CN111184856A - Application of small molecular polypeptide TP-7 in preparation of medicine for treating chronic kidney disease - Google Patents
Application of small molecular polypeptide TP-7 in preparation of medicine for treating chronic kidney disease Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
Abstract
The invention relates to a small molecule polypeptide TP-7 with an effect of antagonizing extracellular matrix tenascin C (TNC) and promoting renal fibers, in particular to application of the small molecule polypeptide TP-7 in preparing a medicament for treating chronic renal disease (CKD). The amino acid sequence of the polypeptide TP-7 is shown as SEQ ID NO. 1. In a plurality of mouse renal fibrosis models, TP-7 has obvious curative effect, can obviously recover renal function and inhibit the renal fibrosis development, and has no obvious toxic or side effect. Therefore, the TP-7 can be prepared into a medicinal preparation for treating chronic kidney diseases.
Description
Technical Field
The invention relates to application of a small molecular polypeptide, wherein the polypeptide is a partial peptide segment of Tenascin C (Tenascon C, TNC), is named as TP-7, and particularly relates to application of the small molecular polypeptide TP-7 in preparation of a chronic renal disease drug.
Background
Chronic Kidney Disease (CKD) refers to a Chronic disorder of the Kidney structure and function caused by various causes. In recent years, the incidence and prevalence of the disease are rapidly increasing, and the disease seriously harms human health after cardiovascular and cerebrovascular diseases, diabetes and malignant tumors. Worldwide, the population of chronic kidney disease progressing to End stage kidney disease (ESRD) is increasing at a rate of 5-6% per year, and patients need dialysis treatment to maintain life, which costs a lot and causes heavy economic burden to families and society. How to effectively inhibit and delay the occurrence and development of chronic kidney diseases is a problem which needs to be urgently solved, but no effective medicine and intervention means for preventing and treating the chronic kidney diseases exist in the clinical practice at present.
Kidney fibrosis is a common pathway for all chronic kidney diseases to progress to end-stage kidney diseases, and is mainly characterized by inflammatory cell infiltration, myofibroblast activation, extracellular matrix overexpression and deposition, destruction of normal kidney structures, formation of scar tissues, loss of renal tubules and capillary networks, progressive loss of renal function, and finally renal failure. In the pathological process, the abnormal expression of the extracellular matrix glycoprotein tenascin C and the formed fibrosis-promoting microenvironment play a crucial role in the initiation and acceleration of the development of renal fibrosis. How to inhibit the formation of a fibrosis-promoting microenvironment and effectively block the regulation and control effect of tenascin C has very important theoretical significance and clinical value for inhibiting the occurrence and development of renal fibrosis.
tenascin-C is a biological macromolecule, has multiple functional structural domains, can be combined with different molecules, and plays multiple biological functions. The invention cuts FN III segment of main functional structural domain of tenascin C to obtain 12 small molecular polypeptides, and animal in vitro and animal in vivo screening finds that one small molecular polypeptide TP-7 consisting of 30 amino acids can effectively inhibit the function of promoting renal fibrosis of tenascin C and play a role of renal protection. As a brand-new protective small molecular 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 new application of a small molecular polypeptide TP-7, in particular to an application of the small molecular polypeptide TP-7 in preparing a medicament for treating chronic kidney diseases.
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 invention, the chronic renal disease is renal interstitial fibrosis, preferably renal interstitial fibrosis caused by unilateral ischemia/reperfusion or ureteral obstruction.
According to a further feature of the use of the present invention, the medicament comprises: a therapeutically effective dose of small molecule polypeptide TP-7 and pharmaceutically acceptable excipients.
The experiment result shows that compared with a Unilateral Ureteral Obstruction (UUO) or unilateral ischemia/reperfusion injury model group, the kidney fibrosis degree of a tail vein injection TP-7 peptide segment group is obviously reduced after the model is made, the interstitial collagen deposition of the kidney is obviously reduced, the expression quantity of smooth muscle actin α, fibronectin and tendon protein C is obviously reduced, and the TP-7 can effectively inhibit the generation and development of mouse kidney tissue fibrosis.
