CN111184856B - Application of small molecule polypeptide TP-7 in preparation of medicine for treating chronic kidney disease - Google Patents
Application of small molecule polypeptide TP-7 in preparation of medicine for treating chronic kidney disease Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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Abstract
The invention relates to a small molecule polypeptide TP-7 with an extracellular matrix tenascin C (TNC) kidney-promoting fiber antagonizing effect, in particular to an application of the small molecule polypeptide TP-7 in preparing a medicament for treating Chronic Kidney Disease (CKD). The amino acid sequence of the polypeptide TP-7 is shown as SEQ ID NO. 1. In a plurality of mouse kidney fibrosis models, the TP-7 has obvious curative effect, can obviously restore kidney function and inhibit the progress of kidney fibrosis, and has no obvious toxic or side effect. Therefore, the TP-7 can be prepared into a pharmaceutical preparation for treating chronic kidney diseases.
Description
Technical Field
The invention relates to application of a small molecular polypeptide, which is a partial peptide segment of Tenascin C (TNC) and is named as TP-7, in particular to application of the small molecular polypeptide TP-7 in preparation of chronic kidney disease medicines.
Background
Chronic kidney disease (Chronic Kidney Disease, CKD) refers to a disorder of chronic kidney structure and function caused by various causes. In recent years, the incidence and prevalence of the disease have increased dramatically, and the disease is a disease which is serious in harming human health after cardiovascular and cerebrovascular diseases, diabetes and malignant tumors. Worldwide, the population suffering from chronic kidney disease to end-stage kidney disease (End stage renal disease, ESRD) is increasing at a rate of 5-6% per year, and patients need dialysis treatment to sustain life, which is costly and places a heavy economic burden on the home and society. How to effectively inhibit and delay the occurrence and development of chronic kidney diseases is an urgent problem to be solved, but no effective prevention and treatment drugs and intervention means exist clinically at present.
Kidney fibrosis is a common pathway for all chronic kidney disease to progress to end-stage kidney disease and is mainly characterized by inflammatory cell infiltration, myofibroblast activation, extracellular matrix overexpression and deposition, disruption of the normal structure of the kidney, scar tissue formation, loss of tubular and capillary network, progressive loss of kidney function, and ultimately renal failure. Recent studies have found that in the above pathological processes, the abnormal expression of the extracellular matrix glycoprotein tenascin-C and the resulting pro-fibrotic microenvironment play a critical role in initiating and accelerating the development of renal fibrosis. How to inhibit the formation of a pro-fibrosis microenvironment, effectively block the regulation and control effect of tenascin C, and has very important theoretical significance and clinical value for inhibiting the occurrence and development of kidney fibrosis.
Tenascin C is a biological macromolecule with multiple functional domains that can bind to different molecules and perform multiple biological functions. According to the invention, the main functional domain FN III segment of tenascin C is cut to obtain 12 small molecule polypeptides, and animal in-vivo and in-vitro screening discovers that one small molecule polypeptide TP-7 consisting of 30 amino acids can effectively inhibit the kidney-promoting fibrosis effect of tenascin C and play a kidney protection function. As a brand new and protective small molecule polypeptide, TP-7 is expected to be used for developing clinical medicines for delaying or treating chronic kidney diseases.
Disclosure of Invention
The invention aims to provide a novel application of a small molecule polypeptide TP-7, in particular to an application of the small molecule polypeptide TP-7 in preparing a medicament for treating chronic kidney disease.
The amino acid sequence of the polypeptide TP-7 is shown as SEQ ID NO. 1.
SEQ ID NO.1：EWRNGKAAIDSYRIKYAPISGGDHAEVDVP。
According to a further feature of the use of the present invention, the chronic kidney disease is renal interstitial fibrosis, preferably that caused by unilateral ischemia/reperfusion of the kidney or ureteral obstruction.
According to a further feature of the use of the present invention, the medicament includes: a therapeutically effective dose of small molecule polypeptide TP-7 and pharmaceutically acceptable auxiliary materials.
Experiments prove that the small molecular polypeptide TP-7 has no obvious toxic or side effect in the mouse and animal experiments. The inventors performed two kidney disease model experiments on TP-7, unilateral ureteral obstruction (UUUO) and unilateral ischemia/reperfusion injury (UIRI). The experimental results show that: compared with a unilateral ureteral obstruction model group or a unilateral ischemia/reperfusion injury model group, the renal fibrosis degree of the tail vein injection TP-7 peptide group after modeling is obviously reduced, the deposition of interstitial collagen of the kidney is obviously reduced, and the expression amounts of smooth muscle actin alpha, fibronectin and tenascin C are obviously reduced, so that the TP-7 can effectively inhibit the occurrence and development of renal tissue fibrosis of mice.
