Effects Of Polygona-polysaccharose On Ferroptosis in Diabetic Nephropathy Rats

Keywords: diabetic nephropathy; polygona-polysaccharose; ferroptosis; transferrin; FTH1; GPX4

 

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes mellitus [1] DN is a serious microvascular complication of diabetes mellitus [1], usually presenting as microalbuminuria in the early stages, followed by progressive development of The early stage usually presents with microalbuminuria, followed by progressive development of massive proteinuria, progressive decrease in glomerular filtration rate, and progressive increase in blood creatinine [2]. progressive increase in creatinine[2] . If left untreated, the disease can lead to chronic disease. If left untreated, it can lead to chronic progressive renal damage and eventually end-stage disease. If left untreated, it can lead to chronic progressive renal injury and eventually end-stage renal failure[3] . Iron death Iron death is an iron-dependent accumulation of unsaturated fatty acid iron death is an iron-dependent accumulation of unsaturated fatty acid phospholipid oxidation leading to cell death, mainly manifested by the The main manifestation of iron death is the accumulation of iron-dependent intracellular lipid peroxides[4] . [4] . Studies It has been found that renal tubular epithelial cell injury is closely associated with iron death[5] . Wang et al.

Wang et al[6] found that glutathione peroxidase 4 (GPX4) expression in renal tubular interstitial (GPX4) expression is closely related to the severity and progression of DN disease. GPX4, as an important target for triggering iron death program, is a sensor of oxidative stress and cell death signaling, and reduced GPX4 expression leads to a significant increase in live GPX4 expression decreases, resulting in a significant increase in live oxygen in the body[7] . . Therefore, inhibition of renal tubular epithelial cell death through the regulation of iron death Therefore, the inhibition of renal tubular epithelial cell death through the regulation of iron death to alleviate DN renal injury may be an effective strategy to treat DN Therefore, inhibition of renal tubular epithelial cell death through regulation of iron death may be an effective strategy for DN treatment.
The polysaccharide of C. flavus is the most abundant and important pharmacological It is one of the most abundant and important pharmacological components of C. flavus. The experimental study found that it plays an important role in improving the renal function of DN and alleviating the inflammatory response. Li et al[8] . Li et al[9] . found that Li et al[9] found that the intervention of C. flavus polysaccharide significantly improved glucolipid metabolism disorders and insulin tolerance in diabetic mice. Li et al. Another study found that the glucose, 24 h glucose and insulin tolerance of mice with diabetic nephropathy were significantly improved after the intervention of yellow extract polysaccharide. Another study found that blood glucose, 24-h urine microalbumin, blood creatinine (SCr) and blood urea nitrogen (BUN) in diabetic nephropathy model mice were significantly improved after the intervention of Fructus flavus polysaccharide. and blood urea nitrogen (BUN) levels were significantly reduced in a diabetic nephropathy model[10] . The treatment of DN with polysaccharide from Flos Chrysanthemi However, the specific mechanism of its action is not clear. Therefore, the present In this study, a high-fat diet combined with streptozotocin (STZ) injection was used to establish a DN rat model.
Therefore, in this study, we established a DN rat model by high-fat diet combined with streptozotocin (STZ) injection, and observed the effect of C. flavus polysaccharide on iron death in rats. Therefore, the present study was conducted to observe the effect of C. flavus polysaccharide on iron death in rats fed with high-fat diet combined with streptozotocin (STZ) injection, and to clarify the mechanism of action of C. flavus polysaccharide on DN.

Materials and methods
1.1 Animals
Fifty healthy male SD rats with a body mass of (200±20) g were housed at the Institute of Radiology, Chinese Academy of Medical Sciences, Beijing, China, under a 12-h light-dark cycle at a temperature of (25±2) °C and relative humidity of (50±15) %, and fed ad libitum. High sugar and high fat diet (10% lard, 20% sucrose, 2.5% cholesterol, 67.5% conventional feed), (4.8% fat, 20% protein and 59.4% total sugar), Beijing Huafukang Biotechnology Co. This experiment was approved by the Cangzhou Hospital of Integrative Medicine (CZX2022-KY-023).

1.2 Drug and preparation
Polysaccharide of Flos Astragalus (purity 70%, batch number S27804), Shanghai Yuanye Biotechnology Co. Ltd. Ltd. was prepared with saline at the concentration of 200, 800 mg/mL, respectively. 800 mg/mL solution. Irbesartan tablets (lot 8A411), Sanofi (Hangzhou) Pharmaceutical Co. (Hangzhou) Pharmaceutical Co., Ltd. was prepared in saline at 30 mg/mL. solution.

