阅读说明：本技术 C16在制备防治慢性肾间质纤维化药物中的用途 (Application of C16 in preparing medicine for preventing and treating chronic renal interstitial fibrosis ) 是由 游然 贾占军 张玥 张爱华 李延伟 周维 姜彧滕 于晓文 于 2021-03-23 设计创作，主要内容包括：本发明公开了C16在治疗慢性肾间质纤维化中的应用,具体涉及C16通过抑制肾小管上皮细胞及成纤维细胞纤维化来治疗慢性肾间质纤维化的应用。C16能减轻慢性肾间质纤维化小鼠模型的病理变化,降低小鼠肾脏组织纤维化因子的表达水平,保护纤维化模型中的肾小管上皮细胞,逆转其转分化作用,并抑制肾成纤维细胞活化。(The invention discloses an application of C16 in treating chronic renal interstitial fibrosis, and particularly relates to an application of C16 in treating chronic renal interstitial fibrosis by inhibiting renal tubular epithelial cell and fibroblast fibrosis. C16 can relieve pathological changes of chronic renal interstitial fibrosis mouse model, reduce expression level of mouse kidney tissue fibrosis factor, protect renal tubular epithelial cells in the fibrosis model, reverse its transdifferentiation effect, and inhibit renal fibroblast activation.)

1. The application of a specific Protein Kinase (PKR) inhibitor C16 shown as a formula I in preparing a medicament for preventing and treating chronic renal fibrosis.

2. The use of claim 1, wherein the C16 has effects of preventing and treating chronic renal fibrosis by protecting renal tubular epithelial cells and inhibiting renal interstitial fibroblast activation in chronic renal fibrosis.

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of a compound C16 in preparation of a medicine for preventing and treating chronic renal interstitial fibrosis.

Background

Chronic Kidney Disease (CKD) is a worldwide public health problem with an incidence of between 8% and 16% based on epidemiological investigations in different regions. In our country, about 1.2 million people suffer from CKD. Although the pathogenesis and intervention strategy of CKD are widely explored for decades, effective means for preventing and treating chronic kidney diseases are still lacked so far, and a considerable number of CKD patients finally progress to end-stage kidney diseases, need expensive kidney replacement therapy and bring great burden to families and society. Although the etiology of chronic kidney diseases is complex, the final common pathological development direction is tubulointerstitial fibrosis, and the fibrosis degree is also an important index influencing the prognosis of CKD. The search for effective drugs for blocking the occurrence and the progression of tubulointerstitial fibrosis is a problem to be solved urgently in the field of kidney diseases at present.

Renal interstitial fibrosis is a complex dynamic process in which activation of renal interstitial fibroblasts and injury and fibrosis of renal tubular epithelial cells are central events in the development of renal interstitial fibrosis. Renal interstitial fibroblasts, when activated, produce large amounts of extracellular matrix, directly resulting in renal interstitial fibrosis. Tubular Epithelial Cells (RTECs), which are kidney resident cells with vigorous metabolic activity and potential proliferative capacity, are highly susceptible to functional and structural damage under disease conditions. It is currently believed that non-lethally damaged RTECs can undergo maladaptive repair processes (maladaptive repair) in which proliferation is activated, extracellular matrix proteins are synthesized, and various chemokines and growth factors are secreted. Abnormally activated tubular epithelial cells are therefore a key link in mediating renal interstitial fibrosis and are a major cause of impaired renal function.

The chemical structure of C16 is:

c16(Imoxin, CAS 608512-97-6) is an inhibitor of RNA-dependent protein kinase (PKR, also known as Eif2ak2), whose inhibition of PKR is mediated through ATP binding sites. C16 may function to inhibit apoptosis by inhibiting the apoptotic PKR/eIF2a signaling pathway. The known application of C16 includes neuroprotective effect, anti-inflammatory effect and antitumor effect. No research reports about the role of C16 in renal fibrosis, so that the role of C16 in renal interstitial fibrosis in chronic renal diseases needs to be verified by systematic animal experiments and cell experiments. The research proves that C16 can inhibit the activation of renal interstitial fibroblasts and relieve the fibrosis of renal tubular epithelial cells to prevent and treat chronic renal fibrosis from in vivo and in vitro experiments. There is no report of the use of C16 in the treatment of chronic renal interstitial fibrosis.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides the application of C16 in the preparation of the medicine for preventing and treating chronic renal fibrosis, and the application solves the technical problems that the pharmacological activity of C16 in renal diseases is not found in the prior art, and the medicine is lack of enough proper medicine for inhibiting the activation of renal interstitial fibroblasts, reversing the fibrosis of renal tubular epithelial cells and treating chronic renal fibrosis.

