Use of orange-yellow piperamide in the preparation of a medicament for the treatment of acute kidney injury

Abstract

This invention discloses the application of orange-yellow piperine in the preparation of a drug for treating acute kidney injury. It belongs to the field of biomedical technology. The molecular formula of orange-yellow piperine is C2. 25 H 26 N2O3 relates to a new pharmaceutical use of orange-yellow piperine, specifically that orange-yellow piperine can effectively reduce the inflammatory response and kidney injury level induced in an in vitro model of acute kidney injury; it can also reduce the expression levels of inflammatory factors IL-6, TNF-α and chemokine MCP-1 in the serum of animal models of kidney injury, improve the pathological condition of kidney tissue in animals with acute kidney injury, inhibit the protein expression of KIM1 in kidney tissue, and reduce the levels of creatinine and urea nitrogen in serum of kidney tissue, thus showing potential therapeutic effects on acute kidney injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of orange-yellow piperamide in the preparation of drugs for treating acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a common clinical condition characterized by a sudden (within 1-7 days) and sustained (>24 hours) decline in renal function, defined as an increase in serum creatinine (SCr) of at least 0.5 mg / dL. Symptoms include azotemia, electrolyte and acid-base imbalances, and systemic symptoms, which may be accompanied by oliguria (<400 ml / 24 h or 17 ml / h) or anuria (<100 ml / 24 h). Based on the location and cause of the lesion, AKI can be classified into three main categories: prerenal, renal, and postrenal. The main causes of AKI include ischemia / reperfusion (I / R), sepsis, and toxic injury. Treatment primarily includes removing the underlying cause, maintaining homeostasis, nutritional support, managing complications, and blood purification therapy. Most patients who survive AKI recover normal renal function, but about 5% do not, requiring maintenance renal replacement therapy; this proportion can be as high as 16% in elderly patients. Approximately 5% of patients, while experiencing a recovery in kidney function, will gradually develop chronic kidney damage. This manifests as persistent hypertension despite a return to normal serum creatinine (Scr) levels, with or without proteinuria, possibly related to compensatory glomerular hypertrophy and secondary focal segmental glomerulosclerosis. Furthermore, elderly patients, those with complications such as sepsis, multiple organ dysfunction syndrome, and those experiencing acute kidney injury after cardiac surgery have high mortality rates. Increasing epidemiological studies indicate that the mortality rate for AKI patients is as high as 60%-80%, yet there are currently no effective treatments or strategies. Therefore, the search for nephroprotective drugs that can reduce tissue damage, promote kidney repair, and prevent chronic fibrosis is of great significance.

[0003] Aurantiamide is an important dipeptide compound isolated from plants such as *Veratrum nigrum*, *Zanthoxylum bungeanum* stems, and *Smilax china*. Previous studies have shown that aurantiamide has a wide range of biological activities, such as anti-inflammatory, antimalarial, and rheumatoid arthritis prevention effects, but its pharmacological effects in the prevention and treatment of acute kidney injury have not yet been reported.

[0004] Chinese patent application CN105769837A discloses the use of capsanthin in the preparation of malaria treatment drugs and provides a pharmaceutical composition containing the capsanthin and a pharmaceutically acceptable carrier. The active ingredient capsanthin comprises 5-95% by weight in the pharmaceutical composition. In vitro antimalarial activity assays showed that capsanthin has good in vitro antimalarial activity against Plasmodium. Therefore, it can be used to prepare malaria treatment drugs and has significant clinical application value. However, this patent does not disclose the use of capsanthin in the preparation of drugs for treating acute kidney injury. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to provide a new pharmaceutical use for orange-yellow piperamide, specifically the use of orange-yellow piperamide in the prevention and treatment of acute kidney injury.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] This invention proposes the application of orange-yellow piperine in the preparation of a drug for treating acute kidney injury, the structural formula of which is:

[0008]

[0009] Beneficial effects: Orange-yellow piperamide has been used to prepare drugs for the treatment of acute kidney injury, demonstrating that it can reduce the expression and secretion levels of cellular inflammatory factors; it can also reduce the expression levels of inflammatory factors IL-6, TNF-α and chemokine MCP-1 mRNA in animal models of acute kidney injury, improve the pathological condition of kidney tissue in animals with acute kidney injury, inhibit the expression of kidney injury factor KIM1 protein in kidney tissue, and reduce the levels of serum creatinine and urea nitrogen in kidney tissue, thus showing potential therapeutic effects on acute kidney injury.

