CN109771648A - Application of inhibitor of deubiquitinase USP11 in preparation of drug for preventing or treating uric acid renal disease - Google Patents
Application of inhibitor of deubiquitinase USP11 in preparation of drug for preventing or treating uric acid renal disease Download PDFInfo
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- CN109771648A CN109771648A CN201910178798.1A CN201910178798A CN109771648A CN 109771648 A CN109771648 A CN 109771648A CN 201910178798 A CN201910178798 A CN 201910178798A CN 109771648 A CN109771648 A CN 109771648A
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- uric acid
- inhibitor
- usp11
- acid nephropathy
- renal
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Abstract
本发明提供了去泛素化酶USP11的抑制剂在制备预防或治疗尿酸性肾病药物中的用途。所述的去泛素化酶USP11的抑制剂为米托蒽醌。所述的尿酸性肾病为肾炎或者肾脏纤维化。本发明还提供了去泛素化酶USP11作为诊断尿酸性肾病的生物标记物的用途。本发明明确了去泛素化酶USP11在尿酸性肾病的发生发展中起关键性作用，证实其抑制剂米托蒽醌可以有效缓解尿酸性肾病引起的肾脏损伤，抑制肾纤维化，改善肾脏炎症，保护肾功能。相比其它现有药物而言，米托蒽醌可以从多方面来阻碍尿酸性肾病的发病及其进展，并对肾纤维化的发生有明显地抑制作用，从而保护肾脏功能，为临床制备米托蒽醌相关的防治尿酸性肾病药物提供理论依据。
The invention provides the use of an inhibitor of deubiquitinating enzyme USP11 in the preparation of medicaments for preventing or treating uric acid nephropathy. The inhibitor of the deubiquitinating enzyme USP11 is mitoxantrone. The uric acid nephropathy is nephritis or renal fibrosis. The present invention also provides the use of the deubiquitinating enzyme USP11 as a biomarker for diagnosing uric acid nephropathy. The present invention clarifies that the deubiquitinating enzyme USP11 plays a key role in the occurrence and development of uric acid nephropathy, and confirms that its inhibitor mitoxantrone can effectively relieve kidney damage caused by uric acid nephropathy, inhibit renal fibrosis, and improve renal inflammation , Protect kidney function. Compared with other existing drugs, mitoxantrone can hinder the onset and progress of uric acid nephropathy in many ways, and can significantly inhibit the occurrence of renal fibrosis, thereby protecting kidney function and providing a clinical preparation for rice Toxantrone-related drugs to prevent and treat uric acid nephropathy provide a theoretical basis.
Description
Technical field
The invention belongs to field of pharmacology, are related to a kind of deubiquitinating enzymes USP11, in particular to deubiquitinating enzymes
Purposes of the inhibitor of USP11 in preparation prevention or treatment uric acid nephropathy drug.
Background technique
Uric acid is the dead end product of purine metabolism, and the intracorporal uric acid of people is substantially deposited in the form of the free state of monosodium urate salt
It is in blood, wherein 80% is generated by Nuclear extract catabolism, 20% is rich in the food catabolism of purine by taking in
It generates.The uric acid that human body is generated and drained daily is 600~700mg, wherein 1/3 is discharged by enteron aisle, 2/3 by kidney.Positive reason
Under condition, the generation and excretion of the daily uric acid of human body are kept substantially dynamic equilibrium.But as uric acid synthesis increase, row in human body
Reduction is let out, will make uric acid concentration in blood is in hypersaturated state, and male's empty stomach serum uric acid level is higher than 420 μm of ol/L, Nv Xinggao
In 360 μm of ol/L, as hyperuricemia (hyperuricemia).