In particular to the application of C16 in preparing a medicament for inhibiting renal interstitial fibrosis and renal tubule protection action of a patient with chronic renal fibrosis, thereby providing a new candidate compound for treating the chronic renal fibrosis.

The above-mentioned application, specifically, C16 can significantly reduce renal fibrosis pathology of chronic renal fibrosis patient, inhibit fibroblast activation, and reverse renal tubular fibrosis.

In addition, C16 can be prepared into a composition of a medicine for preventing and treating chronic renal fibrosis.

We used C16 on a mouse model of renal fibrosis induced by the unilateral ureteral ligation (UUO) model to study its protective effects on renal interstitial fibrosis.

The results show that the intervention treatment of the chronic renal fibrosis in the UUO model by using C16 can obviously reduce the renal interstitial fibrosis of the mice.

C16 can inhibit the activation of renal interstitial fibroblasts, and reduce the fibrosis process of renal tubular epithelial cells under the stimulation of tissue growth factor (TGF-beta 1), thereby reducing renal interstitial fibrosis and improving the pathological damage of kidney.

Our discovery will most likely provide a new effective therapeutic drug for preventing and treating chronic renal fibrosis.

Therefore, the C16 has obvious effects on inhibiting the activation of renal interstitial fibroblasts and protecting renal tubular epithelial cells, and particularly relieving the renal interstitial fibrosis degree of chronic renal fibrosis patients.

Therefore, the application of C16 in preparing the medicine for preventing and treating chronic renal fibrosis is provided; the use of C16 in the manufacture of a medicament for the protection of renal tubular epithelial cells; use of C16 in the manufacture of a medicament for inhibiting the activation of renal interstitial fibroblasts.

Drawings

Figure 1 shows that the therapeutic dose of C16 has no toxic side effects on mice.

FIG. 1A shows that C16 does not affect the concentration of serum urea nitrogen, a normal mouse renal function marker, at an in vivo therapeutic dose of 300 μ g/kg;

FIG. 1B shows that C16 does not affect the concentration of serum creatinine, a normal mouse renal function marker, at a therapeutic dose of 300 μ g/kg in vivo;

FIG. 1C shows that C16 does not affect the concentration of serum glutamate pyruvate transaminase, a normal mouse liver function marker, at a therapeutic dose of 300 μ g/kg in vivo;

FIG. 1D shows that C16 did not affect the serum glutamic-oxaloacetic transaminase concentration of liver and heart function markers in normal mice at the in vivo therapeutic dose of 300. mu.g/kg;

FIG. 1E shows that C16 does not affect the concentration of serum lactate dehydrogenase, a marker of normal mouse cardiac function, at an in vivo therapeutic dose of 300. mu.g/kg;

FIG. 1F shows that C16 does not affect the serum creatine kinase concentration, a marker of normal mouse cardiac function, at an in vivo therapeutic dose of 300 μ g/kg;

figure 2 shows that C16 improves pathology in the mouse UUO model; masson staining was shown to indicate that C16 reduced renal interstitial fibrosis in the UUO model, with a more pronounced therapeutic effect at the 300 μ g/kg dose than at the 100 μ g/kg dose;

figure 3 shows that C16 decreased the expression level of an indicator of fibrosis in kidney tissue in the mouse UUO model.