[0010] Preferably, the concentration of the orange-yellow piperidine is 0.1-100 μM.

[0011] Preferably, the concentration of the orange-yellow piperidine is 6-100 μM.

[0012] Preferably, the concentration of the orange-yellow piperidine is 12.5-50 μM.

[0013] Preferably, the drug for treating acute kidney injury is a drug that can reduce the expression and secretion levels of cellular inflammatory factors.

[0014] Preferably, the drug for treating acute kidney injury is capable of reducing the expression of the intracellular kidney injury factor KIM1.

[0015] Preferably, the drug for treating acute kidney injury is a drug that can reduce the expression levels of inflammatory factors and chemokines in the serum of animals infected with acute kidney injury.

[0016] Preferably, the drug for treating acute kidney injury is a drug that can inhibit the pathological features of kidney tissue in animals with acute kidney injury.

[0017] Preferably, the drug for treating acute kidney injury is a drug that can reduce serum creatinine and blood urea nitrogen in the kidney tissue of animals with acute kidney injury.

[0018] Preferably, the medicament for treating acute kidney injury further includes a pharmaceutically acceptable carrier or excipient.

[0019] The advantages of this invention are:

[0020] Beneficial effects: Orange-yellow piperamide has been used to prepare drugs for the treatment of acute kidney injury, demonstrating that it can reduce the expression and secretion levels of cellular inflammatory factors; it can also reduce the expression levels of inflammatory factors IL-6, TNF-α and chemokine MCP-1 mRNA in animal models of acute kidney injury, improve the pathological condition of kidney tissue in animals with acute kidney injury, inhibit the expression of kidney injury factor KIM1 protein in kidney tissue, and reduce the levels of serum creatinine and urea nitrogen in kidney tissue, thus showing potential therapeutic effects on acute kidney injury. Attached Figure Description

[0021] Figure 1 A comparative graph showing the effect of different concentrations of AA on the viability of HK2 cells;

[0022] Figure 2 A comparative diagram showing the effects of different concentrations of AA on cisplatin-induced damage to HK2 renal tubular epithelial cells in vitro.

[0023] Figure 3 The figure shows the effect of AA on the mRNA expression levels of LPS-induced kidney injury factors, chemokines, and inflammatory factors in HK2 cells, as determined by Real-time PCR.

[0024] Figure 4 The graph shows the changes in the response of AA to LPS-induced kidney injury factor KIM1 in HK2 cells as determined by Western blot.

[0025] Figure 5 This is a staining map of the kidney injury factor KIM1 in an in vitro AA-treated LPS-induced acute kidney injury model using the IF method.

[0026] Figure 6The figure shows the effect of AA on the mRNA expression levels of kidney injury factors, chemokines, and inflammatory factors induced by HR in HK2 cells, as determined by Real-time PCR.

[0027] Figure 7 The graph shows the changes in the response of AA to HR-induced kidney injury factor KIM1 in HK2 cells as determined by Western blot.

[0028] Figure 8 This is a staining map of the kidney injury factor KIM1 in an in vitro AA-treated HR-induced acute kidney injury model using the IF method.

[0029] Figure 9 The figure shows the effect of real-time PCR on the mRNA expression levels of renal injury factors, chemokines, and inflammatory factors in a CLP mouse model.

[0030] Figure 10 The graph shows the changes in the response of AA to the kidney injury factor KIM1 in a CLP mouse model, as determined by Western blot.

[0031] Figure 11 The image shows the staining results of kidney tissue from CLP mouse models and AA-treated CLP mouse models using HE staining.

[0032] Figure 12 The image shows the staining of the kidney injury factor KIM1 in an in vivo CLP mouse model and an AA-treated CLP-induced acute kidney injury model using the IF method.

[0033] Figure 13 The image shows the staining of macrophage marker F4-80 in an in vivo CLP mouse model and an AA-treated CLP-induced acute kidney injury model using immunohistochemistry.

