CN115844884B - Application of compound EP9 in preparation of medicines for resisting renal fibrosis of diabetic nephropathy - Google Patents
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Abstract
The invention relates to an application of a compound EP9 in preparing a medicament for resisting diabetic nephropathy renal fibrosis, belonging to the technical field of diabetic nephropathy. The invention provides a pharmaceutical composition, which takes a compound shown in a formula EP9 or pharmaceutically acceptable salt thereof as an active ingredient or main active ingredient and is assisted with a pharmaceutically acceptable carrier. ACSL1 expression in urine of diabetic nephropathy patients is obviously increased, compared with db/m group, ACSL1 expression in kidney tissue of db/db mice at 24 weeks of age is also obviously increased, and in HK-2 cells stimulated by high sugar, compared with normal group, ACSL1 expression in high sugar group is obviously increased, and indexes of EMT related proteins are regulated. The compound EP9 has the same effect as that of knocking down ACSL1, and provides a new thought for the research and development of the medicament for resisting the renal fibrosis of the diabetic nephropathy by targeting the ACSL1 as the medicament for resisting the renal fibrosis of the diabetic nephropathy.
Description
Technical Field
The invention relates to the technical field of diabetic nephropathy, in particular to application of a compound EP9 in preparing medicines for resisting diabetic nephropathy renal fibrosis by inhibiting ACSL1 expression.
Background
Diabetic nephropathy is the primary cause of end stage renal disease. The mechanism responsible for diabetic nephropathy is complex, and tubular interstitial fibrosis is a common route for most kidney injuries and one of the main pathological features of chronic kidney disease. Kidney fibroblasts play an important role in maintaining the homeostasis of the interstitial matrix and adjacent tissues under physiological conditions. The process of kidney fibrosis is involved in many cells, and the main cause of tubular interstitial fibrosis is from fibroblast activation and expansion, production and deposition of a large number of extracellular matrix (ECM) components, and changes in both the tubules and microvasculature. Continuous exposure to high sugar (HG) environments, tubular epithelial cells are induced to undergo epithelial-mesenchymal transition (EMT), which in turn leads to interstitial fibrosis, and accumulation of extracellular matrix proteins in the mesangial matrix, such as that caused by accumulation of collagen and fibronectin. The current control of its development has been to prevent, alleviate or even reverse DKD by addressing the interactions between hemodynamic and metabolic pathways, to use principal drugs with a blocking effect on the renin-angiotensin-aldosterone system by controlling blood glucose, blood pressure and blood lipids, and to treat dyslipidemia, as well as an effective strategy for preventing diabetic nephropathy. However, in spite of the control of blood glucose, blood lipid and blood pressure, many patients develop diabetic nephropathy. By 2040, the prevalence of diabetes is expected to break through 6 million people, of which more than 2 thousand people will develop DKD. Therefore, there is an urgent need to search for potential therapeutic targets of DKD to slow down the development of DKD.
acyl-CoA synthetase Long chain family member 1 (acsl 1) is an enzyme that is the initial step of fatty acid beta oxidation, playing a key role in lipid biosynthesis and fatty acid degradation. Fatty acids are essential nutrients for the human body, and can be obtained from the daily diet, or from the de novo synthesis and breakdown of cell Triacylglycerols (TAGs) and phospholipids. They are also involved in energy metabolism and cellular signaling pathways to maintain physiological function as important components of the body. Long chain acyl-coa synthetases (ACSL) activate fatty acids for intracellular metabolism, also involved in regulation of uptake. However, the imbalance in fatty acid metabolism leads to excessive lipid biosynthesis and deposition, ultimately making the body susceptible to metabolic disorders.
However, little research has been conducted on the role of ACSL1 in diabetic nephropathy, whether it can be a potential therapeutic target for DKD to slow down the development of DKD is of paramount importance.
Disclosure of Invention
The invention aims at providing an application of a compound EP9 in preparing medicines for resisting diabetic nephropathy and renal fibrosis based on the prior art.
It is a further object of the present invention to provide the use of compound EP9 as a medicament for inhibiting the expression of ACSL 1.
