CN108309986B

CN108309986B - Application of PCN in preparation of medicine for treating 1-type diabetic nephropathy related diseases

Info

Publication number: CN108309986B
Application number: CN201810121259.XA
Authority: CN
Inventors: 贾占军; 于晓文; 张爱华; 许曼; 孟霞; 张学娟; 公伟; 李树珍; 于婧; 游然
Original assignee: Nanjing Childrens Hospital of Nanjing Medical University
Current assignee: Nanjing Childrens Hospital of Nanjing Medical University
Priority date: 2018-02-07
Filing date: 2018-02-07
Publication date: 2020-09-18
Anticipated expiration: 2038-02-07
Also published as: CN108309986A

Abstract

Use of PCN in the manufacture of a medicament for the treatment of a condition associated with type 1 diabetic nephropathy. The invention discloses a new indication of pregnenolone-16a-carbonitrile (PCN). The invention provides a type 1 diabetic nephropathy related disease induced by PCN (PCN) with light chain urea zotocin (STZ), which comprises diseases such as acute tubular injury, tubular cell apoptosis, renal interstitial fibrosis, renal function, urine volume, inflammatory factor expression, proteinuria and the like.

Description

Application of PCN in preparation of medicine for treating 1-type diabetic nephropathy related diseases

Technical Field

The present invention relates to a novel use of PCN, in particular to the use of PCN in the manufacture of a medicament for the alleviation of STZ-induced type 1 diabetic nephropathy-related disorders.

Background

Diabetic Nephropathy (DN) is one of the common complications of diabetes and one of the leading causes of end-stage renal disease, and is characterized by proteinuria, glomerulosclerosis, and progressive loss of renal function. More than one third of diabetic patients suffer from DN, and once advanced to end stage renal disease, are often more troublesome to treat than other renal diseases. In China, the incidence of diseases is also on the rise. Therefore, the method has extremely important significance for preventing and controlling the generation and development of DN.

Type 1 diabetes is insulin dependent diabetes mellitus, and most patients with type 1 diabetes die from DN complications. That is, DN is the most dangerous complication of type 1 diabetes. DN is present in a proportion of up to 1/3 in type 1 diabetic patients and is first detected as a slightly increased indicator of proteinuria (microalbuminuria) followed by continued proteinuria resulting in tubulointerstitial inflammation, scarring, and progressive loss of renal function.

Glomerular hyperfiltration and reduced reabsorption of the proximal convoluted tubules are two major determinants of proteinuria. Studies have indicated that in the early stage of DN, affected by Proximal Tubular Epithelial Cells (PTECs), impaired tubular resorption is a possible cause of proteinuria development, but the intrinsic molecular mechanisms remain unclear.

PCN (pregnenolone-16a-carbonitrile) is an agonist of the Pregnane X Receptor (PXR). PXRs, as endogenous and exogenous activation receptors, play important biological regulatory roles and "detoxification" functions in the defense mechanisms of the body. In addition, a great deal of research also indicates that PXR is widely involved in the substance and energy metabolism of the organism by regulating the expression of downstream target genes and plays an important role in the occurrence and development of certain diseases. The research on PXR is mainly focused on liver lipid metabolism, cardiovascular diseases, obesity and the like, and recently, a small amount of research on PXR for regulating liver carbohydrate metabolism is carried out. In terms of DN, a recent study found that PXR expression was upregulated in db/db mice, and that Slco2b1 (soluble carrier transporter), Rgc32 (complement activating gene) and Pck1 (phosphoenolpyruvate carboxykinase 1) were also upregulated, suggesting that PXR may play a bad role in type 2 DN. In 2012, an article suggests that PXR may be related to type 1 diabetes, but no related report is found later. At present, there is no report that PCN can combat STZ-induced type 1 DN.

Disclosure of Invention

The invention aims to provide application of PCN in preparing a medicament for relieving STZ (streptozotocin) -induced DN 1 related symptoms.

The invention discovers the application of PCN in medicines for relieving STZ-induced DN 1-related diseases through in-vivo animal experiments.

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

The invention also provides application of the PCN in preparing a medicament for relieving acute renal tubule apoptosis and injury induced by STZ.

In addition, the invention also provides application of the PCN in preparing a medicament for relieving the STZ-induced renal interstitial fibrosis.

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

Meanwhile, the invention also finds the application of the PCN in preparing the medicines for reducing the STZ-induced DN 1 urine volume, the kidney/body weight ratio and the microalbumin content.

Drawings

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

FIG. 2 is a QPCR and Western blot method for studying the influence of PCN on STZ-induced acute renal tubule apoptosis and injury related molecules;

FIG. 3 is a QPCR and Western blot method for detecting the effect of PCN on STZ-induced renal interstitial fibrosis;

FIG. 4 is a QPCR study of the effect of PCN on STZ-induced renal inflammatory molecule expression;

FIG. 5 is a graph showing the effect of PCN on STZ-induced renal urine volume, urinary microalbumin content and kidney/body weight ratio.

