CN108309986B - Application of PCN in the preparation of medicaments for the treatment of type 1 diabetic nephropathy-related conditions - Google Patents

Abstract

Application of PCN in the preparation of a medicament for treating a disorder related to type 1 diabetic nephropathy. The invention discloses a new indication of pregnenolone-16a-carbonitrile (PCN). The present invention proposes that PCN can alleviate streptozotocin (STZ)-induced type 1 diabetic nephropathy-related conditions, including acute renal tubular injury, renal tubular cell apoptosis, renal interstitial fibrosis, renal function, urine output, and inflammatory factors expression, proteinuria and other symptoms.

Description

Application of PCN in the preparation of medicaments for the treatment of type 1 diabetic nephropathy-related conditions

technical field

The present invention relates to a new application of PCN, in particular to the application of PCN in the preparation of a medicament for alleviating STZ-induced type 1 diabetic nephropathy-related conditions.

Background technique

Diabetic nephropathy (DN) is one of the common complications of diabetes and one of the leading causes of end-stage renal disease, characterized by proteinuria, glomerulosclerosis, and progressive loss of renal function. More than one-third of diabetic patients suffer from DN, and once it progresses to end-stage renal disease, it is often more difficult to treat than other kidney diseases. In my country, its incidence is also on the rise. Therefore, the prevention and control of the occurrence and development of DN is of great significance.

Type 1 diabetes is insulin-dependent diabetes, and most people with type 1 diabetes die from complications of DN. In other words, the most dangerous complication of type 1 diabetes is DN. DN accounts for up to 1/3 of patients with type 1 diabetes, and its earliest detectable feature is a slight increase in proteinuria (microalbuminuria), followed by persistent proteinuria causing tubulointerstitial inflammation, scarring, and eventually kidney disease. function is gradually lost.

Glomerular ultrafiltration and reduced proximal tubule reabsorption are two major determinants of proteinuria. Some studies have pointed out that in the early stage of DN, affected by proximal tubular epithelial cells (PTECs), impaired tubular reabsorption is a possible reason for the development of proteinuria, but the underlying molecular mechanism remains unclear.

PCN (pregnenolone-16a-carbonitrile) is an agonist of pregnane X receptor (PXR). As an endogenous and exogenous activated receptor, PXR plays an important role in biological regulation and "detoxification" in the body's defense mechanism. In addition, a large number of studies have also shown that PXR is widely involved in the body's material and energy metabolism by regulating the expression of downstream target genes, and plays an important role in the occurrence and development of certain diseases. At present, the research on PXR mainly focuses on liver lipid metabolism, cardiovascular, obesity, etc., and there are also a few studies on the regulation of hepatic glucose metabolism by PXR recently. In terms of DN, a recent study found that in db/db mice, PXR expression was up-regulated, and Slco2b1 (soluble carrier transporter), Rgc32 (complement activation gene) and Pck1 (phosphoenolpyruvate carboxykinase 1) ) was also up-regulated, suggesting that PXR may play a bad role in type 2 DN. In 2012, an article suggested that PXR may be related to type 1 diabetes, but no relevant reports were found later. At present, there is no report that PCN can fight against STZ-induced type 1 DN.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an application of PCN in the preparation of a medicament for alleviating STZ (streptozotocin)-induced type 1 DN-related disorders.

The present invention finds the application of PCN in medicines for alleviating STZ-induced type 1 DN-related disorders through in vivo animal experiments.

Specifically, the present invention proposes the use of PCN in the preparation of a medicament for alleviating STZ-induced type 1 DN renal function.

The invention also proposes the application of PCN in the preparation of medicine for reducing STZ-induced acute renal tubular apoptosis and damage.

In addition, the present invention also proposes the application of PCN in the preparation of a medicament for reducing STZ-induced renal interstitial fibrosis.

Further, the present invention finds the application of PCN in the preparation of a medicament for reducing the expression of STZ-induced renal inflammatory molecules.

At the same time, the present invention also discovers the application of PCN in the preparation of a medicine for reducing STZ-induced type 1 DN urine volume, kidney/body weight ratio and microalbumin content.

Description of drawings

Figure 1 shows the results of renal function and glycogen staining (PAS) after modeling with STZ and treatment with PCN;

Figure 2 shows the effects of PCN on STZ-induced acute tubular apoptosis and damage-related molecules by QPCR and Western blot;

Figure 3 shows the effect of PCN on STZ-induced renal interstitial fibrosis detected by QPCR and Western blot;

Figure 4 is a QPCR method to study the effect of PCN on STZ-induced renal inflammatory molecule expression;

Figure 5 shows the effect of PCN on STZ-induced renal urine volume, urine microalbumin content and kidney/body weight ratio.