Recently as the increase of animal protein absorption ratio in China's resident's dietary structure, the crowd of hyperuricemia
Increasing.One national studies have shown that prevalence of hyperuricemia is 13% in population of China, in male up to
18.5%, hence it is evident that be higher than women (8.0%).The prevalence of hyperuricemia in southern area and rural area is higher than northern area and city
City.And the study found that Patients with Hyperuricemia often merges multiple cardiovascular risk factors.2~3 in Patients with Hyperuricemia
The merger ratio of a cardiovascular risk factors is 30.5%~32.3%, hence it is evident that higher than the merging in normal serum uric acid level crowd
Rate (14.5%~22.5%).Long-term hyperuricemia can cause renal function to damage again.At home, Patients with Hyperuricemia
In, the illness rate of Nephropathy is up to 15.1%, and the probability that renal failure occurs as 505.8 μm of ol/L of blood uric acid > is blood
8 times when uric acid is normal value.Serum uric acid concentration often increases 59.5 μm of ol/L, and the risk that chronic kidney disease occurs increases by 7%
~11%.
When uric acid in blood excessive concentration, when being in hypersaturated state, uric acid, which is deposited on nephridial tissue, can cause uric acid kidney
Sick (hyperuricemic nephropathy), can finally cause renal insufficiency.Between the renal lesions of uric acid nephropathy are mainly
Matter ephritis and renal tubular function are impaired, and renal damage is directly proportional to the raised horizontal and duration length of blood uric acid.High lithemia
The mechanism of mass formed by blood stasis induction kidney damage mainly includes the coup injury and indirect injury of uric acid.The direct kidney injury of uric acid is main
It is largely deposited in renal tubule and renal interstitial for mono-salt-hydrate made of uric acid conversion or urate crystal, causes kidney
Material injury, such as the formation of inflammatory reaction and fibrosis.Calculus urate is formed by obstruction for initiation to kidney simultaneously
A series of complication such as infects, bleeding, ponding, pernicious change.The indirect kidney injury of uric acid specifically includes that blood uric acid increases
The activation of Re-A-A (RAS) system, hyperuricemia stimulation are caused to the damage of blood vessel endothelium, high lithemia
Inflammatory cascade reaction, hyperuricemia cause the generation of organism metabolism syndrome, hyperuricemia to cause kidney renal tubular epithelial thin
Dysuria with lower abdominal colic is divided into renal interstitial fibroblast etc., aggravates chronic kidney disease progress.
The major pathologic features of uric acid nephropathy are kidney region fibrosis, and main mechanism is that renal tubular epithelial has occurred is thin
Matrix converts (Epithelial mesenchymal transmition, EMT), this process is dynamic along with α smooth muscle flesh
Albumen (α-smooth muscle actin, α-SMA) and collagenous fibres expression increase, extracellular matrix accumulation and tubule are thin
Born of the same parents block the G2/M phase.In studies have shown that hyperuricemia renal damage rats model, Snail1, α-SMA, Vimentin albumen table
Up to up-regulation, E-cadherin protein expression is lowered.Snail1 is the central transcription factor for inducing epithelial-mesenchymal conversion, it can be with
The expression for inhibiting E-cadherin, causes the close connection between epithelial cell to disappear, and renal cells interstitial is promoted to turn
Differentiation.In the renal cells cultivated in vitro, uric acid stimulation can also equally cause renal cells interstitial and turn to divide
Change, the significant protein expression of EMT increases.Research confirms that hyperuricemia may be turned by induction renal cells phenotype
Change, promotes the progress of kidney region fibrosis.