In conclusion, TP-7 has the effects of inhibiting kidney tissue fibrosis and chronic kidney disease progression, has no obvious toxic or side effects, and can be used for preparing medicines for treating chronic kidney disease, in particular medicines for inhibiting renal interstitial fibrosis caused by pathological factors such as ischemia/reperfusion or ureteral obstruction, and the mechanism is by inhibiting renal interstitial fibroblast activation and tubular epithelial cell transdifferentiation caused by pathological factors such as ischemia/reperfusion or ureteral obstruction.
Drawings
FIGS. 1A to 1E show that TP-7 inhibits proliferation of fibroblast activation by tenascin C. Wherein, FIG. 1A is a diagram showing the detection of the expression change of fibroblast proliferation activating molecule by Western Blot (WB) method; FIGS. 1B to 1E are statistical results of the variation in the expression of each molecular protein in FIG. 1A.
FIGS. 2A-2B show that TP-7 can restore renal decline caused by unilateral ischemia/reperfusion (UIRI) injury to the kidneys of mice. Wherein, figure 2A shows biochemical method for detecting changes in serum creatinine levels in mice; FIG. 2B shows biochemical methods for detecting changes in serum urea nitrogen levels in mice.
FIGS. 3A-3B show that TP-7 inhibits fibrosis of kidney tissue resulting from unilateral ischemia/reperfusion injury in the kidney of mice. Wherein, FIG. 3A shows that the expression of extracellular matrix protein fibrinectin and alpha-SMA and collagen deposition of kidney tissue of mice caused by ischemia/reperfusion injury can be inhibited after TP-7 is injected into the mice by using a Pinus Margaret staining method and an immunohistochemical staining method; FIG. 3B further demonstrates that TP-7 inhibits the expression of extracellular matrix protein Fibronectin, TNC and alpha-SMA using Western immunoblotting.
FIGS. 4A-4B show that TP-7 inhibits cell proliferation caused by unilateral ischemia/reperfusion injury in the kidneys of mice. FIG. 4A shows that proliferation index Ki-67 and PCNA staining positive cells are increased after unilateral ischemia/reperfusion operation by adopting an immunohistochemical staining method, and the number of activated and proliferated fibroblasts is obviously reduced after TP7 is injected; FIG. 4B shows the detection of the expression change of proliferation-related gene c-fos and PCNA protein by Western immunoblotting.
Fig. 5A-5B show that TP-7 can inhibit renal tissue fibrosis caused by Unilateral Ureteral Obstruction (UUO) in mice. Wherein, FIG. 5A shows that after TP-7 is injected into a mouse body by adopting a Pinus Margaritifera staining method and an immunohistochemical staining method, the expression of mouse kidney tissue collagen deposition, extracellular matrix protein fibrinectin and alpha-SMA caused by ureteral obstruction can be inhibited; FIG. 5B further demonstrates that TP-7 inhibits the expression of extracellular matrix protein Fibronectin, TNC and α -SMA using Western immunoblotting.
FIGS. 6A-6B show that TP-7 inhibits cell proliferation caused by unilateral ureteral obstruction in mice. FIG. 6A shows that after unilateral ureteral obstruction operation, proliferation indexes Ki-67 and PCNA dye positive cells are increased, and after TP7 is injected, the number of activated and proliferated fibroblasts is obviously reduced by adopting an immunohistochemical staining method; FIG. 6B shows the detection of the expression change of proliferation-related gene c-fos and PCNA protein by Western immunoblotting.
Detailed Description
The invention will now be further illustrated by way of example only with reference to the accompanying drawings.
Embodiment one: sources of Small molecule polypeptide TP-7
According to the partial amino acid composition of the FNIII 4 domain of tenascin C (TNC), a small molecule polypeptide consisting of 30 amino acids, designated TP-7, was artificially synthesized by Hangzhou Dangang Biotechnology Co.
The amino acid sequence of the small molecule polypeptide is as follows:
SEQ ID NO.1：EWRNGKAAIDSYRIKYAPISGGDHAEVDVP。
the small molecule polypeptide TP-7 synthesized in this example was used in the experiments of the following examples by sequencing.
Embodiment two: in vitro experiments TP-7 inhibited proliferation of fibroblast activation by tenascin C (TNC)
1. Experimental materials
And (3) cells: rat kidney fibroblasts (NRK-49F).