Figure 3A shows that C16 dose-dependently decreased the protein level of fibrauctin, an index of increased fibrosis in the UUO model;

fig. 3B shows that C16 significantly decreased mRNA expression levels of fibrosis index fibrinectin in the UUO model with a dose-dependent decrease;

fig. 3C shows that C16 significantly reduced mRNA expression levels of fibrosis index α -SMA in the UUO model and presented dose-dependence;

fig. 3D shows that C16 significantly reduced mRNA expression levels of the fibrosis index Collegen I in the UUO model and presented a dose dependence.

FIG. 4 shows that C16 inhibits TGF-. beta.induced fibrosis of tubular epithelial cells in vitro cultured RTEC mice in the tubular epithelial cell line.

FIG. 4A Western blot results show that C16 significantly reduced the protein level of TGF- β -induced renal tubular epithelial cell expression fibrosis index fibrinectin;

figure 4B qPCR results show that C16 significantly reduced TGF- β induced mRNA levels of renal tubular epithelial cell expression fibrosis index Collagen I;

figure 4C qPCR results show that C16 significantly reduced TGF- β induced mRNA levels of renal tubular epithelial cell expression fibrosis index Collagen III.

FIG. 5 shows that C16 significantly reduced TGF-. beta.induced fibroblast activation in NRK-49F rat fibroblast cell line cultured in vitro.

FIG. 5A shows that C16 significantly decreased the protein level of extracellular matrix Fibronectin expressed by TGF-. beta.induced fibroblasts;

FIG. 5B shows that C16 significantly decreased TGF- β -induced mRNA levels of fibroblast cells expressing Fibronectin, an indicator of fibrosis;

figure 5C shows that C16 significantly reduced TGF- β induced mRNA levels of fibroblasts expressing fibrosis index α -SMA;

FIG. 5D shows that C16 significantly decreased the mRNA level of fibroblast expression fibrosis indicator Vimentin in the control and TGF- β induced model groups;

FIG. 5E shows that C16 significantly decreased the fibroblast expression fibrosis index Collagen I mRNA levels in the control and TGF- β induced model groups;

fig. 5F shows that C16 significantly reduced the mRNA levels of the fibroblast expression fibrosis index Collagen III in the control and TGF- β induced model groups.

Detailed Description

The present invention is described in detail below with reference to the drawings and examples, but the present invention is not limited thereto. The biological materials and reagents used in the examples of the present invention are commercially available unless otherwise specified.

EXAMPLE 1 therapeutic doses of C16 have no toxic side effects on mice

1 Experimental materials and methods

1) Administration, raising and sampling of mice

The male mice (8 weeks old and 20-24g in weight) of the C57BL/6 species used in the invention are purchased from Nanjing medical university laboratory animal center, are raised in the SPF level barrier environment of the Nanjing medical university laboratory animal center, and are free to eat, and the circadian rhythms of 12-hour light and 12-hour dark are maintained. Laboratory temperature: 20-25 ℃ and humidity of 50 +/-5%. The mice were randomly divided into a control group (n-10) and a C16 group (n-10) after 1 week of acclimatization.

The present invention uses C16 (purity > 99%) from Santa Cruz. The vehicle was physiological saline. The dosage of mice in the administration group is respectively 300 mug/kg, and the administration is carried out by intraperitoneal injection, and the administration volume is 0.1ml per 10g of body weight; the control mice were given the same volume of the vehicle by intraperitoneal injection once a day for 7 days. Serum of the mice is collected 24h after the last administration, and the serum is subjected to biochemical analysis on the concentration of markers such as renal function, liver function and cardiac function in the serum so as to evaluate the influence of C16 on the renal, liver and cardiac functions of normal mice.

2) Results of the experiment

In order to study the safety of repeated administration of C16 in the high-dose group, after repeated administration of 300 mug/kg of C16 continuously for 7 days in mice, the change of biochemical indexes of each organ in serum is analyzed to evaluate the influence of C16 on the functions of kidney, liver and heart. From the results, it can be seen that C16 had no effect on serum urea nitrogen (fig. 1A) and serum creatinine (fig. 1B) as renal function markers, glutamic-pyruvic transaminase (fig. 1C) and glutamic-oxaloacetic transaminase (fig. 1D) as hepatic function markers, and glutamic-oxaloacetic transaminase (fig. 1D) as cardiac function markers, and lactic dehydrogenase (fig. 1E) and creatine kinase (fig. 1F) as cardiac function markers, indicating that C16 had no significant toxicity to kidney, liver and heart at the therapeutic dose of 300 μ g/kg.