The occurrence and development of renal interstitial fibrosis are related to many A signal pathways and cell factor.(1) transforming growth factor
(TGF-β) signal path.TGF-β is a kind of more function cell factors, takes part in cell Proliferation, differentiation, apoptosis, wound repair and fibre
The regulation of the various kinds of cell process such as dimensionization.Clinical data shows, in the kidney sample and urine of renal disease patient, TGF-β 1
Content obviously rises, and is positively correlated with renal fibrosis degree in obvious, animal model of this phenomenon in a variety of kidney troubles
In be proved.It includes: 1. to pass through to rely on or do not depend on Smad3 mechanism that TGF-β 1, which induces the main mechanism of renal fibrosis, from
Transcriptional level directly induces the synthesis of the ECM such as collagen I, fibronectin；2. inducing metal protease
(metalloproteinases, MMPs) and Timp (tissue inhibitor of
Metalloproteinase, TIMPs) loss of equilibrium, prevent the degradation of ECM；3. the direct effect to the intrinsic cell of kidney.Example
Such as, mesangial cell proliferation and collagen secretion be can induce；Promote epithelial cell and Podocytes in Renal Tissue, aggravates inflammation and secondary fibrosis
Deng；4. promoting the myofibrosis mother cell transdifferentiation in a variety of sources such as fibrocyte, epithelial cell, endothelial cell, macrophage
And proliferation, mediate fiberization.(2) Wnt/ β-catenin signal path.Wnt/ β-the catenin in normal adult's kidney
With respect to silencing, but after tissue damaged, this signal paths will be activated signal path, major regulatory cell differentiation,
Organize homeostasis and injury repair.Research at present has shown that the sustained activation of Wnt/ β catenin signal path is with kidney fibrosis
Closely related, mechanism includes: that the activation of 1. Wnt/ β-catenin β can induce Snail1 in renal cells and podocytic process
The expression of cell causes the close connection between epithelial cell to disappear so that E-cadherin albumen be inhibited to generate, and promotes kidney small
Pipe epithelial cell interstitial transdifferentiation；2. matrix metalloproteinase-7 MMP-7 is Wnt/ β-catenin signal path under another
Target spot is swum, it can degrade extracellular matrix, promote the release of inflammatory mediator, activate other protease, promote progression of fibrosis.
(3) Notch1/Jagged-1 signal path is upper highly conserved signal path of evolving, can regulate and control embryonic development and at
Body tissue stable state.Its induce renal fibrosis main mechanism include: 1. Notch1/Jagged-1 access regulation lymphocyte,
The development and activation of macrophage play important proinflammatory, rush Fibrosis parameters.2. promoting the expression of the related gene of EMT, promote
Into tubular epithelial transdifferentiation, fibrosis is aggravated.
The degradation of intracellular protein mainly passes through Ubiquitin-Proteasome Pathway.Protein is in a series of ubiquitination enzymes
By ubiquitin tag under the action of (ubiquitin kinase E1, ubiquitin binding enzyme E2, ubiquitin ligase E3), then dropped by proteasome
Solution.Protein carries out posttranslational modification by ubiquitination mode, not only can change the stability of intracellular protein, moreover it is possible to adjust
Its positioning and activity are saved, the function of target protein is influenced, to play crucial regulating and controlling effect to cell function.Ubiquitination is one
Reversible process, deubiquitinating enzymes (deubiquitinating enzymes, DUBs) destroy poly ubiquitin by hydrolysis
Change chain between or the thioester bond between ubiquitin and its substrate, remove ubiquitin molecule, to protect target protein not by proteasome institute
Hydrolysis.
Ubiquitin-specific protease family (ubiquitin-specific proteases, USPs) USPs is to find so far
Maximum deubiquitinating enzymes subfamily, member are more than 50, and participation cell cycle, Apoptosis, genetic transcription, DNA damage are repaired
The important cell activities such as multiple.
USP11 (Ubiquitin-specific protease11) belongs to cysteine proteinase, is ubiquitin-specific egg
One of the important member of white enzyme family, it is widely distributed in tissue.It is sub- that USP11 gene is located at 1st area of X chromosome galianconism, 1 band 3
Band, overall length 3300bp include 22 exons.USP11 albumen is made of 963 amino acid, nucleus is primarily located within, thin
Cytoplasm also has a small amount of distribution.Recent study shows that USP11 is adjustable the activity and function of intracellular numerous protein substrates, including
GAP-associated protein GAP etc. in DNA repair protein, viral RNA replication-associated protein and TGF β, NF- κ B signal access, in the hair of disease
It plays an important role in hair tonic exhibition.
Mitoxantrone (MTX) mitoxantrone, can inhibit ubiquitin-specific protease USP11, chemical formula 1,
Bis- [[2- [(2- ethoxy) amino] ethyl] amino] -9, the 10- anthraquinones of 4- dihydroxy -5,8-, molecular weight 444.481, molecular formula
C22H28N4O6, black-and-blue crystallization is odorless, 203-205 DEG C of fusing point, is slightly soluble in water, is slightly soluble in ethyl alcohol, does not dissolve in chloroform and third
Ketone.