It is a third object of the present invention to provide the use of compound EP9 as a medicament for inhibiting the expression of ACSL1 and modulating EMT-related proteins under high sugar stimulation.
The technical scheme of the invention is as follows:
the application of a compound shown in the formula EP9 or pharmaceutically acceptable salt thereof in preparing medicines for resisting diabetic nephropathy and renal fibrosis,
in a preferred embodiment, the present invention provides a pharmaceutical composition for the preparation of an anti-diabetic nephropathy renal fibrosis, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient or as a primary active ingredient, together with a pharmaceutically acceptable carrier. Further, the pharmaceutical composition may be formulated as a liquid, solid or semi-solid formulation. Furthermore, the pharmaceutical composition can be prepared into injection, tablet, capsule or oral liquid. Pharmaceutically acceptable auxiliary materials play a key role in the whole preparation technology development process, and the auxiliary materials account for most of the preparation, so that the properties of the auxiliary materials determine the properties of the preparation to a great extent. The good auxiliary materials can enhance the stability of the main medicine and prolong the effective period of the medicine; the release speed of the main medicine in vivo and in vitro can be regulated and controlled; can change the absorption of the medicine in the body and increase the bioavailability, and the specific auxiliary material type is not limited.
The use of a compound of formula EP9 or a pharmaceutically acceptable salt thereof as a medicament for inhibiting the expression of ACSL1,
in a preferred embodiment, the present invention provides a pharmaceutical composition for inhibiting the expression of ACSL1, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient or a primary active ingredient, together with a pharmaceutically acceptable carrier. Further, the pharmaceutical composition may be formulated as a liquid, solid or semi-solid formulation. Furthermore, the pharmaceutical composition can be prepared into injection, tablet, capsule or oral liquid.
The compound shown in the formula EP9 or pharmaceutically acceptable salt thereof can be used as a medicament for inhibiting the expression of ACSL1 and regulating and controlling EMT related proteins under the stimulation of high sugar,
in a preferred embodiment, the present invention provides a pharmaceutical composition for inhibiting the expression of ACSL1 and modulating EMT-related proteins under high sugar stimulation, which comprises the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, together with a pharmaceutically acceptable carrier. Further, the pharmaceutical composition may be formulated as a liquid, solid or semi-solid formulation. Furthermore, the pharmaceutical composition can be prepared into injection, tablet, capsule or oral liquid.
By adopting the technical scheme of the invention, the advantages are as follows:
the present invention demonstrates the important role of compound EP9 in the anti-diabetic nephropathy renal fibrosis. Animal experiments show that ACSL1 expression is obviously increased in urine of diabetic nephropathy patients, compared with db/m group, ACSL1 expression is also obviously increased in kidney tissue of db/db mice at 24 weeks of age, and in HK-2 cells stimulated by high sugar, ACSL1 expression is obviously increased in high sugar group compared with normal group, and indexes of EMT related proteins are regulated. In vitro experiments show that: EP9 has the same effect as that of knocking down ACSL1, and provides a new thought for the research and development of the medicament for resisting diabetic nephropathy renal fibrosis by targeting ACSL1 as the medicament for resisting diabetic nephropathy renal fibrosis.
Drawings
FIG. 1 shows high expression of ACSL1 in urine shed cells and urine supernatant of diabetic patients;
FIG. 2 shows high expression of ACSL1 in kidney tissue of 24-week-old db/db mice; wherein, a in fig. 2 is the staining of the kidney tissue PAS of the mouse; FIG. 2B is a masson stain of kidney tissue from mice; FIG. 2C shows the expression level of ACSL1 protein in kidney tissue of mice; fig. 2D is mouse kidney tissue immunohistochemistry;
FIG. 3 shows ACSL1 is highly expressed in high sugar stimulated HK-2 cells and is involved in the regulation of EMT-related proteins, wherein A in FIG. 3 is the ACSL1 protein expression level in high sugar stimulated HK-2 cells; FIG. 3B shows the change of EMT-related protein under high sugar stimulation; FIG. 3C shows the protein expression level of knock-down ACSL 1; in FIG. 3D, the regulation of EMT-related proteins by knock-down ACSL1 is shown.