In summary, TP-7 has the functions of inhibiting the kidney tissue fibrosis and the chronic kidney disease progression, and has no obvious toxic or side effect, so the TP-7 can be used for preparing the medicine for treating the chronic kidney disease, in particular the medicine for inhibiting the renal interstitial fibrosis caused by pathological factors such as ischemia/reperfusion or ureteral obstruction, and the mechanism is the activation of renal interstitial fibroblasts and the transdifferentiation of renal tubular epithelial cells caused by pathological factors such as ischemia/reperfusion or ureteral obstruction.
Drawings
FIGS. 1A to 1E show that TP-7 inhibits tenosin C-induced fibroblast activation proliferation. Wherein, FIG. 1A is a method for detecting the expression change of fibroblast proliferation activating molecules by adopting a protein immunoblotting (Western Blot, WB) method; FIGS. 1B to 1E are the results of statistics of the expression changes of the respective molecular proteins in FIG. 1A.
FIGS. 2A-2B show that TP-7 can restore reduced renal function in mice caused by unilateral ischemia/reperfusion (UIRI) injury of the kidney. Wherein, FIG. 2A shows the biochemical method for detecting the serum creatinine level change of the mouse; FIG. 2B shows the biochemical detection of changes in serum urea nitrogen levels in mice.
FIGS. 3A to 3B show that TP-7 can inhibit the fibrosis of kidney tissue caused by unilateral ischemia/reperfusion injury of mouse kidney, wherein, FIG. 3A is the method of Masson's staining and immunohistochemical staining to confirm that the collagen deposition, extracellular matrix protein fibrinectin and α -SMA can be inhibited in mouse kidney tissue caused by ischemia/reperfusion injury after the mouse is injected with TP-7 in vivo, and FIG. 3B is the method of Western blotting to further confirm that TP-7 can inhibit the expression of extracellular matrix protein fibrinectin, TNC and α -SMA.
FIGS. 4A-4B show that TP-7 inhibits cell proliferation in mice caused by unilateral ischemia/reperfusion injury of the kidney. FIG. 4A shows that proliferation indexes Ki-67 and PCNA staining positive cells are increased after unilateral ischemia/reperfusion surgery, and the number of activated and proliferated fibroblasts is obviously reduced after TP7 injection; FIG. 4B shows the detection of the expression changes of the proliferation-associated genes c-fos and PCNA protein by Western blotting.
FIGS. 5A to 5B show that TP-7 can inhibit fibrosis of kidney tissue caused by Unilateral Ureteral Obstruction (UUO) in mice, wherein FIG. 5A shows that collagen deposition and expression of extracellular matrix proteins, namely, fibrinectin and α -SMA are inhibited in the kidney tissue of the mice caused by ureteral obstruction by using a Masson staining and immunohistochemical staining method after mice are injected with TP-7 in vivo, and FIG. 5B shows that TP-7 can inhibit expression of extracellular matrix proteins, namely, fibrinectin, TNC and α -SMA by using a Western blotting method.
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 positive cells are increased, and the number of activated and proliferated fibroblasts is obviously reduced after TP7 is injected; FIG. 6B shows the detection of the expression changes of the proliferation-associated genes c-fos and PCNA protein by Western blotting.
Detailed Description
The invention will now be further described, by way of example only, with reference to the accompanying drawings.
The first embodiment is as follows: source of small molecule polypeptide TP-7
According to the FN III 4 domain of tenascin C (TNC), a small molecule polypeptide consisting of 30 amino acids was artificially synthesized by Hangzhou Dangang Biotechnology limited and named as TP-7.
The amino acid sequence of the small molecular polypeptide is as follows:
SEQ ID NO.1：EWRNGKAAIDSYRIKYAPISGGDHAEVDVP。
the synthesized small molecule polypeptide TP-7 of this example was used in the experiments of the following examples by sequencing.
Example two: TP-7 inhibits activation and proliferation of fibroblasts caused by tenascin C (TNC) in vitro experiments
1. Experimental Material
Cell: rat kidney fibroblasts (NRK-49F).
Culture medium: DMEM/F12(1:1) medium containing 10% FBS.
The culture conditions are as follows: 5% CO at 37 ℃2An incubator.