Summary of the invention
For above-mentioned uric acid nephropathy clinical settings and its harmfulness, and it clinically there is no effectively radical cure at present or alleviate
The drug of uric acid nephropathy kidney fibrosis lesion, the present invention provides the inhibitor of deubiquitinating enzymes USP11 preparation prevention or
Treat the purposes in uric acid nephropathy drug.This purposes will solve kidney function caused by clinical treatment uric acid nephropathy
It can the technical problems such as damage and kidney fibrosis lesion.
The present invention provides the inhibitor of deubiquitinating enzymes USP11 in preparation prevention or treatment uric acid nephropathy drug
Purposes.
Further, the inhibitor of the deubiquitinating enzymes USP11 is mitoxantrone.
Further, the uric acid nephropathy is ephritis or renal fibrosis.
The present invention also provides purposes of the deubiquitinating enzymes USP11 in the biomarker of diagnosis uric acid nephropathy.
The present invention utilizes Oteracil Potassium and adenine suspension oral gavage induced rat uric acid nephropathy model, probes into USP11
Mechanism of action in uric acid nephropathy.First, it has been found that it is big that USP11 inhibitor MTX can effectively inhibit uric-acid nephropathy
The generation of mouse α smooth muscle actin and collagen fibrous proteins reduces extracellular matrix accumulation.The uric acid of MTX is injected intraperitoneally
Nephrotic rats are substantially reduced compared to untreated rat, Vimentin, SLUG, Snail1, and E-cadherin is increased, this
Prompt USP11 plays an important role in kidney tubular epithelial transdifferentiation, inhibits USP11 that can effectively reverse this pathology mistake
Journey.Secondly, it has been found that USP11 inhibitor can significantly inhibit a series of stream signal for causing renal fibrosis logical
Road, such as TGF-β/smad signal path, the work of Wnt/ β-catenin signal path and Notch1/Jagged-1 signal path
Property.And, it has been found that USP11 inhibitor MTX can inhibit lymphocyte and macrophage to ooze out, and reduce inflammatory reaction.
In summary, it is believed that USP11 plays an important role in the generation and progress of uric acid nephropathy.The suppression of USP11
The generation of renal inflammation caused by uric acid nephropathy and fibrosis can effectively be alleviated and be treated to preparation mitoxantrone, and change
Kind renal function.This lays the foundation to understand uric acid nephropathy pathogenesis in depth, also treats uric acid nephropathy for clinical development
Newtype drug provides foundation.
The present invention is compared with prior art, and technological progress is significant.Innovative point and beneficial effect of the invention exists
The key effect in clear deubiquitinating enzymes USP11 is in the occurrence and development of uric acid nephropathy, it was demonstrated that its inhibitor rice support anthracene
Kidney injury caused by uric acid nephropathy can be effectively relieved in quinone, inhibit kidney fibrosis, improve renal inflammation, protect renal function.
For other existing drugs, mitoxantrone can hinder the morbidity and its progress of uric acid nephropathy from many aspects, and right
Kidney fibrosis has significantly inhibiting effect, to protect renal function, prepares the relevant prevention and treatment of mitoxantrone for clinic
Uric acid nephropathy drug provides theoretical foundation.
Detailed description of the invention
Fig. 1 shows that MTX inhibitor improves renal tissue of uric acid nephropathy rats and damages and lower USP11 expression quantity.
Fig. 2 shows that MTX inhibitor improves uric acid nephropathy rat renal damage and kidney region fibrosis.
Fig. 3 shows that MTX inhibits the generation of rat uric acid nephropathy renal fibrosis.
Fig. 4 shows that MTX inhibitor blocks epithelial cell in renal tissue of uric acid nephropathy rats-mesenchyma conversion (EMT)
Occur.
Fig. 5 shows that MTX inhibitor blocks the activation of TGF-β 1/Smad3 signal path in renal tissue of uric acid nephropathy rats.
