CN108309986A - Applications of the PCN in preparing the drug for treating type 1 diabetes nephrosis associated disease - Google Patents
Applications of the PCN in preparing the drug for treating type 1 diabetes nephrosis associated disease Download PDFInfo
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Abstract
Applications of the PCN in preparing the drug for treating type 1 diabetes nephrosis associated disease.The invention discloses a kind of new indications of pregnenolone 16a carbonitrile (PCN).The present invention, which proposes PCN, can mitigate the illnesss such as the type 1 diabetes nephrosis associated disease, including expression, the albuminuria of acute renal tubular injury, rat tubular cell apoptosis, kidney region fibrosis, renal function, urine volume, inflammatory factor of streptozotocin (STZ) induction.
Description
Technical field
The present invention relates to the new opplications of PCN, and the type 1 diabetes kidney for mitigating STZ inductions is being prepared more particularly to PCN
Application in the drug of sick associated disease.
Background technology
Diabetic nephropathy (diabetic nephropathy, DN) is common one of the complication of diabetes, is also caused
One of main reason of end-stage renal disease, feature include that albuminuria, glomerulosclerosis and renal function are gradually lost.Surpass three
/ mono- diabetic suffers from DN, once end-stage renal disease is developed to, often more than the treatment of other kidney troubles
It is intractable.And in China, incidence is also in rising trend.Therefore the occurrence and development of prevention DN have extremely important meaning.
Type 1 diabetes are insulin-dependent diabetes mellitus, and type 1 diabetes patient dies of DN complication mostly.That is 1
The maximum complication harm of patients with type Ⅰ DM is exactly DN.Ratios of the DN in type 1 diabetes patient is up to 1/3, can examine earliest
Go out to be characterized in that proteinuria has increased slightly (Microalbuminuria), then lasting albuminuria cause renal tubular interstitium inflammation,
Scar, until renal function is gradually lost.
Glomerulus hyperfiltration and the reabsorption of proximal convoluted tubule reduction are two big determinants of albuminuria.There is research to point out DN
In early days, it is influenced by proximal tubular epithelial cells (proximal tubular epithelial cells, PTECs), impaired is small
Pipe re-absorption is the possible cause of albuminuria development, but inherent molecular mechanism is still unknown.
PCN (pregnenolone-16a-carbonitrile) be Pregnane X Receptor (pregnane X receptor,
PXR agonist).PXR plays important biological regulation as endogenous and allogene activated receptor in the defense mechanism of body
Effect and ＂ removing toxic substances ＂ functions.In addition, numerous studies also show PXR by adjusting the expression of downstream target gene by wide participation body
Substance and energetic supersession, and play a significant role in the occurrence and development of certain diseases.At present about the more collection of the research of PXR
In in Liver Lipid Metabolism, angiocarpy, obesity etc., also have recently on a small quantity about PXR regulation and control liver glycometabolism research.
And in terms of DN, there is a research to find in db/db mouse recently, PXR up-regulated expressions, and Slco2b1 (soluble charge materials
Transport protein), Rgc32 (complement activation gene) and Pck1 (phosphoenolpyruvate carboxykinase 1) also raise therewith, prompt
PXR may play bad effect in 2 type DN.2012, there is article to propose that PXR may be related to type 1 diabetes, but below
It can not find out relevant report.Currently, also there has been no the reports that PCN can fight 1 type DN caused by STZ.
Invention content
The purpose of the present invention is to provide a kind of PCN to prepare the 1 type DN phases for mitigating STZ (streptozotocin) inductions
Application in the drug of related disorders.
The present invention has found PCN in the drug for the 1 type DN associated diseases for mitigating STZ inductions by interior animal experiment
Using.
Specifically, the present invention proposes PCN answering in the drug for preparing the 1 type DN renal functions for mitigating STZ inductions
With.
The invention also provides PCN in preparing the acute tubular apoptosis for mitigating STZ inductions and the drug of damage
Application.
In addition, the invention also provides PCN in the drug for preparing the renal interstitial fibrosis for mitigating STZ inductions
Using.
Further, present invention finds PCN in the medicine for preparing the expression for mitigating the renal inflammation molecule that STZ is induced
Application in object.
Meanwhile it has also been found that PCN prepare the 1 type DN urine volume induced for reducing STZ, kidney/weight ratio and
Application in the drug of microalbumin content.
