood circulation can effectively improve the condition of patients with chronic renal failure, improve blood microcirculation, and inhibit inflammatory reactions in the body [5-6]. However, the therapeutic mechanism of the method of tonifying the kidney and activating blood circulation for chronic renal failure is not completely clear. Therefore, this study established a rat model of chronic renal failure to further observe the effects and mechanisms of the self-formulated formula of tonifying the kidney and activating blood circulation, intending to provide a reference basis for the clinical application of the method of tonifying the kidney and activating blood circulation for the treatment of chronic renal failure. The research results are reported as follows.

Chinese medicinal materials such as Cistanche deserticola

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1 Materials and Methods

1.1 Experimental animals

40 SPF-level healthy 6-week-old male SD rats, with a body weight of 130~170 g, were purchased from Beijing Innokai Technology Co., Ltd. with an animal production license number of SCXK (Beijing) 2019 - 0002. Before the experiment, the rats were fed adaptively for 2 weeks, fed freely with water, with an ambient temperature of (24 ± 1) ℃, relative humidity of 40% to 60%, and a light cycle of 12 hours light and 12 hours dark. This research scheme has been approved by the Ethics Committee of Haikou Traditional Chinese Medicine Hospital.

1.2 Drugs, reagents, and instruments

The prescription for tonifying the kidney and activating blood circulation consists of 10 g of cooked rehmannia, 10 grams of cistanche deserticola, 10 g of Xianling spleen, 15 g of raw astragalus, 5 g of wine rhubarb, 10 g of salvia miltiorrhiza, and 15 g of angelica. All traditional Chinese medicine materials are purchased from Haikou Traditional Chinese Medicine Hospital. Conventional decoction, filtration, concentration to 4 g/mL of crude drug, preservation for standby. Adenine (Shanghai Jizhi Biochemical Technology Co., Ltd., batch number: A800684); Serum creatinine (SCr) test kit (Beijing Lingbao Technology Co., Ltd.); Urea nitrogen (BUN) and uric acid (UA) detection kit (Beijing Boaze Technology Co., Ltd.); Malondialdehyde (MDA) and superoxide dismutase (SOD) detection kit (Shanghai Qizheng Biotechnology Co., Ltd.); Rabbit anti intracellular phosphatidylinositol 3 kinases (PI3K) polyclonal antibody, rabbit anti-protein kinase B (Akt) polyclonal antibody (Shanghai Jinsui Biotechnology Co., Ltd.); Rabbit anti-B lymphocyte tumor 2 genes (Bcl-2) antibody, rabbit anti-Bcl-2 related X protein (Bax) antibody (Guangzhou Chuanrong Biotechnology Co., Ltd.); Goat anti-rabbit IgG secondary antibody (Shanghai Biyuntian Biotechnology Co., Ltd.). MPR A100 enzyme marker [Yasuwang (Shanghai) Trading Co., Ltd.]; Gel imager (Bio-Rad Company, USA); ADVIA2400 fully automatic biochemical analyzer (Siemens Company).

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1.3 Modeling, grouping, and administration

Select 10 rats as the normal group, and the remaining 30 rats to construct a chronic renal failure model. Determine whether the model is successful based on the serum level of renal function indicators and renal pathological changes in the model rats [7]. Method of modeling [8]: 250 mg/kg adenine was administered by gavage once a day for 28 consecutive days. One rat died during the modeling process. The rats successfully modeled were randomly divided into the model group (9 rats), low dose group of Bushen Huoxue Formula (10 rats), and high dose group of Bushen Huoxue Formula (10 rats). After the completion of modeling, the daily dose of adults was converted into the dose of rats according to the "Pharmacological Experimental Methods", and was set as a low dose of Kidney Tonifying and Blood Activating Formula. The low and high-dose groups of Bushen Huoxue Formula were given 0.52 and 2.08 g/kg of Bushen Huoxue Formula solution by gavage. The normal group and model group were given equal volumes of physiological saline by gavage. Once a day for 28 consecutive days.

1.4 Observation indicators and methods

1. 4. 1. Detection of renal function indicators

After the intervention, blood was collected from the abdominal aorta of the rats, and serum samples were isolated after centrifugation. Enzyme-linked immunosorbent assay was used to detect the levels of SCr, BNU, and UA in rats in each group. The operation was carried out according to the instructions on the kit.

1. 4. 2 HE staining

Rat renal tissue was taken, fixed, dehydrated, paraffin-embedded, sectioned, stained, dehydrated, and sealed again. Pathological changes in rat renal tissue were observed and photographed.

1. 4. 3 Detection of oxidative stress indicators in renal tissue

Take rat kidney tissue, prepare homogenate, centrifuge, and separate the supernatant. According to the reagent instructions, use an enzyme-linked immunosorbent assay to detect the levels of MDA and SOD.