[0034] Figure 14 The graph shows the effect of AA on serum creatinine and blood urea nitrogen levels in CLP model mice. 14(a) represents serum creatinine, and 14(b) represents blood urea nitrogen.

[0035] Figure 15 The figure shows the effect of real-time PCR on the mRNA expression levels of renal injury factors, chemokines, and inflammatory factors in an IR mouse model.

[0036] Figure 16 The graph shows the changes in the response of AA to the kidney injury factor KIM1 in an IR mouse model, as determined by Western blot.

[0037] Figure 17The images show the staining results of kidney tissue from a mouse model with simple IR and a mouse model with IR after AA treatment, using a glycogen (PAS) staining kit.

[0038] Figure 18 The image shows the staining of the kidney injury factor KIM1 in an in vivo IR mouse model and an IR-induced acute kidney injury model treated with AA, using the IF method.

[0039] Figure 19 The image shows the staining of macrophage marker F4-80 in an in vivo IR mouse model and an IR-induced acute kidney injury model treated with AA using immunohistochemistry.

[0040] Figure 20 The graph shows the effect of AA on serum creatinine and blood urea nitrogen levels in IR model mice. 20(a) represents serum creatinine, and 20(b) represents blood urea nitrogen.

[0041] Figure 21 The image shows the staining results of heart, liver, spleen and lung tissues of mice in the drug-only group using the hematoxylin and eosin (H&E) staining kit.

[0042] Figure 22 The images show the effects of ALT and AST staining kits on serum ALT and AST levels in mice in the sham group and the drug-only group. Figure 22(a) shows the effect on ALT levels, and figure 22(b) shows the effect on AST levels. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0045] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0046] The compound used in the following examples is orange-yellow piperidine (AA), whose structure is as follows:

[0047]

[0048] Purchased from APE×BIO Biotechnology Co., Ltd., USA.

[0049] Example 1: Study on the effect of different concentrations of orange-yellow piperidine (AA) on the viability of human renal tubular epithelial cells (HK2 cells) in vitro.

[0050] HK2 cells were cultured in HK2 cell-specific culture medium containing different concentrations of amino acids (AA), and cell proliferation was detected using the CCK8 assay to determine cell viability. HK2 cell-specific culture medium without AA was used as the normal control group (NC). AA concentrations were 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM. The detection results are shown below. Figure 1 As shown in the figure, when the concentration of AA is 0.1-100 μM, AA does not show obvious toxicity to HK2 cells.

[0051] An in vitro cisplatin (Cisplatin) cell model was constructed using HK2 cells. HK2 cells were cultured in HK2 cell-specific medium containing different concentrations of AA + CIS, and cell proliferation was assessed using the CCK8 assay to determine cell viability. HK2 cell-specific medium without AA + Cisplatin served as the normal control (NC), while HK2 cell-specific medium without AA but containing Cisplatin served as the damage control (CIS). The concentration of Cisplatin was 20 μM, and the concentrations of AA were 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM. The results are shown below. Figure 2 As shown, from Figure 2 The results showed that capsaicinamide (AA) could alleviate cisplatin-induced renal tubular epithelial cell damage in vitro, and the mitigation effect was dose-dependent with increasing AA concentration, resulting in less renal tubular epithelial cell damage. CCK8 results indicated that capsaicinamide significantly reduced the growth-inhibiting effect of cisplatin on HK2 cells within the range of 6.25-100 μM.

[0052] Example 2: Study on the effect of AA on LPS (lipopolysaccharide)-induced in vitro cellular inflammatory response

[0053] HK2 cells were cultured in different experimental media, which were grouped as follows:

[0054] The normal control group (NC), the high-dose AA group (AA 100 μM), the LPS model group (LPS 1 μg / ml), the LPS model + low-dose AA group (LPS 1 μg / ml + AA 25 μM), the LPS model + medium-dose AA group (LPS 1 μg / ml + AA 50 μM), and the LPS model + high-dose AA group (LPS 1 μg / ml + AA 100 μM) were designated as NC, A, B, C, D, and E groups, respectively.