Detailed Description

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

Western blot：

total kidney tissue protein was extracted from the tissue lysate, protein concentration was determined by BCA assay, 30. mu.g protein load, 8% or 12% polyacrylamide gel electrophoresis (SDS-PAGE), 300mA × 1.5h wet transfer to PVDF membrane, blocking buffer for 1h at room temperature, TBST elution followed by addition of primary antibody, Bax (Cell Signaling Technology), Bcl-2(Cell Signaling Technology), Caspase3(Cell Signaling Technology), Cleaved Caspase3(Cell Signaling Technology), Fibronectin (Abcam), GAPDH (Cell Signaling Technology), overnight incubation at 4 ℃. The membranes were washed 5 times with TBST, 5min each time, incubated with the corresponding secondary antibody for 1h at room temperature, and washed 5 times with TBST. The antigen-antibody complex is displayed by an enhanced chemiluminescence method (ECL), a darkroom X-ray film is exposed and scanned, the protein quantification adopts gray value analysis of a target band, and the relative expression quantity of the target protein is expressed by the gray value of the target band/GAPDH gray value.

Real-time fluorescence Quantitative PCR (QPCR):

total RNA of kidney tissues is extracted, and the concentration and purity of the RNA solution are determined by spectrophotometry. Mu.g of gRNA was reverse-transcribed into cDNA using a reverse transcription kit (Takara, DaLian), and the change of different genes was examined according to the following reaction system.

a. Reaction system

PCR thermal cycling parameters

PAS dyeing:

the tissue was fixed with 4% paraformaldehyde for 48h, sectioned with paraffin, deparaffinized to water, rinsed with distilled water, and rinsed 3 times with 70% alcohol. Soaking in periodic acid alcoholic solution for 10min (the temperature of the solution is 17-20 deg.C), washing with 70% alcohol, adding into reducing solution for 1min (the temperature of the solution is 17-20 deg.C), washing with 70% alcohol, adding colorless salt base fuchsin solution for 1-1.5h, and placing into 37 deg.C incubator in winter when the room temperature is low. Washing with flowing water for 10min, re-staining cell nucleus with Mayer \ s hematoxylin re-staining solution for 3-5min, differentiating with 1% hydrochloric acid alcohol, washing with flowing water, dehydrating, and sealing.

Masson staining:

fixing the tissue with 4% paraformaldehyde for 48h, embedding the section in paraffin, and dewaxing to water; washing with tap water and distilled water sequentially for 3 times; staining nuclei with Weibert hematoxylin liquid for 5-10min, and washing with running water for 10 min; adding Masson ponceau acid red recovering solution for 5-10 min; soaking and washing the fabric for a moment by using 2% glacial acetic acid aqueous solution; differentiating by 1% phosphomolybdic acid water solution for 3-5 min; directly dyeing for 5min with aniline blue or light green liquid without washing; soaking and washing the fabric for a moment by using 0.2% glacial acetic acid aqueous solution; 95% alcohol, anhydrous alcohol, xylene transparent, and neutral gum sealing.

The present invention will be described in detail below with reference to specific examples.

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

Male C57BL/6 mice weighing 18-22 g were divided into 3 groups, i.e., a control group, an STZ model group, and a PCN treatment group (n-10). The left kidney was removed from all 3 groups and was recovered for one week after surgery.

Control group: intraperitoneal injection of an equal volume of medium (sodium citrate) is performed 1 time a day for 5 days;

STZ model group: injecting STZ into abdominal cavity at a dose of 50mg/kg for 5 days;

PCN treatment group: the treatment is carried out by intraperitoneal injection of STZ (50 mg/kg), continuous administration for 5 days, administration of PCN (intraperitoneal injection, 50mg/kgPCN, 200 μ L/time), 1 time per day for 4 weeks, collecting 24h urine volume with metabolism cage every week, and collecting blood and kidney tissue after 4 weeks.

Blood samples were centrifuged (20min, 3000r/min) and serum Creatinine and Urea nitrogen were measured using Creatinine Kit (Creatine Assay Kit (cat: K625-100, biorars)) and Urea nitrogen Kit (QuantiChrrom Urea Assay Kit (cat: DIUR-500, Hayward, Calif.)), and the results are shown in FIG. 1A.

Fig. 1B shows the results of PAS staining after STZ modeling and PCN treatment, and it can be seen from the results that the success of STZ modeling significantly increased serum creatinine and urea nitrogen, indicating kidney damage. After the PCN is used for treatment, the renal function can be obviously improved, the creatinine and urea nitrogen levels are obviously reduced compared with a model group, and p is less than 0.01. According to the PAS staining results, the STZ group renal tubular structure is destroyed and protein casts are formed. The PCN treatment group was able to significantly improve STZ-induced renal tubular injury.

Figure 1C is the results of Masson staining after molding with STZ and treatment with PCN. As can be seen from the results, the STZ modeling was successful, the renal interstitial collagen deposition and the Masson staining positive area were significantly increased. The PCN treatment group was able to significantly reduce Masson positive areas, suggesting that PCN improved the extent of STZ-induced fibrosis of the tubulointerstities.