Culture medium: DMEM/F12 (1:1) medium containing 10% FBS.
Culture conditions: 37 ℃ C. Contains 5% CO 2 An incubator.
2. Experimental treatment
Rat kidney fibroblasts (NRK-49F) were cultured at 1.5X10 6 After 1 day of cultivation in 6cm dishes, cultivation was performed with serum-free DMEM/F12 (1:1) mediumAfter culturing for 12 hours, cells are stimulated by adding tenascin C recombinant protein (50 ng/ml), and simultaneously small molecule polypeptide TP-7 (50 ng/ml) is added for incubation for 48 hours, protein is collected, and Western blot experiments are carried out.
3. Experimental results
As shown in FIGS. 1A-1E, the TP-7 small molecule polypeptide can effectively inhibit the activation proliferation of fibroblasts caused by tenascin C (TNC).
Embodiment III: kidney protecting effect of small molecule polypeptide TP-7 on mouse kidney unilateral ischemia/reperfusion model
1. Experimental animal
BABL/c mice, male, 8 weeks old, body weight 20-22g, SPF grade.
Animals are weighed and numbered by ear nails, 18 healthy mice with the weight of 20-22g are selected and randomly divided into 3 groups, and 6 mice in each group are respectively a sham operation group, a kidney unilateral ischemia/reperfusion model group and a kidney unilateral ischemia/reperfusion model group, and then TP-7 groups are injected.
2. Experimental grouping
1) Group of sham operations: after anesthetizing the mice with 1 ml/kg body weight of 1% sodium pentobarbital at 26 degrees celsius, a median incision in the abdomen was selected; after local disinfection, the skin, subcutaneous, muscular and peritoneal layers are incised layer by layer, and the left renal pedicel is found and then sutured layer by layer. After local disinfection, the ear nail marks are verified and placed in the corresponding squirrel cage.
2) Renal unilateral ischemia/reperfusion model group: and anesthesia and disinfection are carried out simultaneously. Skin, subcutaneous, muscular layer and peritoneum are cut layer by layer, surrounding connective tissue is slightly separated after a left renal pedicle is found, the left renal pedicle is rapidly blocked by using a nondestructive miniature arterial clip, the kidney is changed from bright red to purple black, a white ischemia ring appears around the renal pedicle, the blocking is successful, a mouse is placed on a metal heating plate at 37.5 ℃, a micro-wet gauze is covered on the abdomen for moisturizing, the arterial clip is gently removed after timing for 35 minutes, and renal blood flow reperfusion is observed, and the purple black gradually changes into bright red. And suturing layer by layer after the operation is finished. After local sterilization, the markers are verified and placed in the corresponding squirrel cage. Right side nephrectomy, with anesthesia and sterilization, was performed on day 10 post-operation. Making 1-2cm incision on the back of the right side, removing the envelope of the right kidney after finding out the right kidney, ligating the kidney pedicle of the right side, cutting the kidney of the right side, and suturing layer by layer after the operation is finished. After local sterilization, the markers are verified and placed in the corresponding squirrel cage.
3) Renal unilateral ischemia/reperfusion model injection TP-7 group: and anesthesia and disinfection are carried out simultaneously. Skin, subcutaneous, muscular layer and peritoneum are cut layer by layer, surrounding connective tissue is slightly separated after a left renal pedicle is found, the left renal pedicle is rapidly blocked by using a nondestructive miniature arterial clip, the kidney is changed from bright red to purple black, a white ischemia ring appears around the renal pedicle, the blocking is successful, a mouse is placed on a metal heating plate at 37.5 ℃, a micro-wet gauze is covered on the abdomen for moisturizing, the arterial clip is gently removed after timing for 35 minutes, and renal blood flow reperfusion is observed, and the purple black gradually changes into bright red. And suturing layer by layer after the operation is finished. After local sterilization, the markers are verified and placed in the corresponding squirrel cage.