Fig. 6 shows that MTX inhibitor blocks Notch1/Jagged-1 and Wnt1/ β-in renal tissue of uric acid nephropathy rats
The activation of catenin signal path.
Fig. 7 shows that MTX inhibits CD68 in renal tissue of uric acid nephropathy rats+Macrophage and CD3+The infiltration of lymphocyte.
Specific embodiment
1 material of embodiment and method
1) reagent consumptive material
P-Smad3, Smad3, E-cadherin, Notch1, Jagged-1, Vimentin, SLUG, Snail1, Wnt1, β-
Catenin antibody is purchased from cell signaling Technology company.USP11, Fibronectin antibody are purchased from Abcam
Company.Collagen I (A2), TGF-β RI, CD68, CD3, GAPDH antibody are purchased from Santa Cruz company.MTX is purchased from
Selleckchem company.α-SMA antibody and other reagents are purchased from Sigma company.
2) the uric acid nephropathy model of rat and experimental group
The uric acid nephropathy model of rat:
SPF grades male SD rat 32, (250 ± 20) g is provided by Shanghai Slac Experimental Animal Co., Ltd., raising
In Tongji University's Experimental Animal Center.According to rat body weight with the ratio of adenine 100mg/kg, Oteracil Potassium 1500mg/kg,
Be made the suspension of final concentration of 80g/L after the two being mixed with distilled water, with 20ml/kg/d gastric infusion in two times daily,
Continuous 21 days.All zooperies abide by Chinese experimental the care of animal and use regulations.
Experimental group:
A.sham group (n=8 is only): the same dose of 0.9% physiology of the same dose of distilled water+intraperitoneal injections of gastric infusion
Salt water continues 21 days；
B.sham+MTX group (n=8 is only): the same dose of distilled water of gastric infusion+intraperitoneal injection 0.35mg/kg MTX,
Continue 21 days；
C.HN group (n=8 is only): according to suspension+intraperitoneal injection of rat body weight gastric infusion adenine and Oteracil Potassium
The same dose of 0.9% physiological saline continue 21 days；
D.HN+MTX group (n=8 is only): according to suspension+abdominal cavity of rat body weight gastric infusion adenine and Oteracil Potassium
0.35mg/kg MTX is injected, continues 21 days.
3) blood specimen leave and take and biochemical indicator detection
Fasting 12h before blood is taken, angular vein takes blood 2ml or according to rat body weight according to 1ml/kg dosage intraperitoneal injection 3%
Abdominal aortic blood 5ml after yellow Jackets anesthesia.Room temperature 2500rpm is centrifuged 15min, and serum is taken to manage to sterile Eppendorf
In, -80 DEG C freeze blood biochemistry index to be measured.Blood specimen and urine specimen by automatic clinical chemistry analyzer measure serum uric acid,
Creatinine, urea nitrogen and microdose urine protein.
4) nephridial tissue leave and take and the preparation of paraffin section
Speed takes the double kidneys of separation after rat is put to death.Left kidney peplos are peelled off, are placed in 10% neutral formalin fixer solid
It is fixed, make paraffin section.The thin slice of left nephridial tissue sample interception thickness 2mm or so is placed in 10% neutral formalin fixer
It is fixed for 24 hours after, then respectively by 70% ethyl alcohol for 24 hours, 80% ethyl alcohol 12h, 95% ethyl alcohol overnight, 100% subgradient of ethyl alcohol 1h × 2
Dehydration, the transparent 30-60min of dimethylbenzene, waxdip is overnight, embeds into paraffin mass.The thin slice of 3 μ m-thicks is cut into slicer (Leica),
It is affixed in the clean glass slide impregnated through 1 ‰ (v/v) poly-D-lysines, number, and stayed overnight in 37 DEG C of ovens, deposits in drying and keep away
At light, analyzed for pathomorphism.