FIG. 4 shows that EP9 inhibits ACSL1 expression and modulates EMT-related proteins under high sugar stimulation; wherein a in fig. 4 is the effect of different concentrations of EP9 on the expression level of ACSL1 under high sugar stimulation; in FIG. 4B is the modulation of EMT-related proteins by EP9 under high sugar stimulation.
Detailed Description
In order that the technical scheme of the invention may be more clearly understood, the invention is further illustrated by the following examples, which are not to be construed as limiting the invention in any way.
1. ELISA for detecting ACSL1 expression in urine
Experimental materials: urine flow
The invention was carried out in a medical university of Xuzhou affiliated hospital 3 to 6 months 2022. 84 urine samples are collected altogether, wherein 20 urine samples of healthy people of a health management center of an affiliated hospital of Xuzhou medical university, 30 urine samples of diabetics of endocrinology and 34 urine samples of diabetic nephropathy patients are collected. Basic information and clinical parameters of each patient, including sex, age, medication, diabetes course, blood pressure, blood glucose level, serum creatinine, were recorded, and a 10ml urine sample was taken from the first morning urine flow. Urine specimens were centrifuged at 3000g for 15 minutes and stored at-80 ℃. The supernatant was divided into 1.5ml aliquots and immediately stored at-80 ℃. Urine samples were centrifuged at 10000g for 15 minutes, urine exfoliating cells were retained and immediately stored at-80 ℃.
ELISA kit, the procedure was as follows:
(1) The strips required for the experiment were removed from the sealed bag equilibrated to room temperature;
(2) Reserving blank holes;
(3) Adding samples or human ACSL1 standard substances with different concentrations (0 ng/ml hole and standard substance diluent) into corresponding holes (100 mu L/hole), and incubating in an incubator at 37 ℃ for 90min;
(4) Preparing a working solution of the biotinylated human ACSL2 antibody 30min in advance;
(5) Washing the plate for 2 times;
(6) Adding biotinylated human ACSL1 antibody working solution (100 mu L/hole), and incubating in a incubator at 37 ℃ for 60min;
(7) Preparing an enzyme conjugate working solution 30min in advance; placing at room temperature in a dark place;
(8) Washing the plate for 3 times;
(9) Adding enzyme conjugate working solution (100 mu L/hole) except blank holes, incubating in a incubator at 37 ℃ and in a dark place for 30min;
(10) Washing the plate for 5 times;
(11) Adding TMB color development working solution (comprising blank holes) into the mixture to form 100 mu L/hole, incubating the mixture in a incubator at 37 ℃ and in a dark place, wherein the standard curve has a deep color with high concentration, and the mixture can be terminated when the standard curve has an obvious color gradient; the experimental color reaction does not exceed 30 minutes;
(12) Stop solution (including blank wells) was added at 100. Mu.L/well and OD (450 nm) values (within 10 minutes) were measured immediately after mixing.
The experimental results are as follows:
the expression of ACSL1 in urine supernatant of normal person (NC), diabetic patient (DM), diabetic nephropathy patient (DKD) was detected by ELISA kit (as shown in FIG. 1). The results showed that the expression of ACSL1 in urine supernatant of DKD group was significantly increased compared to NC and DM groups.
2. ACSL1 is highly expressed in db/db mouse kidney tissue
Male C57BLKS/J background Lepdb/Lepdb (db/db) (7 weeks old, 30-35 g) and non-diabetic control Lepdb/m (db/m) mice (7 weeks old, 15-20 g) were purchased from Nanjing university model animal research center (Jiangsu, china). All animals were placed in a shielded environment and were free to be served with food and water before and during the experiment. All experimental procedures were approved by the animal ethics committee of the university of Xuzhou medical science. Db/m and db/db mice were fed to 24 weeks of age, and blood, urine and kidney tissue were collected and observed for expression of db/m and db/db mice.
