The Effects Of Cistanche Shenqiwan On Renal Damage, GRP78, And Autophagy-related Factors in Zucker Diabetic Fatty Rats With Type 2 Diabetes Mellitus

amount of normal saline. Rats were continuously administered for 4 weeks. The general condition of rats was observed regularly. The fasting blood glucose (FBG) and 24-hour urinary microalbumin (24 h U-mAlb) levels were measured. Renal tissue was stained and morphology was observed. The ultra-microstructure was observed by transmission electron microscopy. The mRNA levels of GRP78, Beclin-1, and LC3 in rat renal tissue were examined by real-time PCR and the protein levels were examined by Western blotting.Results Compared with the model group, rats in the Shenqiwan group showed

improved fur gloss, more weight increase, and more activity; both FBG and 24 h U-mAlb levels were decreased (P < 0.05). The kidneys of rats in the model group showed dilated glomerular capillaries, irregularly thickened basement membranes, widened mesangial areas, and segmental sclerosis; renal tubular atrophy and renal interstitial fibrous tissue were also observed in these kidneys. These pathological changes were alleviated in the Shenqiwan group. Compared with the model group, both gene and protein levels of GRP78 were decreased (P < 0.05) in the Shenqiwan group; the protein level of Beclin-1

was decreased (P < 0.05); and the protein level of LC3 was increased (P < 0.05). Conclusion Shenqiwan may alleviate renal damage in ZDF rats and delay the progression of diabetic kidney disease by reducing endoplasmic reticulum stress and enhancing autophagy.

 

Keyword: Shenqiwan; type 2 diabetes mellitus; diabetic kidney disease; endoplasmic reticulum stress; autophagy; GRP78; Zucker diabetic fatty rats

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Diabetic kidney disease (DKD) is an important microvascular complication of diabetes mellitus and a major cause of end-stage renal disease [1]. DKD is an important microvascular complication of diabetes and a major cause of end-stage renal disease [1]. Patients with DKD may present with persistent proteinuria and persistently reduced glomerular filtration rate [2]. The pathogenesis of DKD is complex, and recent studies have shown that endoplasmic reticulum stress (ERS) in the kidney is the most common cause of end-stage renal disease. （The endoplasmic reticulum stress (ERS) and autophagy are also closely related to DKD, and the study of their pathogenesis may be important for the treatment of DKD. DKD is more similar to the "lower extinction" in the category of thirst disorders in Chinese medicine.
The typical symptoms of thirst and excessive drinking and urination are recorded in the Jin Kui Yao (Essentials of the Golden Horoscope), and the Kidney Qi Pill is proposed as the main therapeutic formula. The Jin Kui Yao (金匮要略) recorded the typical symptoms of thirst and excessive urination and proposed the Kidney Qi Pill as the main treatment formula. The treatment of DKD with TCM can indeed effectively protect renal function and has safe and reliable characteristics [5-6]. The treatment of DKD with Chinese medicine can effectively protect kidney function and has safe and reliable characteristics [5-6]. The efficacy of Kidney Qi Pill in tonifying the kidney and helping Yang has been confirmed in related studies [7]. Our previous study also Our previous study also showed that renal qi pills have good protective effects on the structure and function of kidney tissues in diabetic mice [8]. In the present study, we observed the effects of renal qi pill on In the present study, we observed the effects of renal qi pills on renal tissue damage, ERS, and autophagy-associated factors in Zucker diabetic fatty rat (ZDF). In this study, we investigated the possible mechanisms of renal damage in ZDF rats by observing the effects of renal qi pills on renal tissue damage, ERS and autophagy-related factors mediated by renal qi pills.

1 Materials
1.1 Experimental animals
Twenty 12-week-old SPF male ZDF rats (body mass 390-410 g, fed 5C08 high-quality rodent chow for 4 weeks to complete induction of type 2 diabetes) and seven 12-week-old SPF male Zucker Lean (ZL) rats (body mass 350-380 g) were purchased from Beijing Viton Lehua Laboratory Animal Technology Co. All rats were housed in the barrier environment of the Institute of Clinical Medicine, China-Japan Friendship Hospital, license number: SYXK (Beijing) 2016-0043, and the platform provided an independent ventilation system inside the barrier, maintaining a 12-h alternation of light and dark, a room temperature of 23-25 ℃, and a room relative humidity of 50%-60%. All rats were allowed to drink and feed freely during the feeding period.