5) Masson is dyed: tissue is fixed on 4% paraformaldehyde liquid, conventional dehydration embedding, with weight after slice dewaxing to water
Potassium chromate mordant handles tissue 12-18h, and 5-10 minutes flowing water of WeigerShi Garapa uniformly dyeing is slightly washed, 1% hydrochloride alcohol point
Change, flowing water rinses several minutes and returns indigo plant, and Ponceaux acid fuchsin liquid contaminates 5-10 minutes, distilled water short rinse, phosphomolybdic acid aqueous solution
Processing about 3-5 minutes, is not washed with water, is directly redyed 2-5 minutes with aniline blue liquid, and 1% glacial acetic acid is handled 1 minute, 95% alcohol
Repeatedly, absolute alcohol dehydration, dimethylbenzene is transparent, neutral gum sealing for dehydration.
6) immunohistochemistry detects
Immunohistochemical staining: entire colouring method is carried out referring to SP reagent kit product specification, and antibody is with the phosphorus of 0.1M
Phthalate buffer (Phosphate-Buforsiluflon, PBS, pH 7.4) dilution, concentration 1:100 substitute primary antibody with PBS
Make negative control.Citrate buffer antigen retrieval, DAB colour developing, haematoxylin are redyed, neutral gum mounting, om observation.
7)Western-blot
Take one 80 DEG C of freezing peritoneal tissues requirements (about 20mg).Every l00mg tissue plus 200 μ l protein lysates, on ice
Homogenate.4 DEG C, 12000r/min.It is centrifuged 10min, takes supernatant, measures protein concentration with BCA protein quantification kit (sunbio).
8% separation gel and 5% concentration glue, sample-adding are successively made again, and electrophoresis terminates electrophoresis, wet process electrotransfer when bromophenol blue to glue bottom
To pvdf membrane, 90min is put into TBST and washes 3 times 5min/ times, prepares 5% skimmed milk power with TBST, after film is immersed, room temperature is put
Set lh.Primary antibody is diluted in proportion (1:1000) with confining liquid, is incubated for jointly with film, 4 spend night.TBST is washed 3 times, 5min/ times.
With the secondary antibody of confining liquid dilution HRP label, thinner ratio falls 1:2000, is incubated for 1.5h jointly with film.TBST washes 3 times, and 5min/ times,
ECL exposure.Gray analysis is carried out to image with LabWorks software after photograph.The variation of relative amount=destination protein gray scale/
GAPDH gray scale.
8) statistical analysis
Statistical procedures are carried out using SPSS20.0 and 2012 software of EXCEL, all data use means standard deviation
(x- ± s) is indicated, comparison among groups use one-way analysis of variance, using P < 0.05 as statistically significant.
2 MTX inhibitor of embodiment, which improves renal tissue of uric acid nephropathy rats, to be damaged and lowers USP11 expression quantity
This experiment, with the ratio of adenine 100mg/kg, Oteracil Potassium 1500mg/kg, is incited somebody to action according to rat body weight with distilled water
The suspension of final concentration of 80g/L is made after the two mixing, with 20ml/kg/d gastric infusion in two times daily, builds within continuous 21 days
The vertical uric acid nephropathy model of rat, and the USP11 inhibitor MTX of 0.35mg/kg is injected intraperitoneally, observe MTX inhibitor pair
The protective effect of renal tissue of uric acid nephropathy rats damage and renal fibrosis.The blood of blood biochemistry uric acid nephropathy rat as the result is shown
Uric acid, serum creatinine, plasma wrea values of nitrogen might have significant raising, and after injecting MTX inhibitor, the serum content of three drops to normally
Horizontal (Figure 1A -1C).In addition, MTX can also lower abnormal raised Microalbuminuria (figure caused by uric acid nephropathy
1D).Immunoblot results are shown, USP11 is hardly expressed in normal rat kidney, and exception is high in renal tissue of uric acid nephropathy rats
USP11 is expressed, and after injecting MTX, expression quantity drops to normal level (Fig. 1 E-1F).Result above prompts uric acid nephropathy
High expression USP11, can significantly inhibit the expression quantity of kidney USP11 after injecting MTX inhibitor, and improve uric acid in rat kidney
Nephrotic rats kidney injury.