Description of the drawings
Fig. 1 is the renal function and staining for glycogen (PAS) result after treating with STZ modelings and with PCN；
Fig. 2, which is that QPCR, Western blot method research PCN are related to the STZ acute tubular apoptosis induced and damage, to be divided
The influence of son；
Fig. 3 is QPCR, Western blot methods detect influences of the PCN to the STZ renal interstitial fibrosis induced；
Fig. 4 is influences of the QPCR method research PCN to the STZ renal inflammation developed by molecule induced；
Fig. 5 is the shadow for counting kidney urine volume, microdose urine protein content and kidney/weight ratio that PCN induces STZ
It rings.
Specific implementation mode
Western blot, real-time fluorescence quantitative PCR, the concrete operation step of PAS dyeing are as follows in the present invention：
Western blot：
Tissue lysates extract renal tissue total protein, measure protein concentration using BCA methods, take 30 μ g albumen loadings,
8% or 12% polyacrylamide gel electrophoresis (SDS-PAGE), 300mA × 1.5h is wet to walk around to pvdf membrane, confining liquid room temperature
1h is closed, primary antibody, Bax (Cell Signaling Technology), Bcl-2 (Cell is added after TBST elutions
SignalingTechnology), Caspase3 (Cell SignalingTechnology), Cleaved caspase3 (Cell
SignalingTechnology), Fibronectin (Abcam), GAPDH (Cell SignalingTechnology), 4 DEG C incubate
It educates overnight.TBST washes film 5 times, each 5min, is incubated at room temperature 1h with corresponding secondary antibody, TBST washes film 5 times.Antigen-antibody complex
It is shown with Enhanced chemiluminescence (ECL), darkroom X-ray film is exposed and scanned, and quantification of protein, which uses, carries out purpose band
Gray value is analyzed, and the relative expression quantity of destination protein is indicated with purpose band gray value/GAPDH gray values.
Real-time fluorescence quantitative PCR (QPCR)：
Renal tissue total serum IgE is extracted, RNA solution concentration and purity are measured with spectrophotometry.Utilize Reverse Transcriptase kit
1 μ gRNA reverse transcriptions at cDNA, the situation of change of different genes are detected according to following reaction system by (Takara, DaLian).
A. reaction system
B.PCR thermal circulation parameters
PAS is dyed：
4% paraformaldehyde fixing organization 48h, specimens paraffin embedding slices, dewaxing to water, distilled water flushing, 70% alcohol rinse 3
It is secondary.Periodic acid alcoholic solution 10min (this solution temperature is preferably 17-20 degree) is immersed, after 70% alcohol is washed, is entered in reducing solution
1min (this solution temperature is preferably 17-20 degree) after 70% alcohol is washed, enters colourless slag fuchsin solution 1-1.5h, winter room
When temperature is relatively low, 37 degree of incubators can be put into.Flowing water rinses 10min, with Mayer ' s haematoxylins redye liquid and redye nucleus 3-5min,
Broken up again with 1% hydrochloride alcohol, after flowing water rinses, is dehydrated transparent, last mounting.
Masson is dyed：
4% paraformaldehyde fixing organization 48h, specimens paraffin embedding slices, dewaxing to water；Tap water, distilled water flushing 3 are used successively
It is secondary；Core 5-10min is contaminated with Weigert bush sperm, flowing water rinses 10min；With Masson Ponceaux acid fuchsin liquid 5-
10min；A moment is embathed with 2% glacial acetic acid aqueous solution；1% phosphomolybdic acid aqueous solution breaks up 3-5min；Without washing, aniline is directly used
Blue or light green liquor contaminates 5min；A moment is embathed with 0.2% glacial acetic acid aqueous solution；95% alcohol, absolute alcohol, dimethylbenzene is transparent, in
Property natural gum sealing.
Below by specific embodiment, the present invention will be described in detail.
Influences of 1 PCN of embodiment to the STZ diabetic nephropathy renal functions induced.
The male C57BL/6 mouse for taking 18~22g of weight are divided into 3 groups, i.e. control group, STZ model groups and PCN treatments
Group (n=10).3 left kidneys of the equal surgical removal of group, post-operative recovery one week.