Example 2C16 improvement of renal fibrosis in UUO model

1 materials of the experiment

The mice of the C57BL/6 genus and C16 (purity 99%) used in the present invention were the same as those obtained in example 1.

2 method of experiment

2.1 animal drug administration, modeling and sampling

30 male C57BL/6 mice (7 weeks old at the time of purchase, 20-24g in body weight) were housed in the SPF-class barrier environment of the laboratory animal center of Nanjing university of medical sciences, and the animals were fed freely, maintaining a circadian rhythm of 12 hours of light and 12 hours of darkness. Laboratory temperature: 20-25 ℃ and humidity of 50 +/-5%. The mice were randomly divided into a control group and a C16 group 1 week after acclimation. Among them, the C16 group was divided into a low dose group (100ug/kg) and a high dose group (300ug/kg), and each group contained ten mice. After grouping, control mice and C16 mice were each intraperitoneally injected with vehicle or C16 once a day. The specific operation was the same as in example 1. Unilateral ureteral ligation (UUO) is performed 24 hours after the first administration, bilateral kidneys are taken after mice are euthanized 7 days after the operation, renal cortex is taken after renal envelope is removed and preserved at-80 ℃ for extracting RNA and protein, or renal tissues are fixed by paraformaldehyde and then subjected to pathological examination.

2.2 pathological examination of renal cortex

Kidney tissues were fixed with paraformaldehyde, paraffin-encapsulated, and histologically examined after histological sectioning. Staining by a masson trichrome method, and observing the degree of renal interstitial fibrosis. Where the blue areas indicate fibrotic regions.

3 results of the experiment

To evaluate the effect of C16 on renal fibrosis, we examined mouse renal cortex pathology and the degree of interstitial fibrosis using masson trichrome staining. After 7 days of UUO modeling, renal pathological injuries such as obvious interstitial fibrosis and tubular dilatation necrosis appear in renal cortex, and the corresponding renal pathological injuries are reduced after C16 treatment (figure 2). Thus, C16 can significantly reduce renal fibrotic pathology and renal tubular injury. These results indicate that C16 can reduce the pathological changes of tubulointerstitial fibrosis.

Example 3C16 reduction in expression levels of an indicator of fibrosis in Kidney tissue

1. Experimental materials and methods

The origin and use of mice and C16 were as described in example 2. The method for establishing the mouse UUO model and the tissue selection are the same as those in example 2.

1)RT-PCR

After RNA in a sample is extracted by adopting an RNAi sso reagent of Takara company according to the instruction, the RNA is reversely transcribed into cDNA by utilizing a reverse transcription kit of Vazyme company, and the RT-PCR detection is carried out by adopting SYBR green PCR mix and combining with a corresponding primer.

1) Western blotting

The kidney tissue extracts proteins and operates according to literature procedures.

2) Statistical analysis

Data are presented using mean ± SEM. The comparisons between groups were performed using analysis of variance (ANOVA) and the data between groups was performed using T test. P <0.05 is statistically significant.

2. Results of the experiment

In order to further confirm that C16 has a therapeutic effect on renal fibrosis in a UUO model, the expression conditions of renal fibrosis specific molecular indexes, namely, fibrinectin, alpha-sma, collagen I and the like in renal cortex are detected. C16 significantly reduced the protein level of fibrosis index fibrinectin in the UUO model and was dose-dependent (fig. 3A). As shown in the RT-PCR test of FIG. 3, the renal fibrosis indexes such as Fibronectin, alpha-sma, collagen I, etc. are significantly highly expressed in the mouse UUO model renal cortex, while C16 can significantly reduce the expression level (FIGS. 3B-D). Thus, the results of changes in these fibrosis indices at mRNA and protein levels further demonstrate that C16 can reduce renal interstitial fibrosis in the UUO model.

Example 4C16 significantly inhibits TGF-beta induced fibrosis of tubular epithelial cells.