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

The improvement condition of PCN on STZ-induced DN 1 is determined by detecting the expression level of the related molecules of the renal apoptosis and the loss by using a QPCR method, and the result is shown in figure 2A, the expression of the related molecules Bax of the apoptosis of the STZ model group is obviously increased, and the p is less than 0.001; the expressions of the injury related molecules Kim-1 and NGAL are obviously increased, and p is less than 0.001. And the PCN treatment group can obviously reduce the expression levels of Bax, Kim-1 and NGAL, and p is less than 0.05.

The improvement of STZ-induced DN 1 by PCN is determined by detecting the expression level of the related molecules of the renal apoptosis and the loss by using a Western blot method, and the result is shown in figure 2B, the protein level of the cleavage fragment Cleavedcaspase3 of the apoptosis related molecules Bax and Caspase3 in the STZ model group is obviously increased; bcl-2 protein levels were significantly down-regulated. And the PCN treatment group can obviously reduce the expression level of Bax and Cleavedcaspase3 and up-regulate the level of Bcl-2 protein.

The results show that PCN can obviously reduce the expression level of STZ-induced renal tubular cell apoptosis and loss-related molecules.

Example 3 effect of PCN on STZ-induced expression of molecules associated with interstitial fibrosis in the tubules.

The influence of PCN on STZ-induced renal tubular interstitial fibrosis related molecule expression is researched by using QPCR and Western blot method.

As shown in fig. 3A, the effect of PCN on STZ-induced expression of molecules associated with interstitial fibrosis in the tubules was investigated using QPCR. In the STZ-induced DN model, the mRNA levels of fibrosis indexes alpha-SMA, fibrinectin and Collagen III in the STZ model group are obviously increased compared with the control group, and p is less than 0.05. And the PCN treatment group can obviously reduce the mRNA level of alpha-SMA, Fibronectin and Collagen III, and p is less than 0.01.

As shown in FIG. 3B, the effect of PCN on STZ-induced expression of molecules associated with interstitial fibrosis in the tubules was investigated by Western blot. In the STZ-induced DN model, the fibrosis index fibrinectin protein level in the STZ model group is obviously increased compared with that in the control group, while the PCN treatment group can obviously reduce the fibrinectin protein level.

The results show that PCN can significantly reduce the expression level of fibrosis-associated molecules in the STZ-induced type 1 DN model.

Example 4 effect of PCN on STZ-induced expression of molecules of inflammation of the kidney.

The effect of PCN on STZ-induced renal inflammatory molecule expression was studied using QPCR method.

As shown in fig. 4, the effect of PCN on STZ-induced renal injury molecule expression was investigated using QPCR. In the STZ-induced DN model of type 1, the expression levels of inflammatory factors IL-6 and TNF-alpha in the STZ model group are obviously increased compared with the control group, and p is less than 0.05. And the PCN treatment group can obviously reduce the expression levels of IL-6 and TNF-alpha, and p is less than 0.01.

The results show that PCN can obviously reduce the expression level of IL-6 and TNF-alpha in the STZ-induced DN model type 1.

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

24h urine was collected from mice in metabolism cages at fixed time weekly, and urine volume was counted for each group. Four collections were made for a total of four weeks. As a result, as shown in FIG. 5A, the STZ model was successfully molded, and the urine volume was greatly increased. Compared with the STZ model group, the PCN treatment group can obviously reduce urine volume and has good treatment effect in the first week of treatment; after four weeks, the kidneys and body weights of the mice were weighed and counted, and the results are shown in fig. 5B, wherein the kidney/body weight ratio of the STZ model group was significantly increased, and P was less than 0.001. Compared with the STZ model group, the PCN treatment group can obviously reduce the kidney/body weight ratio, and P is less than 0.05. The urine at the fourth week is mixed evenly, diluted according to a certain proportion, and the content of microalbumin in the urine is measured by an ELISA method, and the result is shown in figure 5C, wherein the content of microalbumin in the urine of the STZ model group is obviously increased, and P is less than 0.001. Compared with the STZ model group, the PCN treatment group can obviously reduce the content of urine microalbumin, and P is less than 0.001.

The results show that in the STZ-induced DN model type 1, the urine volume is greatly increased, the kidney/body weight ratio is obviously up-regulated, and proteinuria occurs. PCN treatment with PXR agonist reduces urine volume, down-regulates kidney/body weight ratio, and reduces urine microalbumin content.

Claims

Use of a PCN in the manufacture of a medicament for the reduction of STZ-induced type 1 diabetic nephropathy-associated condition, wherein the PCN is pregnenolone-16a-carbonitrile, which is an agonist of the pregnane X receptor, the STZ-induced type 1 diabetic nephropathy-associated condition is STZ-induced renal interstitial fibrosis, and the reduction of STZ-induced type 1 diabetic nephropathy-associated condition is the reduction of STZ-induced renal urine volume, urinary microalbumin content and kidney/body weight ratio.