Control group：Isometric medium (sodium citrate) is injected intraperitoneally one time a day, totally 5 days；
STZ model groups：STZ, 50mg/kg, successive administration 5 days is injected intraperitoneally；
PCN treatment groups：STZ is injected intraperitoneally, 50mg/kg, successive administration is after 5 days, PCN administrations (intraperitoneal injection, 50mg/
KgPCN, 200 μ L/ times), one time a day, treats 4 weeks, collect urine volume for 24 hours with metabolic cage weekly, take blood after 4 weeks, leave and take nephridial tissue.
Blood specimen is centrifuged into (20min, 3000r/min), with creatinine reagent box (Creatinine Assay Kit (cat:
K625-100, biomars)), urea nitrogen kit (QuantiChrom Urea Assay kit (cat:DIUR-500,
Hayward, CA)) serum creatinine, urea nitrogen are measured, experimental result is shown in Figure 1A.
Figure 1B uses STZ as can be seen from the results for the PAS coloration results after being treated with STZ modelings and with PCN
Modeling success, serum creatinine, urea nitrogen significantly increase, and kidney is prompted to be damaged.And after using PCN treatments, it can be obviously improved
Renal function, creatinine, urea nitrogen levels are substantially reduced compared with model group, p<0.01.According to PAS coloration results, STZ group renal tubules
Structure is destroyed, and protein cast is formed.PCN treatment groups can significantly improve the renal damage of STZ inductions.
Fig. 1 C are the Masson coloration results after treating with STZ modelings and with PCN.As can be seen from the results, STZ groups
Modeling success, renal interstitial collagen deposition, Masson stained positives region showed increased.PCN treatment groups can significantly reduce
Masson stained positives region prompts PCN to improve the fibrosis of the renal tubular interstitium of STZ inductions.
Influences of 2 PCN of embodiment to the STZ rat tubular cell apoptosis induced and kidney injury relevant molecule.
1 type that PCN induces STZ is measured using the expression that QPCR methods detect kidney apoptosis and lose relevant molecule
The improvement situation of DN, as a result as shown in Figure 2 A, the apoptosis relevant molecule Bax expression of STZ model groups are significantly raised, p<0.001；Damage
It is significantly raised to hinder the expression of relevant molecule Kim-1, NGAL, p<0.001.And PCN treatment groups can significantly reduce Bax, Kim-1,
The expression of NGAL, p<0.05.
PCN is measured to STZ using the expression that Western blot methods detect kidney apoptosis and lose relevant molecule
The improvement situation of 1 type DN of induction, as a result as shown in Figure 2 B, apoptosis relevant molecule Bax, Caspase3 of STZ model groups is cut
It is significantly raised to be sliced section Cleavedcaspase3 protein levels；Bcl-2 protein levels are obviously lowered.And PCN treatment groups can show
Writing reduces the expression of Bax, Cleavedcaspase3, raises Bcl-2 protein levels.
The result shows that PCN can be substantially reduced the rat tubular cell apoptosis of STZ inductions and lose the expression water of relevant molecule
It is flat.
Influences of 3 PCN of embodiment to the STZ renal interstitial fibrosis relevant molecule expression induced.
The renal interstitial fibrosis relevant molecule table that STZ is induced using QPCR and Western blot method research PCN
The influence reached.
As shown in Figure 3A, using QPCR method research PCN to the STZ renal interstitial fibrosis relevant molecule expression induced
It influences.In the DN models of STZ inductions, fibrosis indices in hepatic α-SMA, Fibronectin, Collagen III of STZ model groups
MRNA level in-site it is significantly raised compared with the control group, p<0.05.And PCN treatment groups can significantly reduce α-SMA,
The mRNA level in-site of Fibronectin, Collagen III, p<0.01.
As shown in Figure 3B, the renal interstitial fibrosis correlation point STZ induced using Western blot method research PCN
The influence that sublist reaches.STZ induction DN models in, the fibrosis indices in hepatic Fibronectin protein levels of STZ model groups with it is right
According to group compared to apparent increase, and PCN treatment groups can significantly reduce Fibronectin protein levels.
The result shows that PCN can be substantially reduced the expression of fibrosis relevant molecule in 1 type DN models of STZ inductions.
Influences of 4 PCN of embodiment to the STZ renal inflammation developed by molecule induced.