1.2 Ethical review
The animal experiments were approved by the Experimental Animal Ethics Sub-committee of the Academic Committee of Beijing University of Traditional Chinese Medicine (BUCM) with the approval number BUCM-4-2021102801-4034.
1.3 Main drugs and reagents
The tablets (Shu Di Huang, Shan Yu Fei, Shan Yao, Mudan Pi, Fu Ling, Cistanche, Ze Xie, Gunner's Fructus, Gui Zhi) for the decoction of Kidney Qi Pill were purchased from Beijing Tong Ren Tang Co. Physiological saline, specification: 500 mL, provided by Shandong Hualu Pharmaceutical Co. Silver Staining Solution (Beijing Regen Biotechnology Co., Ltd., Lot No. DG0090), Magic SYBR Mixture (Jiangsu Kangwei Century Biotechnology Co., Ltd.), mRNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc.), RNA TRIzol Reagent (RNA TRIzol Reagent), RNA TRIzol Reagent (RNA TRIzol Reagent). RNA TRIzol Reagent (Beijing Solabao Technology Co., Ltd.), Rat Urine Microalbumin ELISA Kit (Jiangsu Kote Biotechnology Co., Ltd., Lot No. KT8560-A), GRP78 BiP Antibody, LC3B Antibody and Beclin-1 Antibody (Abcam, UK) (batch numbers: ab108613, ab192890, ab207612), rabbit anti-GAPDH polyclonal antibody (Proteintech, USA), goat anti-rabbit IgG(H&L)-HRP (Beijing Baiao Yijie Technology Co. Ltd.).

 

Blood glucose meter, blood glucose test strips (Shanghai Roche Diagnostic Products Co. Ltd.), low-temperature high-speed centrifuge (Eppendorf, Germany), fluorescence quantitative PCR instrument, double vertical electrophoresis instrument and transfer electrophoresis instrument (Burroughs Life Medical Products Co. Ltd.), gel imager (Protein Simple, USA), light microscope (Leica, Germany), transmission electron microscope (Leica, Germany), and gel imager. (Leica, Germany), transmission electron microscope (Hitachi, Japan).

2 Methods
2.1 Preparation of experimental drugs
The decoction of Kidney-Qi Pill was prepared by converting the dose of Kidney-Qi Pill in the Jin Kui Yao [9] according to the ancient and modern ratios of the Han system of metrology in the first edition of "Study on the Dose and Proportion of Typhoid Prescriptions" [2015], and the dose of the decoction required for the experiment was as follows: Shu Di Huang 24 g, Shan Yu Fei 12 g, Cistanche 10 g, Shan Yao 12 g, Mudan Pi 9 g, Fu Ling 9 g, Ze Xie 9 g, Gunnera 3 g, and Gui Zhi 3 g. The drugs were soaked in distilled water for 2 h. The decoction was then concentrated for 30 min and filtered through sterile gauze. Decoct the herbs for 30 min, then add the remaining drugs and decoct for 1 h. After the temperature of the solution is lowered to room temperature, the solution is concentrated and filtered through sterile gauze, and the filtrate is packed and stored at -20 ℃. The drug should be stored for no more than 1 week, and the solution should be brought to room temperature before administration.

2.2 Establishment and grouping of animal models
The ZDF rats were induced with 5C08 high-fat diet for 4 weeks, and blood glucose was measured by tail-tip blood collection at 12 weeks of age. The rats in the kidney qi pill group were given aqueous decoction of kidney qi pill, and the daily dose of 8.12 g/kg per rat was calculated according to the formula in "Disease models in experimental animals" [10], and the dosing concentration was 0.812 g/mL, and the rats in the model and control groups were given saline in the corresponding volume for 4 weeks.

2.3 General condition and fasting blood glucose (FBG) test of rats
The general conditions of the rats were observed and recorded daily, and the body masses were weighed regularly. At the end of 12 weeks and 16 weeks of age, blood was collected from the tail tip of rats after 8 h of fasting, and FBG was measured by Roche blood glucose meter and test strips. 2.4 The 24-h urinary microalbumin (24-h U-mAlb) was measured at the end of 16 weeks of age, and the 24-h U-mAlb concentration was measured according to the instructions of LISA kit.

2.5 Kidney tissue specimen collection
At the end of 16 weeks of age, all rats fasted for 8 h. After anesthesia, the abdominal cavity was opened and the bilateral kidneys were exposed, and the peritoneum was removed intact and peeled off. The kidney cortex was separated and partly fixed in 4% paraformaldehyde for the preparation of 3 μm paraffin sections; partly cut into 1 mm3 tissue blocks and fixed in 2.5% glutaraldehyde for the preparation of ultrathin sections. The remaining kidney tissues were stored at -80 ℃ for Realtime PCR and Western blotting assays.

 

2.6 Application of morphological observation of kidney tissues
PAS staining, Masson staining, and hexaammonium silver-hematoxylin-eosin staining were used to observe the renal histopathological changes under a light microscope and to acquire images. Transmission electron microscopy was applied to observe the ultrastructural changes of kidney tissues and images were collected.

 

2.7 Real-time PCR assay
Frozen kidney tissues were ground on ice and TRIzol reagent was added to extract total RNA, and centrifuged at 4 ℃ with a radius of 9.5 cm. The supernatant was extracted by centrifugation at 12 000 r/min for 10 min, and then centrifuged for 15 min with chloroform. The supernatant was centrifuged again for 15 min at room temperature for 10 min, and the supernatant was washed with 75% ethanol. expression at the mRNA level. The primers were designed using Primer Premier 5.0 software and synthesized by Shanghai Bioengineering Co. Ltd. The primer sequences are as follows.