Detailed ways

The specific operation steps of Western blot, real-time fluorescence quantitative PCR, and PAS staining in the present invention are as follows:

Western blot:

The total protein of kidney tissue was extracted from the tissue lysate, the protein concentration was determined by BCA method, 30 μg protein was loaded, 8% or 12% polyacrylamide gel electrophoresis (SDS-PAGE), 300mA×1.5h wet transfer to PVDF membrane , the blocking solution was blocked at room temperature for 1h, and the primary antibody was added after TBST elution, Bax (Cell Signaling Technology), Bcl-2 (Cell Signaling Technology), Caspase3 (Cell Signaling Technology), Cleaved caspase3 (Cell Signaling Technology), Fibronectin (Abcam), GAPDH (Cell Signaling Technology) ) and incubated overnight at 4°C. The membrane was washed 5 times with TBST, 5 min each time, incubated with the corresponding secondary antibody for 1 h at room temperature, and the membrane was washed 5 times with TBST. Antigen-antibody complexes were displayed by enhanced chemiluminescence (ECL), darkroom X-ray film was exposed and scanned, and protein quantification was performed by gray value analysis of the target band, and the target band gray value/GAPDH gray value was used to express the target. relative protein expression.

Real-time quantitative PCR (QPCR):

Total RNA from kidney tissue was extracted, and the concentration and purity of RNA solution were determined by spectrophotometry. Using reverse transcription kit (Takara, DaLian), 1 μg RNA was reverse transcribed into cDNA, and the changes of different genes were detected according to the following reaction system.

a. Reaction system

b. PCR thermal cycling parameters

PAS staining:

The tissues were fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, deparaffinized to water, rinsed with distilled water, and rinsed with 70% alcohol for 3 times. Immerse in periodate alcohol solution for 10min (the temperature of this solution is preferably 17-20 degrees), after washing with 70% alcohol, put it into the reducing solution for 1min (the temperature of this solution is preferably 17-20 degrees), after washing with 70% alcohol, Into the colorless basic fuchsin solution for 1-1.5h, when the room temperature is low in winter, it can be put into a 37-degree incubator. Rinse with running water for 10 min, counterstain the nuclei with Mayer's hematoxylin counterstain solution for 3-5 min, then differentiate with 1% hydrochloric acid alcohol, rinse with running water, dehydrate and clear, and finally mount.

Masson Stain:

The tissue was fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and dewaxed to water; washed with tap water and distilled water for 3 times in turn; stained with Weigert hematoxylin semen for 5-10 min, rinsed with running water for 10 min; Red solution for 5-10min; immersion in 2% glacial acetic acid aqueous solution for a moment; 1% phosphomolybdic acid aqueous solution for differentiation for 3-5min; directly dyed with aniline blue or light green solution for 5min without washing; immersion in 0.2% glacial acetic acid aqueous solution Moment; 95% alcohol, anhydrous alcohol, xylene clear, neutral gum mounting.

The present invention will be described in detail below through specific examples.

Example 1 Effects of PCN on renal function in STZ-induced diabetic nephropathy.

Male C57BL/6 mice weighing 18-22 g were taken and divided into 3 groups, namely control group, STZ model group and PCN treatment group (n=10). The left kidney was surgically removed in all three groups, and the patients recovered for one week after operation.

Control group: an equal volume of media (sodium citrate) was injected intraperitoneally once a day for 5 days;

STZ model group: intraperitoneal injection of STZ, 50 mg/kg, continuous administration for 5 days;

PCN treatment group: intraperitoneal injection of STZ, 50 mg/kg, after continuous administration for 5 days, PCN administration (intraperitoneal injection, 50 mg/kg PCN, 200 μL/time), once a day, treatment for 4 weeks, collected in metabolic cages for 24 hours per week Urine volume, blood was collected after 4 weeks, and kidney tissue was collected.

The blood samples were centrifuged (20min, 3000r/min), and the creatinine kit (Creatinine Assay Kit (cat: K625-100, biomars)), urea nitrogen kit (QuantiChrom Urea Assay kit (cat: DIUR-500, Hayward, CA) )) to measure serum creatinine and blood urea nitrogen, and the experimental results are shown in Figure 1A.

Figure 1B shows the results of PAS staining after modeling with STZ and treatment with PCN. It can be seen from the results that the modeling with STZ was successful, and serum creatinine and blood urea nitrogen were significantly increased, indicating that the kidneys were damaged. However, after PCN treatment, renal function could be significantly improved, and the levels of creatinine and blood urea nitrogen were significantly lower than those in the model group, p<0.01. According to the results of PAS staining, the tubular structure of STZ group was destroyed, and protein casts were formed. The PCN treatment group could significantly improve STZ-induced tubular damage.

Figure 1C shows the results of Masson staining after modeling with STZ and treatment with PCN. It can be seen from the results that the STZ group was successfully modeled, the renal interstitial collagen was deposited, and the Masson staining positive areas were significantly increased. The masson staining-positive area was significantly reduced in the PCN treatment group, suggesting that PCN ameliorated the STZ-induced tubulointerstitial fibrosis.