1. 4. 4 Protein Immunoblotting Test

After taking homogenate from rat kidney tissue, add RIPA to extract total protein, centrifuge at 3000 r/min (centrifugation radius: 15 cm) for 10 minutes, take the supernatant and measure the protein content. After protein separation by gel electrophoresis, membrane transfer, sealing, and TBST cleaning, the primary antibodies PI3K, Akt, Bcl-2, Bax, and β- Actin (diluted by volume ratio of 1:1000) was incubated overnight at 4 ℃, the film was washed, and a second antibody (diluted by volume ratio of 1:5000) was added to continue to incubate at room temperature for 2 hours. ECL was exposed for development, and the gray value of the electrophoretic bands was analyzed using ImageJ software. Results Based on the target protein and internal reference protein（ β- Action) grayscale value ratio representation.

1.5 Statistical methods

SPSS 25.0 statistical software was used for data analysis. The measurement data were expressed as mean ± standard deviation (x ± s). Univariate analysis of variance was performed for comparison between multiple groups, and a t-test was used for comparison between the two groups. The difference was statistically significant when P<0.05 was used.

2 Results

2.1 Comparison of renal function index levels of rats in each group

Table 1 shows that compared with the normal group, the levels of BUN, SCR, and UA in the model group increased (P<0.05); Compared with the model group, the levels of BUN, SCr, and UA in the low and high dose groups of Bushen Huoxue Formula decreased (P<0.05) in a dose-dependent manner. Comparison of renal histopathological changes of rats in each group Figure 1 shows that the renal tissue structure of rats in the normal group is complete, with an orderly arrangement of glomeruli and renal tubules, without significant abnormal changes; Compared with the normal group, the model group had mild necrosis and inflammatory cell infiltration in the renal tissue, renal tubular dilation, glomerular deformation, and other pathological changes; Compared with the model group, the pathological conditions of the low and high dose groups of Bushen Huoxue Recipe were improved.

Table 1  Comparison of the levels of renal function indicators among various groups of rats （x ± s）

Note: ① P ＜ 0.05, compared with the normal group; ② P<0.05, compared with the model group; ③ P<0.05, compared with the low dose group of Bushen Huoxue Formula

Figure 1  Comparison of histopathological changes of rat kidney tissues among various groups （HE staining，×400）

Comparison of oxidative stress index levels in renal tissue of rats in each group

Table 2 shows that compared with the normal group, the MDA level in the renal tissue of the model group rats increased, while the SOD level decreased (P<0.05); Compared with the model group, the MDA level in the kidney tissue of rats in the low and high dose groups of Bushen Huoxue Recipe decreased, while the SOD level increased (P<0.05).

The expression level of PI3K and Akt proteins in renal tissue of rats in each group

Comparing Table 3 and Figure 2, the results showed that compared with the normal group, the expression levels of PI3K and Akt proteins in the renal tissue of the model group were significantly increased (P<0.05); Compared with the model group, the expression levels of PI3K and Akt proteins in the kidney tissue of rats in the low and high dose groups of Bushen Huoxue Formula decreased (P<0.05).

Table 2  Comparison of the levels of oxidative stress indicators in renal tissues among various groups of rats （x ± s）

Note: ① P ＜ 0.05, compared with the normal group; ② P<0.05, compared with the model group; ③ P<0.05, compared with the low dose group of Bushen Huoxue Formula

Table 3   Comparison of protein expression levels of PI3K and Akt in renal tissues among each groups of rats （x ± s）

Note: ① P ＜ 0.05, compared with the normal group; ② P<0.05, compared with the model group; ③ P<0.05, compared with the low dose group of Bushen Huoxue Formula

Expression levels of Bcl-2 and Bax proteins in renal tissues of rats in each group

Comparing the results in Table 4 and Figure 3, it is shown that compared with the normal group, the expression level of Bcl-2 protein in the renal tissue of rats in the model group significantly decreased, while the expression level of Bax protein significantly increased (both P<0.05); Compared with the model group, the expression level of Bcl-2 in the kidney tissue of rats in the low and high dose groups of Bushen Huoxue Formula increased, while the expression level of Bax decreased (both P<0.05).

Figure 2   Western Blot electropherograms of PI3K and Akt proteins in renal tissues in each groups of rats

Table 4   Comparison of protein expression levels of Bcl-2 and Bax in renal tissues among various groups of rats （x ± s）

Note: ① P ＜ 0.05, compared with the normal group; ② P<0.05, compared with the model group; ③ P<0.05, compared with the low dose group of Bushen Huoxue Formula

Figure 3   Western Blot electropherograms of Bcl-2 and Bax proteins in renal tissues in each groups of rats

3 Discussion

Adenine induced chronic renal failure model is a common method for screening therapeutic drugs for chronic renal failure and clarifying the mechanism of drug action [9]. Muscle metabolite SCr, purine metabolite end product UA, and plasma nitrogen-containing compound BUN accumulate in the blood during renal function damage and are important indicators for detecting renal function [10-11]. In this study, a chronic renal failure model was established by adenine-induced renal damage in rats. The modeling results showed that in the model group, the levels of SCr, BUN, and UA in the blood increased, inflammatory cell infiltration occurred in the renal tissue, renal tubular dilation, glomerular deformation, and other pathological changes, indicating that the modeling was successful. After treatment with different concentrations of Bushen Huoxue Formula, the levels of SCr, BUN, and UA in the blood of rats decreased, and the renal tissue structure and physiological function were improved to varying degrees, suggesting that Kidney Tonifying and Blood Activating Formula Composed of Cistanche deserticola and Other Prescriptionshas a protective effect on the kidneys of rats with chronic renal failure.