2. Experimental treatment
Rat kidney fibroblasts (NRK-49F) were cultured at 1.5X 106After being planted in a 6cm culture dish and cultured for 1 day, the culture dish is cultured for 12 hours by using a serum-free DMEM/F12(1:1) culture medium, tenonin C recombinant protein (50ng/ml) is added to stimulate cells, meanwhile, micromolecular polypeptide TP-7(50ng/ml) is added to carry out incubation for 48 hours, and then protein is collected for Western blot experiment.
3. Results of the experiment
As shown in fig. 1A to fig. 1E, TP-7 small-molecule polypeptide can effectively inhibit activation and proliferation of fibroblasts caused by tenascin c (tnc).
Example three: kidney protection effect of small molecular polypeptide TP-7 on mouse kidney unilateral ischemia/reperfusion model
1. Laboratory animal
BABL/c mice, male, 8 weeks old, body weight 20-22g, SPF grade.
Weighing animals, numbering by ear nails, selecting 18 healthy mice with the weight of 20-22g, randomly dividing the mice into 3 groups of 6 mice each, and injecting TP-7 groups after a pseudo-operation group, a kidney unilateral ischemia/reperfusion model group and a kidney unilateral ischemia/reperfusion model are respectively selected.
2. Experiment grouping
1) The sham operation group: after anesthetizing a mouse with 1 ml/kg body weight of 1% sodium pentobarbital at 26 ℃, selecting a median abdominal incision; after topical sterilization, the skin, subcutaneous, muscular and peritoneal membranes were dissected layer by layer and the left renal pedicle was found and then sutured layer by layer. After local sterilization, the ear tag was verified and placed in the corresponding squirrel cage.
2) Renal unilateral ischemia/reperfusion model group: the anesthesia and disinfection are performed as above. The skin, subcutaneous tissue, muscular layer and peritoneum are incised layer by layer, the surrounding connective tissue is slightly separated after the left renal pedicle is found, the left renal pedicle is quickly blocked by a non-invasive micro artery clamp, the kidney is changed from bright red to purple black, a white ischemic ring appears around the renal pedicle to indicate successful blocking, a mouse is placed on a metal heating plate at the temperature of 37.5 ℃, the abdomen is covered with a piece of slightly wet gauze to preserve moisture, the artery clamp is gently taken down after the time is counted for 35 minutes, the renal blood flow is observed to be re-perfused, and the black purple color is gradually changed into bright red. And suturing layer by layer after the operation is finished. After partial sterilization, the markers were verified and placed in the corresponding cages. On day 10 after surgery, the right kidney was excised, anesthetized and disinfected as above. Making an incision of 1-2cm on the back of the right side, removing the envelope after finding the right kidney, ligating the pedicle of the right kidney, cutting off the right kidney, and suturing layer by layer after the operation is finished. After partial sterilization, the markers were verified and placed in the corresponding cages.
3) Kidney unilateral ischemia/reperfusion model TP-7 group was injected: the anesthesia and disinfection are performed as above. The skin, subcutaneous tissue, muscular layer and peritoneum are incised layer by layer, the surrounding connective tissue is slightly separated after the left renal pedicle is found, the left renal pedicle is quickly blocked by a non-invasive micro artery clamp, the kidney is changed from bright red to purple black, a white ischemic ring appears around the renal pedicle to indicate successful blocking, a mouse is placed on a metal heating plate at the temperature of 37.5 ℃, the abdomen is covered with a piece of slightly wet gauze to preserve moisture, the artery clamp is gently taken down after the time is counted for 35 minutes, the renal blood flow is observed to be re-perfused, and the black purple color is gradually changed into bright red. And suturing layer by layer after the operation is finished. After partial sterilization, the markers were verified and placed in the corresponding cages.