Example 2 Effects of PCN on STZ-induced renal tubular cell apoptosis and kidney injury-related molecules.

QPCR was used to detect the expression levels of apoptosis and loss-related molecules in the kidneys to determine the improvement of PCN on STZ-induced type 1 DN. As shown in Figure 2A, the expression of apoptosis-related molecules Bax in the STZ model group was significantly increased, and p <0.001; the expressions of damage-related molecules Kim-1 and NGAL were significantly increased, p<0.001. The PCN treatment group could significantly reduce the expression levels of Bax, Kim-1 and NGAL, p<0.05.

Western blot was used to detect the expression levels of apoptosis and loss-related molecules in the kidney to determine the improvement of PCN on STZ-induced type 1 DN. The results are shown in Figure 2B. The protein level of fragment Cleavedcaspase3 was significantly increased; the protein level of Bcl-2 was significantly decreased. The PCN treatment group could significantly reduce the expression levels of Bax and Cleavedcaspase3, and up-regulate the protein level of Bcl-2.

The results showed that PCN could significantly reduce STZ-induced renal tubular cell apoptosis and the expression levels of loss-related molecules.

Example 3 Effects of PCN on STZ-induced expression of tubulointerstitial fibrosis-related molecules.

The effects of PCN on the expression of STZ-induced tubulointerstitial fibrosis-related molecules were investigated by QPCR and Western blot.

As shown in Figure 3A, the effect of PCN on the expression of STZ-induced tubulointerstitial fibrosis-related molecules was investigated by QPCR. In the STZ-induced DN model, the mRNA levels of fibrosis indicators α-SMA, Fibronectin, and Collagen III in the STZ model group were significantly higher than those in the control group, p<0.05. The PCN treatment group could significantly reduce the mRNA levels of α-SMA, Fibronectin, and Collagen III, p<0.01.

As shown in Figure 3B, the effect of PCN on the expression of STZ-induced tubulointerstitial fibrosis-related molecules was investigated by Western blot. In the STZ-induced DN model, the fibrosis index Fibronectin protein level in the STZ model group was significantly increased compared with the control group, while the PCN treatment group could significantly reduce the Fibronectin protein level.

The results showed that PCN could significantly reduce the expression levels of fibrosis-related molecules in STZ-induced type 1 DN model.

Example 4 The effect of PCN on STZ-induced renal inflammatory molecule expression.

The effect of PCN on STZ-induced renal inflammatory molecule expression was investigated by QPCR.

As shown in Figure 4, the effect of PCN on the expression of STZ-induced kidney injury molecules was studied by QPCR method. In the STZ-induced type 1 DN model, the expression levels of inflammatory factors IL-6 and TNF-α in the STZ model group were significantly higher than those in the control group, p<0.05. The PCN treatment group could significantly reduce the expression levels of IL-6 and TNF-α, p<0.01.

The results showed that PCN could significantly reduce the expression levels of IL-6 and TNF-α in STZ-induced type 1 DN model.

Example 5 Effects of PCN on STZ-induced renal urine volume, urine microalbumin content and kidney/body weight ratio.

The 24h urine of mice was collected in metabolic cages at a fixed time every week, and the urine volume of each group was counted. A total of four weeks, collected four times. The results are shown in Figure 5A, the STZ model group was successfully modeled, and the urine volume was significantly increased. Compared with the STZ model group, the PCN treatment group can significantly reduce the urine volume, and has a good therapeutic effect in the first week of treatment; after four weeks, the kidneys and body weight of the mice were weighed and counted, and the results were shown in Figure 5B As shown, the kidney/body weight ratio was significantly up-regulated in the STZ model group, P<0.001. Compared with the STZ model group, the PCN treatment group could significantly down-regulate the kidney/body weight ratio, P<0.05. The urine of the fourth week was mixed and diluted in a certain proportion, and the content of microalbumin in the urine was determined by ELISA. The results are shown in Figure 5C, the content of urine microalbumin in the STZ model group was significantly increased, P<0.001 . Compared with the STZ model group, the PCN treatment group could significantly down-regulate the urinary microalbumin content, P<0.001.

The results showed that in the STZ-induced type 1 DN model, the urine output was greatly increased, the kidney/body weight ratio was also significantly up-regulated, and proteinuria was present. Treatment with the PXR agonist PCN reduced urine output, down-regulated the kidney/body weight ratio, and decreased urinary microalbumin content.

Claims (1)

1. The application of PCN in the preparation of a medicine for alleviating STZ-induced type 1 diabetic nephropathy-related conditions, wherein the PCN is pregnenolone-16a-carbonitrile, an agonist of pregnane X receptors, and the STZ-induced Type 1 diabetic nephropathy-related disorder is STZ-induced renal interstitial fibrosis, and the reduction of STZ-induced type 1 diabetic nephropathy-related disorder is reduction of STZ-induced renal urine volume, urine microalbumin content and kidney/body weight ratio.

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