3. Experimental procedure
The TP-7 powder was dissolved in 0.01mol/L glacial acetic acid to give a stock solution having a concentration of 100ug/ul, and diluted with sterile physiological saline to give a solution having a concentration of 10 ug/ul. Cage feeding of each component of the experiment. The sham group was only observed. The renal unilateral ischemia/reperfusion model group was given saline tail vein injection only. The group of injected TP-7 peptide was given a tail vein of TP-7 peptide starting on day 4 after unilateral ischemia/reperfusion operation, and continuously injected for 6 days, with a physiological saline tail vein containing TP-7 peptide of 50ug/kg body weight each day. Each group of mice was sacrificed on day 11 post-operation, left kidneys were taken, the tissues were fixed with 4% neutral buffered formaldehyde, the tissues were frozen with liquid nitrogen, and the frozen tissues were stored with OCT. Formaldehyde-fixed tissue is subjected to three-color dyeing and multi-index immunohistochemical dyeing after dehydration, embedding, slicing and tabletting, and the indexes comprise smooth muscle actin alpha (alpha-SMA), fibronectin (Fibronectin) and the like, and cell proliferation related indexes Ki-67, PCNA and the like. Protein was extracted after homogenization of frozen tissues, and the expression levels of smooth muscle actin alpha (alpha-SMA), fibronectin, tenascin C (TNC), C-fos, and PCNA were examined by immunoblotting (Western blot).
4. Experimental results
1) TP-7 reduces decline in renal function caused by unilateral ischemia/reperfusion injury of the kidney
The experimental results are shown in fig. 2A and 2B, and compared with the unilateral ischemia/reperfusion model group, the injection of TP-7 can obviously reduce serum creatinine and serum urea nitrogen rise caused by injury. 2) TP-7 alleviates renal fibrosis lesions in mice caused by unilateral ischemia/reperfusion
(I) Marsonian staining and immunohistochemical staining to detect collagen deposition level in kidney tissue
As shown in FIG. 3A, the results of the Pinus massoniana staining and immunohistochemical analysis show that TP-7 can reduce the deposition of renal interstitial collagen fibers of unilateral ischemia/reperfusion mice, namely, the deposition of renal interstitial collagen of mice injected with TP-7 is obviously less than that of unilateral ischemia/reperfusion model groups, and the expression level of Fibronectin and smooth muscle actin alpha (alpha-SMA) of mice injected with TP-7 is also obviously reduced.
(II) Western blot detection of expression of kidney tissue fibrosis molecules
As shown in FIG. 3B, western blot shows that the expression levels of Fibronectin (fibrinectin), smooth muscle actin alpha (alpha-SMA) and tenascin C (TNC) are obviously reduced in the TP-7 group of mice injected compared with the unilateral ischemia/reperfusion model group.
3) TP-7 inhibits proliferation activation of mouse renal interstitial fibroblasts by unilateral ischemia/reperfusion
(I) Immunohistochemical detection of the extent of proliferation of kidney fibroblasts
The experimental results are shown in FIG. 4A, and the immunohistochemical results show a significant decrease in Ki-67 and PCNA positive cells in the TP-7 injected mice compared to the unilateral ischemia/reperfusion model group.
(II) Western blot detection the results of the expression experiments of kidney tissue proliferation related molecules are shown in FIG. 4B, and Western blot shows that the expression levels of c-fos and PCNA are reduced in the mice injected with TP-7 group compared with the unilateral ischemia/reperfusion model group.
Embodiment four: inhibition effect of small molecule polypeptide TP-7 on kidney fibrosis of mouse unilateral ureteral obstruction model
1. Experimental animal
BALB/c mice, male, 8 weeks old, body weight 20-22g, SPF grade.
Animals were weighed and numbered first, and 15 healthy mice with a weight of 20-22g were selected and randomly divided into 3 groups of 5 animals each. Comprises a false operation group, a unilateral ureteral obstruction model group and a unilateral ureteral obstruction post-molding injection TP-7 group.
2. Experimental grouping
1) Group of sham operations: after anesthetizing a mouse with 1 ml/kg body weight of 1% sodium pentobarbital at 26 degrees celsius, selecting the abdomen center as an incision; after local disinfection, the skin, subcutaneous, muscular and peritoneal layers are incised layer by layer, and after the left ureter is found, the suture is performed layer by layer. After local disinfection, the ear nail marks are checked and placed in the corresponding squirrel cage.
2) Unilateral ureteral obstruction model group: and anesthesia and disinfection are carried out simultaneously. Skin, subcutaneous, muscular and peritoneal layers were incised layer by layer, and after finding the left ureter, 1/3 of the segment above the ureter was ligated and sutured layer by layer. After local disinfection, the ear nail marks are checked and placed in the corresponding squirrel cage.
3) TP-7 group injection after unilateral ureteral obstruction molding: and anesthesia and disinfection are carried out simultaneously. Skin, subcutaneous, muscular and peritoneal layers were incised layer by layer, and after finding the left ureter, 1/3 segment of the ureter was ligated and sutured layer by layer. After local disinfection, the ear nail marks are checked and placed in the corresponding squirrel cage.