3. Procedure of experiment
TP-7 powder was dissolved in 0.01mol/L glacial acetic acid to 100ug/ul stock solution, and diluted with sterile physiological saline to 10ug/ul solution, experimental groups were housed in cages, sham surgery groups were observed, kidney single-side ischemia/reperfusion model groups were given physiological saline tail vein injection only, group injected with TP-7 peptide was injected in tail vein starting from day 4 after single-side ischemia/reperfusion surgery, TP-7 peptide was injected continuously for 6 days, mice were sacrificed on day 11 after surgery, left-side kidneys were taken, 4% neutral buffered formalin-fixed tissue, liquid nitrogen frozen tissue was used, OCT frozen tissue was stored, formalin-fixed tissue was dehydrated, embedded, sliced, stained with masson trichrome and multi-index immunohistochemical staining, these indices include actin indices, fibrosis index α (α - α), Fibronectin (sheet-production), Fibronectin (SMA), and other related cell proliferation indices (tnon protein) were extracted by Western blot, (α) assay, (α).
4. Results of the experiment
1) TP-7 relieves renal function decline caused by unilateral renal ischemia/reperfusion injury
The experimental results are shown in fig. 2A and 2B, and compared with the unilateral ischemia/reperfusion model group, the increase of serum creatinine and serum urea nitrogen caused by injury can be obviously reduced after injection of TP-7. 2) TP-7 alleviates mouse renal fibrotic lesions caused by unilateral ischemia/reperfusion
(I) Method for detecting collagen deposition level of kidney tissue by using masson staining and immunohistochemical staining
As shown in FIG. 3A, the results of masson staining and immunohistochemistry show that TP-7 can reduce renal interstitial collagen fiber deposition in mice with unilateral ischemia/reperfusion, i.e., the renal interstitial collagen deposition in mice injected with TP-7 is significantly less than that in mice injected with unilateral ischemia/reperfusion, and the expression levels of Fibronectin (Fibronectin) and smooth muscle actin α (α -SMA) in mice injected with TP-7 are also significantly reduced.
(II) Western blot detection of expression of kidney tissue fibrosis molecules
The experimental results are shown in FIG. 3B, and Western blot shows that the expression levels of Fibronectin (Fibronectin), smooth muscle actin α (α -SMA) and tenascin C (TNC) are obviously reduced in the TP-7 group mice compared with the unilateral ischemia/reperfusion model group.
3) TP-7 inhibits proliferation and activation of mouse renal interstitial fibroblasts caused by unilateral ischemia/reperfusion
(I) Immunohistochemical detection of degree of proliferation of renal fibroblasts
The experimental results are shown in FIG. 4A, and the immunohistochemical results show that Ki-67 and PCNA positive cells are significantly reduced in mice injected with TP-7 compared to the unilateral ischemia/reperfusion model group.
(II) Western blot detection of expression experiment results of kidney tissue proliferation related molecules as shown in FIG. 4B, Western blot shows that c-fos and PCNA expression levels of mice injected with TP-7 are reduced compared with a unilateral ischemia/reperfusion model group.
Example four: inhibition effect of small molecular polypeptide TP-7 on mouse unilateral ureteral obstruction model kidney fibrosis
1. Laboratory animal
BALB/c mice, male, 8 weeks old, body weight 20-22g, SPF grade.
Animals were weighed and numbered first, and 15 healthy mice weighing 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 injection TP-7 group after model making.
2. Experiment grouping
1) The sham operation group: after the mice were anesthetized with 1 ml/kg body weight of 1% sodium pentobarbital at 26 ℃, the midline of the abdomen was selected as the incision; after local disinfection, the skin, subcutaneous, muscular and peritoneal membranes are incised layer by layer, and the left ureter is discovered and then sutured layer by layer. After local disinfection, the ear nail mark is checked and placed in the corresponding squirrel cage.
2) Unilateral ureteral obstruction model group: the anesthesia and disinfection are performed as above. The skin, subcutaneous layer, muscular layer and peritoneum were incised layer by layer, and after finding the left ureter, the left ureter was ligated to section 1/3 of the ureter and sutured layer by layer. After local disinfection, the ear nail mark is checked and placed in the corresponding squirrel cage.
3) Injecting TP-7 group after the unilateral ureteral obstruction is modeled: the anesthesia and disinfection are performed as above. The skin, subcutaneous layer, muscular layer and peritoneum are incised layer by layer, and after finding the left ureter, the left ureter is ligated with 1/3 segments of ureter, and then sutured layer by layer. After local disinfection, the ear nail mark is checked and placed in the corresponding squirrel cage.