3 MTX inhibitor of embodiment improves uric acid nephropathy rat renal damage and kidney region fibrosis
This experiment establishes the uric acid nephropathy model of rat using adenine and Oteracil Potassium suspension oral gavage 21 days, and gives
The MTX inhibitor of 0.35mg/kg is injected intraperitoneally, by PAS decoration method (Periodic Acid-Schiff stain) and
Masson decoration method observes MTX inhibitor to the inhibiting effect of uric acid nephropathy rat renal damage and kidney region fibrosis.
PAS coloration result and renal damage scoring display normal rat kidney renal tubule result are normal, no expansion and renal tubular epithelial
Cell detachment phenomenon, and apparent tubule dilatation phenomenon can be found in renal tissue of uric acid nephropathy rats, a large amount of renal tubular epithelials
Cell detachment.In HN+MTX group, injection MTX inhibitor can improve renal damage situation, and renal damage scores compared with mould
Type group is decreased significantly (Fig. 2A -2B).It is big that Masson coloration result and positive area quantitative display MTX can inhibit uric acid nephropathy
The generation (Fig. 2 C-2D) of mouse kidney region fibrosis.Result above prompts MTX inhibitor to improve uric acid nephropathy rat renal tubule damage
Wound and kidney region fibrosis, shield to kidney.
The generation of 4 MTX of embodiment inhibition rat uric acid nephropathy renal fibrosis
Protective effect of the MTX inhibitor to uric acid nephropathy renal interstitial fibrosis rat is further looked at, we choose two
A markers of fibrosis index: a-SMA and Fibronectin, with big in the method for immunoblotting and immunohistochemistry detection each group
The degree of mouse renal fibrosis.Immunoblot results show that a-SMA expression quantity is lower in normal rat kidney, uric acid nephropathy mould
High expression a-SMA index in the rat kidney of type, and after using MTX inhibitor, expression quantity is suppressed significantly, and is dropped to just
Ordinary water puts down (Fig. 3 A-3B).Immunohistochemistry and quantitative analysis results show that MTX inhibitor is to abnormal high caused by uric acid nephropathy
The a-SMA and Fibronectin of expression have inhibiting effect (Fig. 3 C-3F).Result above further clarifies MTX inhibitor to urine
Acid nephrotic rats kidney region fibrosis has inhibiting effect.
5 MTX inhibitor of embodiment blocks epithelial cell in renal tissue of uric acid nephropathy rats-mesenchyma conversion (EMT) hair
It is raw
Epithelial cell-mesenchyma conversion, refers to that epithelial cell is converted by specific program with interstitial phenotype cells
Biological process has played important function, main feature in the multiple fiber disease including renal fibrosis
There are the reduction of cell adhesion molecule (such as E- calcium mucin) expression, cytokeratin cytoskeleton to be converted into vimentin
(Vimentin) with the feature etc. of mesenchymal cell on the cytoskeleton and form based on.Therefore, we pick on several
Endothelial cell marker (E-cadherin) and mesenchymal cell marker (Vimentin, SLUG, Snail1) are to study MTX
It is no to delay the generation of renal fibrosis by mediating EMT.Immunoblot results are shown, in uric acid nephropathy model group,
Vimentin, SLUG and Snail1 expression have significant up-regulation, and epithelial cell marker E-cadherin expression is suppressed, and is said
Bright EMT is one of the factor for mediating uric acid nephropathy renal fibrosis to occur.On the contrary, in treatment group, MTX inhibitor can more than
Epithelial cell marker is adjusted, mesenchymal cell marker is lowered, to hinder the generation (Fig. 4 A-4D) of EMT.ImmunohistochemistryResults Results
It has been shown that, Vimentin are hardly expressed in normal rat kidney, the high expression in renal tissue of uric acid nephropathy rats, and are injected
After MTX inhibitor, and the expression quantity (Fig. 4 E-4F) of Vimentin can be significantly inhibited.Result above prompt, MTX inhibitor pass through
EMT is hindered to delay the generation of uric acid nephropathy renal fibrosis.