3. Experimental procedure
The TP-7 powder was dissolved in 0.01mol/L glacial acetic acid to give a stock solution having a concentration of 100ug/ul, and diluted with sterile physiological saline to give a solution having a concentration of 10 ug/ul. Cage feeding of each component of the experiment. The sham group was only observed. The unilateral ureteral obstruction model group was given saline tail vein injection only. Groups injected with TP-7 peptide groups were injected with TP-7 peptide from the tail vein on day 4 after unilateral ureteral obstruction surgery for 6 consecutive days with tail vein injection of physiological saline containing TP-7 peptide of 50ug/kg body weight each day. After 10 days of raising under the same raising condition, each group of mice is sacrificed, the left kidney is taken, and 4% neutral buffer formaldehyde is used for fixing tissues, liquid nitrogen freezing tissues and OCT (optical coherence tomography) preserving frozen tissues respectively. Formaldehyde-fixed tissue is subjected to three-color masson staining and multi-index immunohistochemical staining after dehydration, embedding, slicing and tabletting, and the indexes comprise fibrotic indexes such as smooth muscle actin alpha (alpha-SMA), fibronectin and the like. Protein was extracted after homogenization of frozen tissues, and the expression levels of smooth muscle actin alpha (alpha-SMA), fibronectin, tenascin C (TNC), C-fos, and PCNA were examined by immunoblotting (Western blot).
4. Experimental results
1) TP-7 alleviates mouse kidney fibrosis lesions caused by unilateral ureteral obstruction
(I) Marsonian staining and immunohistochemical staining to detect collagen deposition level in kidney tissue
As shown in FIG. 5A, the results of the Pinus massoniana staining show that TP-7 can reduce the deposition of renal interstitial collagen fibers in mice with unilateral ureteral obstruction, i.e. the deposition of renal interstitial collagen in mice injected with TP-7 is obviously less than that in a control group with unilateral ureteral obstruction model. The immunohistochemical results all show that compared with a unilateral ureteral obstruction model group, the expression level of Fibronectin and smooth muscle actin alpha (alpha-SMA) of the TP-7 group mice is also obviously reduced.
(II) Western blot detection of expression of kidney tissue fibrosis molecules
The experimental results are shown in FIG. 5B, and Western blot results show that the expression levels of mouse Fibronectin (Fibronectin), smooth muscle actin alpha (alpha-SMA) and tenascin C (TNC) of TP-7 groups are obviously reduced compared with a unilateral ureteral obstruction model group.
2) TP-7 inhibits proliferation and activation of mouse renal interstitial fibroblasts caused by unilateral ureteral obstruction
(I) Immunohistochemical detection of the extent of proliferation of kidney fibroblasts
The experimental results are shown in FIG. 6A, and the immunohistochemical results show that the Ki-67 and PCNA positive cells of the mice injected with TP-7 group are obviously reduced compared with the control group of the unilateral ureteral obstruction model.
The result of the expression experiment of the kidney tissue proliferation related molecules detected by Western blot is shown in FIG. 6B, and the Western blot shows that the expression level of c-fos and PCNA of the mice injected with TP-7 groups is reduced compared with a control group of a unilateral ureteral obstruction model.
In conclusion, in vivo and in vitro experiments prove that TP-7 can inhibit proliferation and activation of kidney fibroblasts; can improve renal function decline caused by unilateral renal ischemia/reperfusion injury of mice; has remarkable inhibiting effect on renal fibrosis caused by unilateral renal ischemia/reperfusion injury and unilateral ureteral obstruction of mice. Therefore, it is considered that TP-7 can be a novel drug effective in improving renal function and inhibiting the progress of renal fibrosis due to chronic kidney disease.
SEQUENCE LISTING
<110> southern Hospital at southern medical university
Application of <120> small molecule polypeptide TP-7 in preparation of medicine for treating chronic kidney disease
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> PRT
<213> Synthesis
<400> 1
Glu Trp Arg Asn Gly Lys Ala Ala Ile Asp Ser Tyr Arg Ile Lys Tyr
1 5 10 15
Ala Pro Ile Ser Gly Gly Asp His Ala Glu Val Asp Val Pro
20 25 30
Claims (2)
1. The application of a small molecule polypeptide TP-7 in preparing a medicament for treating renal interstitial fibrosis caused by ischemia reperfusion or ureteral obstruction, wherein the amino acid sequence of the polypeptide TP-7 is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein said medicament comprises: a therapeutically effective dose of small molecule polypeptide TP-7 and pharmaceutically acceptable auxiliary materials.
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