The experimental results are as follows:
PAS staining showed that the db/db mice had more pronounced tubular dilation (A in FIG. 2), suggesting a characteristic of tubular injury, compared to db/m mice. The map positive area distribution was significantly increased in db/db mice compared to db/m mice, indicating collagen accumulation in the renal cortex (B in fig. 2). The results indicate that diabetic mice have reduced renal function. In addition, western blot showed a significant increase in the expression of ACSL1 total protein in db/db mice (C in FIG. 2), and immunohistochemistry showed that ACSL1 was more significantly expressed around the kidney tubules of db/db mice (D in FIG. 2)
3. High sugar stimulates HK-2 cells, compared with NG group, HG group ACSL1 expression is obviously increased, and the high sugar participates in regulating EMT
Human proximal tubular epithelial cell line HK-2 was given away by university of Nanjing. Cells were cultured in DMEM containing 5.56mmol/L glucose (normal glucose, NG), 10% fetal bovine serum (Invitrogen, grand Island, N.Y.) and 1% penicillin-streptomycin were added to the DMEM at 37℃and 5% CO 2 Is cultured in a wet incubator. To induce EMT, cells were cultured in a medium containing 60mmol/L glucose for 72h.5.56mmol/L glucose and mannitol were used for osmotic control (MA). In ACSL1-shRNA experiments, HG-stimulated HK-2 cells were transfected with control lentiviral vector (HG+NC) or ACSL1+shRNA lentiviral vector (HG+ACSL 1-shRNA) for 72h. HK-2 cells were cultured in HG without other treatments as a control (HG).
The experimental results are as follows:
western blot results showed that HG promoted the expression of ACSL1 (A in FIG. 3). Furthermore, HG inhibited the expression of E-cadherein and Zo-1, and increased the expression of α -SMA and Vimentin interstitial proteins (B in FIG. 3). These data indicate that HG can induce EMT of HK-2 cells. To further elucidate the role of ACSL1 in DKD EMT, we knocked down the expression of ACSL1 protein in HG-cultured HK-2 cells (C in FIG. 3), while finding significant upregulation of the expression of epithelial cell markers (E-cadherein and ZO-1). Mesenchymal cell markers (vimentin and α -SMA) expression were down-regulated (D in fig. 3). Taken together, ACSL1 knockout can effectively improve EMT kidney fibrosis caused by diabetic nephropathy.
4. EP9 inhibits ACSL1 expression and regulates EMT-related proteins under high sugar stimulation.
The cells were cultured, plated, given HG stimulation, and then treated with EP9 at final concentrations of 1, 5, 10. Mu. Mol/L for 48 hours, respectively, to determine the inhibitory concentration. Further, small molecule drug compound EP9 reverses the index of EMT-related proteins under HG stimulation by inhibiting the ACSL1 target.
The experimental results are as follows:
as the concentration of EP9 increases, ACSL1 was inhibited more significantly, thereby determining a concentration of 10. Mu. Mol/L (A in FIG. 4). Further studies have found that the hg+ep9 group significantly reversed the index of EMT-related proteins in HG stimulation (B in fig. 4) compared to the HG group, producing the same effect as knock-down of ACSL1, thereby improving diabetic kidney fibrosis.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.
Claims (1)
1. The application of a compound shown in the formula EP9 or pharmaceutically acceptable salt thereof in preparing medicines for resisting diabetic nephropathy and renal fibrosis,
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| CN113456631B (en) * | 2021-08-06 | 2022-06-21 | 徐州医科大学 | Small molecule drug targeting ACSL1 and application thereof in treatment of endometrial cancer |
| CN115716822A (en) * | 2022-11-14 | 2023-02-28 | 徐州医科大学 | Application of benzimidazolyl isoxazole compound in preparation of medicines related to multiple myeloma |
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| CN113456631B (en) * | 2021-08-06 | 2022-06-21 | 徐州医科大学 | Small molecule drug targeting ACSL1 and application thereof in treatment of endometrial cancer |
| CN115716822A (en) * | 2022-11-14 | 2023-02-28 | 徐州医科大学 | Application of benzimidazolyl isoxazole compound in preparation of medicines related to multiple myeloma |
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| Title |
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| 肾小管上皮间充质转化与肾脏纤维化;王来亮等人;中国病理生理杂志;第30卷(第10期);1910-1914、1920 * |
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