Influence using QPCR method research PCN to the STZ renal inflammation developed by molecule induced.
As shown in figure 4, the influence using QPCR method research PCN to the STZ Kidney injury molecule expression induced.It is lured in STZ
In the 1 type DN models led, inflammatory factor IL-6, the TNF-α expression of STZ model groups are significantly raised compared with the control group, p<
0.05.And PCN treatment groups can significantly reduce IL-6, TNF-α expression, p<0.01.
The result shows that PCN can be substantially reduced STZ induction 1 type DN models in IL-6, TNF-α expression.
The influence for kidney urine volume, microdose urine protein content and kidney/weight ratio that 5 PCN of embodiment induces STZ.
The set time collects the urine for 24 hours of mouse with metabolic cage weekly, counts the urine volume of each group.Surrounding altogether collects four
It is secondary.As a result as shown in Figure 5A, STZ model group modelings success, urine volume presentation are significantly raised.PCN treatment groups and STZ model group phases
Than urine volume can be significantly reduced, and just has good therapeutic effect within first week in treatment；After surrounding, Mouse Kidney is weighed
Dirty and weight, and counted, as a result as shown in Figure 5 B, kidney/weight ratio of STZ model groups obviously raises, P<0.001.
PCN treatment groups can obviously lower kidney/weight ratio, P compared with STZ model groups<0.05.By the urine mixing of 4th week,
It dilutes by a certain percentage, the content of microalbumin in urine, as a result as shown in Figure 5 C, STZ model groups is measured using ELISA method
Microdose urine protein content obviously raise, P<0.001.It is micro- can obviously to lower urine compared with STZ model groups for PCN treatment groups
Measure albumin content, P<0.001.
The result shows that in 1 type DN models of STZ inductions, urine volume increases considerably, and kidney/weight ratio also obviously raises,
And there is albuminuria.Urine volume can be reduced after giving the PCN processing of PXR agonists, while lowering kidney/weight ratio, and is subtracted
Oliguresis microalbumin content.
Claims (6)
- Applications of the 1.PCN in the drug for preparing the type 1 diabetes nephrosis associated disease for mitigating STZ inductions.
- 2. application according to claim 1, which is characterized in that the type 1 diabetes nephrosis associated disease of STZ induction is The type 1 diabetes nephrosis renal function of STZ inductions.
- 3. application according to claim 1, which is characterized in that the type 1 diabetes nephrosis associated disease of STZ induction is The acute tubular apoptosis of STZ inductions and damage.
- 4. application according to claim 1, which is characterized in that the type 1 diabetes nephrosis associated disease of STZ induction is The renal interstitial fibrosis of STZ inductions.
- 5. application according to claim 1, which is characterized in that the type 1 diabetes nephrosis related diseases for mitigating STZ inductions Disease is the expression for the renal inflammation factor for mitigating STZ inductions.
- 6. application according to claim 1, which is characterized in that the type 1 diabetes nephrosis related diseases for mitigating STZ inductions Disease is kidney urine volume, microdose urine protein content and kidney/the weight ratio for reducing STZ inductions.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117122603A (en) * | 2023-09-07 | 2023-11-28 | 南方医科大学顺德医院(佛山市顺德区第一人民医院) | Pharmaceutical preparation and application thereof |
-
2018
- 2018-02-07 CN CN201810121259.XA patent/CN108309986B/en active Active
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| ATSUSHI WATANABE, ET AL.: "Aberrant DNA methylation of pregnane X receptor underlies metabolic gene alterations in the diabetic kidney", 《AM J PHYSIOL RENAL PHYSIOL》 * |
| HIROHISA NAGAHORI, ET AL.: "Combining Genomics To Identify the Pathways of Post-Transcriptional Nongenotoxic Signaling and Energy Homeostasis inLivers of Rats Treated with the Pregnane X Receptor Agonist, Pregnenolone carbonitrile", 《JOURNAL OF PROTEOME RESEARCH》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117122603A (en) * | 2023-09-07 | 2023-11-28 | 南方医科大学顺德医院(佛山市顺德区第一人民医院) | Pharmaceutical preparation and application thereof |
| CN117122603B (en) * | 2023-09-07 | 2024-03-19 | 南方医科大学顺德医院(佛山市顺德区第一人民医院) | Pharmaceutical preparation and application thereof |
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