1. Experimental materials and methods

Mouse tubular epithelial cells (RTECs) were cultured in DMEM/F12 medium containing 10% fetal bovine serum at 37 ℃ in 5% carbon dioxide and 95% air. To investigate the effect of PKR and its specific inhibitor C16 on renal tubular epithelial cells, we used TGF- β 1 treatment to induce RTECs fibrosis in vitro. RTECs are pretreated for 2h by 0.5 mu M C16 in a low-serum culture medium, then TGF-beta 1 with the final concentration of 5ng/ml is added to simulate the fibrosis of renal tubular epithelial cells in the process of chronic renal fibrosis, and after 24h, cells are collected to extract total protein for Western Blot detection or RNA is extracted for RT-PCR detection of Collegen I and Collegen III. Fibrosis indicator molecule fibrinectin antibody was purchased from Abcam. The reference protein beta-actin and the secondary antibody were purchased from baaode company, Nanjing. The statistics and analysis of the data were as described in example 3.

2. Results of the experiment

TGF-. beta.1 stimulation of RTECs can mimic tubular epithelial fibrosis in chronic kidney disease in vitro. Under the stimulation of TGF-beta 1, the activation of renal interstitial fibroblasts can be detected by the expression level of extracellular matrix such as fibronectin. C16 significantly inhibited the expression up-regulation of fibrosis gene fibrosis induced by TGF-. beta.1 at the protein level (FIG. 4A). Administration of C16 inhibited TGF-. beta.induced upregulation of mRNA expression of the genes Collegen I and Collegen III in RTECs cells fibrosis (FIGS. 4B & C). These results indicate that C16 inhibits fibrosis of renal tubular epithelial cells.

Example 5C16 significantly inhibited TGF- β induced renal interstitial fibroblast activation.

1. Experimental materials and methods

The rat renal interstitial fibroblast NRK-49F is cultured by a DMEM medium containing 10% fetal bovine serum under the conditions of 37 ℃, 5% carbon dioxide and 95% air. To investigate the effect of C16 on renal interstitial fibroblast activation, we used TGF- β 1 treatment to induce NRK-49F cell activation in vitro, we added NRK-49F to low serum medium with 0.5 μ M C16 pre-treated for 2h, followed by TGF- β 1 at a final concentration of 5ng/ml to mimic fibroblast activation during chronic renal fibrosis, collected total cell protein after 24h for Western Blot assay, or cells were collected, RNA was extracted and then assayed for expression of fibrotic genes, fibrinectin, α -sma, Vimentin, Collagen I and Collagen III using RT-PCR. Fibrosis indicator molecule fibrinectin antibody was purchased from Abcam. The reference proteins GAPDH and secondary antibody were purchased from Baaode, Nanjing. The statistics and analysis of the data were as described in example 3.

2. Results of the experiment

The activation of renal interstitial fibroblasts is a direct cellular mechanism leading to renal interstitial fibrosis, and the activated fibroblasts can produce a large amount of extracellular matrix, such as fibronectin, Collagen I and Collagen III, and the like, directly leading to renal interstitial fibrosis. Under the stimulation of TGF-beta 1, the activation of renal interstitial fibroblasts can be detected by the expression level of extracellular matrix such as fibronectin. The experimental results showed that at the protein level, C16 significantly inhibited TGF- β 1-induced fibrinectin expression (fig. 5A). In agreement with this, C16 also significantly inhibited the expression upregulation of the TGF-. beta.1-induced fibrosis genes, fibrinectin, alpha-sma, Vimentin, Collagen I and Collagen III, at the mRNA level (FIGS. 5B-F). These results indicate that C16 significantly inhibited TGF- β 1-induced renal interstitial fibroblast activation.

In conclusion, the invention provides a new application of the compound C16 in preparing a medicine for preventing and treating chronic renal fibrosis, the inhibitor is administered by an injection route, and the aim of preventing and treating the chronic renal fibrosis is achieved by inhibiting PKR, inhibiting the excessive activation of fibroblasts and directly inhibiting the action mechanism of renal tubular epithelial cell fibrosis and effectively inhibiting the renal fibrosis.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.