6 MTX inhibitor of embodiment blocks the activation of TGF-β 1/Smad3 signal path in renal tissue of uric acid nephropathy rats
TGF-β 1/Smad3 signal path plays in regulation Stem Cell Activity, orga- nogenesis internal organs fibrosis focuses on
The effect wanted.After TGF-β and TGF-β II receptor (TGF-β RII) combine, reactivation raises TGF-β I receptor (TGF-β RI)
The receptor complex of dimeric forms is formed after combination, thus phosphorylation Smad3 albumen, and start downstream cascade pathway.It is immune
Blot results discovery MTX inhibitor can lower the expression quantity of TGF-β RI in renal tissue of uric acid nephropathy rats, and inhibit Smad3 phosphorus
The generation of acidification, but MTX has no effect on total Smad3 level (Fig. 5 A-5C).P- further is carried out to each group rat kidney
Smad3 histochemical staining, p-Smad3 expression quantity is less in normal kidney as the result is shown, in the kidney of uric acid nephropathy rat model
In dirty tissue, p-Smad3 high is expressed in nucleus, and after using MTX treatment, expression quantity is again significant to lower (Fig. 5 D-5E).
Result above prompt, MTX inhibitor block the activation of TGF-β 1/Smad3 signal path in renal tissue of uric acid nephropathy rats, thus
Inhibit the generation of renal fibrosis.
7 MTX inhibitor of embodiment blocks Notch1/Jagged-1 and Wnt1/ β-in renal tissue of uric acid nephropathy rats
The activation of catenin signal path
In addition to TGF-β 1/Smad3, we also study MTX for Notch1/Jagged-1 and Wnt1/ β-catenin this two
The important rush fibrosis signal path of item whether also inhibiting effect having the same.In normal rat kidney, basic detection is not
To the expression of Notch1 and Jagged-1, in renal tissue of uric acid nephropathy rats, the two is in high expression status, shows Notch1/
This signal path of Jagged-1 is activated, and promotes the generation of renal fibrosis.In treatment group, MTX inhibitor can it is significant under
The expression for adjusting Notch1 and Jagged-1, to hinder the activation (Fig. 6 A-6C) of Notch1/Jagged-1.Equally, it is urinating
In acid nephrotic rats kidney, Wnt1/ β-catenin signal path is in state of activation, and after injecting MTX inhibitor, the two
Expression decreased significantly (Fig. 6 D-6F).Result above prompt, MTX inhibitor block in renal tissue of uric acid nephropathy rats
The activation of Notch1/Jagged-1 and Wnt1/ β-catenin signal path, to inhibit the generation of renal fibrosis.
8 MTX of embodiment inhibits CD68 in renal tissue of uric acid nephropathy rats+Macrophage and CD3+The infiltration of lymphocyte
Existing research confirms during renal fibrosis, macrophage-mediated inflammatory reaction and cell mediated
It is immunoreacted important facilitation, therefore we study MTX inhibitor for CD68+Macrophage and CD3+Lymphocyte
The influence of infiltration.Immunohistochemistry and immunoblot results are shown, in renal tissue of uric acid nephropathy rats, CD68 expression quantity is significantly higher than
Normal rat kidney, and after using MTX treatment, expression and positive cell number have significant downward (Fig. 7 A-7D).
In addition, the cell of the CD3 positive is not expressed substantially in normal rat kidney, and in the kidney that uric acid nephropathy is damaged, sun
Property cell number increased significantly.In treatment group, MTX inhibitor can significantly lower the cell number (Fig. 7 E-7F) of the CD3 positive.This says
Bright MTX inhibitor is for CD68 in renal tissue of uric acid nephropathy rats+Macrophage and CD3+Lymphocyte is impregnated with certain inhibition
Effect.
Claims (4)
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| CN114736917A (en) * | 2022-03-27 | 2022-07-12 | 苏州大学 | Application of USP11 in inhibiting the degradation of cytokine IL6 |
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| WO2018064323A1 (en) * | 2016-09-28 | 2018-04-05 | Organovo, Inc. | Use of engineered renal tissues in assays |
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