 

1.3 Main reagents and instruments
Urine protein (batch number C035-2-1), SCr (batch number C011-2-1), BUN (batch number C013-2-1) BUN (batch number C013-2-1), malondialdehyde (MDA, batch number C003-1- 2), glutathione (GSH, lot C006-2-1), total iron ion content (batch number A039-2-1), Nanjing Jiancheng Biological Engineering Research Institute Institute of Biological Engineering, Nanjing, China; Transferrin (batch number ab278498), ferritin Transferrin (lot ab278498), ferritin heavy chain (FTH1, lot ab75973), GPX4 (lot ab252833), and β-ferritin (lot ab252833). ab252833), β-actin (lot ab179467), Abcam, UK. iMark680 multifunctional enzyme marker, Bio-Rad, USA. Fresco17 centrifuge, Thermo, USA; 5810 benchtop frozen centrifuge, Eppndorf, Germany; ABI Prism® 7500 fluorescence quantification Applied Biosystems, USA; DYCZ-24DN vertical electrophoresis tank, Beijing Liuyi Instrument Factory, Beijing, China; SH-523 chemiluminescence imaging Ltd, Hangzhou Shenhua Technology Co.

 

1.4 Modeling, grouping and drug administration
After 1 week of adaptive feeding, 10 rats were randomly selected as normal The remaining 40 rats were referred to the method of literature [11] to establish the DN rat model. The rats were fed with high sugar and high fat diet for 7 weeks, and then fasted for 12 h without water. The rats were fed with high sugar and high fat diet for 7 weeks, then fasted with food and water for 12 h. STZ was injected intraperitoneally at 30 mg/kg. After 72 h, blood was collected from the tail vein to detect random blood glucose, and a blood glucose of ≥16.7 mmol/L was considered as successful modeling of diabetes. The rats were fed with high sugar and high fat diet for 1 week, and 24-h urine was collected from the metabolic cage.

 

 

1.5.5 Western blot assay
Collect kidney tissues, add 150 μL of RIPA lysis buffer, homogenize The protein supernatant was retained after centrifugation. The total protein concentration was determined using the BCA protein assay kit. The total protein concentration was determined by using BCA protein assay kit, and the protein concentration was homogenized. Equal amounts of protein 20 μg, 8%-12% SDS-PAGE was used for protein separation, and the protein was transferred The membranes were closed with 5% skimmed milk powder at room temperature for 2 h. Rabbit anti-rat primary antibody Transferrin (1:2 000), FTH1 (1:3 000) and GPX4 (1:3 000) were added. 3,000), GPX4 (1:3,000), β-actin (diluted 1:5,000), and incubated overnight at 4 ℃. Incubate overnight at 4 ℃. The membrane was washed, secondary antibody (1:9 000) was added and incubated for 2 h at room temperature. The membranes were washed with TBST, developed by ECL chemiluminescence and detected using
Image J software was used to analyze the grayscale values of the bands.

 

1.6 Statistical methods
SPSS Statistics25.0 statistical software was used for analysis. Experiment The results were expressed as xˉ±s, and all data were tested for normality and chi-square. All data were tested for normality and chi-square, and t-tests were used if they were satisfied, and non-parametric tests were used if they were not satisfied. P<0.05 indicates that the difference is statistically significant.

 

2 Results
2.1 Effects of Flavopiridium polysaccharide on body mass and fasting blood glucose of model rats Compared with the normal group, the body mass of rats in the model group was significantly reduced (P<0.01) and FBG was significantly increased (P<0.01); compared with the model group, the body mass of rats in the positive drug group was significantly increased (P<0.05), and the differences in FBG of rats in each group were not statistically significant (P>0.05). See Table 2.

2.2 Effects of Flavopiridium polysaccharide on renal function indexes of model rats
Compared with the normal group, the SCr, BUN level and 24 h urine protein content of rats in the model group were significantly higher (P<0.01); compared with the model group, the Cr, BUN level and 24 h urine protein content of rats in the positive drug group and the high dose group of Flavopiridium polysaccharide were significantly lower (P<0.05, P<0.01). See Table 3.

2.3 Effects of yellow extract polysaccharide on the histopathological morphology of the kidney of model rats
The glomerulus and tubular structure of normal group rats did not show any abnormalities, and the thylakoid membrane and thylakoid stroma were not proliferated, and there was no inflammatory cell infiltration, etc.; the model In the model rats, focal tubular degeneration and atrophy were observed, and the glomerular basement membrane was slightly The rats in the model group showed focal tubular degeneration and atrophy, slightly thickened glomerular basement membrane, thylakoid hyperplasia, and fatty degeneration of glomeruli and tubules. The rats in each drug administration group showed reduced lesions, and the improvement was more obvious in the positive drug group and the high dose of Flavopiridium polysaccharide. The improvement was more obvious in the positive drug group and the high dose of Flavopiridium polysaccharide. See Figure 1.