3. Procedure of experiment
TP-7 powder was dissolved in 0.01mol/L glacial acetic acid to 100ug/ul stock solution, diluted with sterile physiological saline to 10ug/ul solution, experimental groups were housed in cages, sham surgery groups were observed only, single-sided ureteral obstruction model groups were given physiological saline tail vein injection only, group injection of TP-7 peptide was started on day 4 after single-sided ureteral obstruction surgery, TP-7 peptide was injected continuously for 6 days, physiological saline tail vein injection containing 50ug/kg body weight of TP-7 peptide was performed every day, mice were sacrificed after 10 days of same rearing, left kidney was selected, 4% neutral buffered formaldehyde-fixed tissue, liquid nitrogen frozen tissue and OCT-preserved frozen tissue, formaldehyde-fixed tissue was dehydrated, embedded, sliced, and stained with masson trichrome and multi-index immunohistochemical staining, actin indices including fibrotic index smooth muscle α (α - α), Fibronectin (SMA) extraction, Fibronectin (fibrin) extraction, and Fibronectin homogenization (TNon) using fibrin-C (TNF-protein) homogenization (homogeneous blotting), and Fibronectin (TNF-3) expression.
4. Results of the experiment
1) TP-7 alleviating mouse renal fibrosis lesion caused by unilateral ureteral obstruction
(I) Method for detecting collagen deposition level of kidney tissue by using masson staining and immunohistochemical staining
As shown in FIG. 5A, the result of masson staining shows that TP-7 can reduce the deposition of renal interstitial collagen fibers of mice with unilateral ureteral obstruction, i.e., the deposition of renal interstitial collagen of the mice injected with TP-7 is obviously less than that of the mice injected with model of unilateral ureteral obstruction, and the immunohistochemical result shows that the expression levels of Fibronectin (Fibronectin) and smooth muscle actin α (α -SMA) of the mice injected with TP-7 are also obviously reduced compared with the model of unilateral ureteral obstruction.
(II) Western blot detection of expression of kidney tissue fibrosis molecules
The experimental results are shown in FIG. 5B, and the Western blot results show that the expression levels of Fibronectin (Fibronectin), smooth muscle actin α (α -SMA) and tenascin C (TNC) of mice injected with TP-7 group are obviously reduced compared with the 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 degree of proliferation of renal fibroblasts
The experimental results are shown in fig. 6A, and the immunohistochemical results show that Ki-67 and PCNA positive cells of mice injected with TP-7 group are obviously reduced compared with the control group of the unilateral ureteral obstruction model.
(II) Western blot detection results of expression of kidney tissue proliferation-related molecules are shown in FIG. 6B, and Western blot shows that c-fos and PCNA expression levels of mice injected with TP-7 are reduced compared with a unilateral ureteral obstruction model control group.
In conclusion, in vivo and in vitro experiments prove that TP-7 can inhibit proliferation and activation of kidney fibroblasts; can improve the kidney function reduction caused by unilateral kidney ischemia/reperfusion injury of mice; has obvious inhibiting effect on renal fibrosis caused by unilateral renal ischemia/reperfusion injury and unilateral ureteral obstruction of mice. Therefore, TP-7 is considered to be a novel drug effective in improving renal function and inhibiting the progress of renal fibrosis in chronic renal disease.
SEQUENCE LISTING
<110> southern hospital of southern medical university
Application of <120> small molecular 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> Artificial 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
2025 30
Claims (4)
1. The application of the small molecular polypeptide TP-7 in the preparation of the medicine for treating chronic kidney diseases is disclosed, wherein the amino acid sequence of the polypeptide TP-7 is shown as SEQ ID NO. 1.
2. Use according to claim 1, characterized in that: the chronic kidney disease is renal interstitial fibrosis.
3. Use according to claim 2, characterized in that: the chronic kidney disease is renal interstitial fibrosis caused by ischemia/reperfusion or ureteral obstruction.
4. The use of claim 1, wherein the medicament comprises: a therapeutically effective dose of small molecule polypeptide TP-7 and pharmaceutically acceptable excipients.
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| CN115068480A (en) * | 2022-08-09 | 2022-09-20 | 郑州大学第一附属医院 | Application of cyclin 42 small-molecule inhibitor in preparation of medicine for treating chronic kidney disease |
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