Chinese medicinal materials such as cistanche deserticola

Some studies have shown that oxidative stress can promote the progression of renal failure, and the degree of oxidative stress in renal tissue increases with the gradual increase in the degree of renal function damage. SOD is a class of factors that can defend against inflammatory reactions, and MDA is the decomposition product of lipid peroxidation in the body [13]. Therefore, enhancing SOD levels, reducing MDA levels, and reducing oxidative stress levels in renal tissue can effectively inhibit the progression of chronic renal failure. The results of this study also confirmed that after the intervention of Bushen Huoxue Formula, the level of oxidative stress indicators in rats decreased, indicating that Kidney Tonifying and Blood Activating Formula Composed of Cistanche deserticola and Other Prescriptions can play a role in antioxidant stress and anti-inflammatory reactions, and inhibit the development of renal failure.

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PI3K is an important signal transduction pathway in the process of growth factor signal transduction. Akt is a serine protein kinase that belongs to the downstream factor of PI3K. In the process of chronic renal failure, the PI3k/Akt pathway is involved in inflammatory reactions, parenchymal cell apoptosis, differentiation, and collagen formation and deposition. Lie Beifeng et al. [14] found that the expression of phosphorylated Akt in renal tubulointerstitium of diabetes nephropathy model rats increased, indicating that PI3K/Akt signaling pathway may participate in the process of renal tubular lesions. In chronic renal failure model rats, PTEN/PI3k/Akt signaling pathway is involved in the process of renal interstitial fibrosis [15]. Other studies have shown that 5/6 nephrectomized rats may inhibit TGF by β/ PI3K/Akt signaling pathway, thereby effectively reducing SCr, alleviating renal fibrosis, and improving renal function in rats [16]. Runyan et al. [17] found that TGF in glomerular cells- β Stimulating Akt phosphorylation and administering PI3k inhibitors to glomerular cells can reduce TGF- β expression. The results of this study indicate that Bushen Huoxue Formula can inhibit the expression of PI3k/Akt protein in renal tissue of rats with chronic renal failure, inhibit the PI3k/Akt signaling pathway, and thereby delay the progression of the disease in rats with chronic renal failure. For kidney tissue, apoptosis has a dual effect [18]: On the one hand, apoptosis can clear inflammatory invasion cells and abnormal proliferative cells, promoting the repair of renal tissue structure and the recovery of renal function; On the other hand, apoptosis often leads to a decrease in the number of renal cells, which plays a key role in the occurrence of glomerulosclerosis and tubular atrophy, and can exacerbate the progression of renal disease. Research [19] shows that the number of apoptosis in renal tubular cells in patients with renal disease is significantly higher than that in healthy individuals, and the number of apoptosis in renal tissue is closely related to renal interstitial injury index, renal fibrosis index, urinary protein level, and the degree of deterioration of renal function. The Bcl-2 apoptotic protein family can be divided into three categories: proapoptotic promoter proteins, proapoptotic effector proteins, and anti-apoptotic proteins. The antagonism between proapoptotic proteins and anti-apoptotic proteins determines the apoptosis process of these large numbers of cells [20]. Research [18] shows that inducing upregulation of Bcl-2 expression can reduce cell apoptosis. In acute renal injury models and chronic renal failure models, knocking out Bax and Bak genes in proximal renal tubular epithelial cells can reduce apoptosis of renal tubular epithelial cells and alleviate the degree of renal failure [18]. The results of this study indicate that the expression of Bcl-2 protein increases and the expression of Bax protein decreases in chronic renal failure rats treated with Kidney Tonifying and Blood Activating Formula Composed of Cistanche deserticola and Other Prescriptions. This may be because Bushen Huoxue Recipe inhibits the expression of apoptosis-related proteins Bcl-2 and Bax downstream of the PI3K/Akt signaling pathway, inhibits renal cell apoptosis, thereby alleviating renal fibrosis, and delaying the deterioration of renal function.

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In summary, Kidney Tonifying and Blood Activating Formula Composed of Cistanche deserticola and Other Prescriptions can alleviate renal function damage in rats with chronic renal failure, and its mechanism may be related to inhibiting PI3K/Akt signaling pathway and regulating the expression of apoptotic proteins Bcl-2 and Bax. In addition, renal fibrosis and deterioration of renal function in rats with adenine intragastric administration may be more closely related to renal tubular injury, and the deeper mechanisms need to be further studied.

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