[0055] After culturing for 24 hours, HK2 cells were harvested, and RNA was extracted from each group. The expression levels of mRNA in the supernatant of the kidney injury factor KIM1, the chemokine MCP-1, and the inflammatory factor TNF-α were measured using real-time quantitative PCR. The results are shown below. Figure 3 As shown in the figure, the results indicate that AA significantly reduced the expression levels of KIM1, TNF-α, and MCP-1 mRNA in LPS-induced in vitro cells, with the best reduction effect observed at a AA concentration of 100 μM, i.e., group E.

[0056] Western blot analysis was used to measure the level of the kidney injury factor KIM1 protein in HK2 cells cultured in the same NC, A, B, C, D, and E groups as described above. The results are as follows: Figure 4 As shown, AA significantly inhibited LPS-induced KIM1 protein expression in cells.

[0057] The kidney injury factor KIM1 in the supernatant of HK2 cells cultured from normal control group (NC), LPS model group (LPS 1 μg / ml) (i.e., group B), and LPS model + high-dose AA group (LPS 1 μg / ml + AA 100 μM) (i.e., group E) was stained using the IF method. The results are as follows: Figure 5 As shown, consistent with the above results, AA significantly reduced the level of LPS-induced kidney injury in HK2 cells in vivo.

[0058] Example 3: Study on the effect of AA on HR (hypoxia / reoxygenation)-induced in vitro cellular inflammatory response

[0059] HK2 cells were cultured in different experimental media, which were grouped as follows:

[0060] The normal control group (NC), the high-dose AA group (AA100μM), the HR model group, the HR model + low-dose AA group (HR+AA25μM), the HR model + medium-dose AA group (HR+AA50μM), and the HR model + high-dose AA group (HR+AA100μM) are denoted as NC, A, F, G, H, and I groups, respectively.

[0061] The HR model was specifically designed for hypoxia in 0.5% low-glucose medium for 12 hours, followed by reoxygenation in 2% normal medium for 6 hours.

[0062] HK2 cell supernatant was collected, RNA was extracted from the supernatant, and the expression levels of mRNA of kidney injury factor KIM1, chemokine MCP-1, and inflammatory factor IL-6 in the supernatant were determined by real-time quantitative PCR. The results are as follows: Figure 6As shown in the figure, AA significantly reduced the expression levels of KIM1, MCP-1, and IL-6 mRNA in in vitro cells induced by the HR model. The best reduction effect was observed when the AA concentration was 100 μM, i.e., in group I.

[0063] The expression levels of β-actin and KIM1 proteins in HK2 cells cultured in the above groups were measured using Western blot. The results are as follows: Figure 7 As shown, AA significantly inhibited HR-induced KIM1 protein expression in cell supernatant, with the best inhibitory effect observed at a AA concentration of 100 μM, i.e., in group I.

[0064] The kidney injury factor KIM1 in the supernatant of HK2 cells cultured from the normal control group (NC), the HR model group (F group), and the HR model + high-dose AA group (I group) was stained using the IF method. The results are as follows: Figure 8 As shown, AA significantly inhibited the expression of the kidney injury factor KIM1 protein in HK2 cells in the HR model, reducing the level of HR-induced kidney injury in vivo.

[0065] Example 4: Study on the effects of AA on untreated mouse heart, liver, spleen, lung, and kidney tissues

[0066] Healthy male C57BL / 6J mice (20-22g) aged 6-8 weeks were selected and housed in a pathogen-free environment (23±2°C and 55±5% humidity). All mice had free access to food and water under a 12-hour light / 12-hour dark cycle.

[0067] Constructing a mouse model of simple AA (anti-AA) treatment: Six mice were administered the drug three times by gavage, with each gavage interval of 24 hours. The drug concentration was 10 mg / kg of AA. Twenty-four hours after the last administration, the mice were sacrificed along with the model group. The heart, liver, spleen, and lungs were harvested, embedded, and stained with hematoxylin and eosin (HE). Figure 21 As shown, there are no obvious vacuolar structures in the heart, liver, spleen, and lung tissues, indicating that AA at this dose has no obvious toxicity to other organs.

[0068] Example 5: Study on the effects of AA on serum indicators reflecting renal function and levels of damaging and inflammatory factors in renal tissue of mice in CLP and IR models.

[0069] Administration method in mice: The drug was administered by gavage three times before surgery, with each administration 24 hours apart. Surgery was performed 24 hours after the last gavage administration.

[0070] CLP model construction: Mice were randomly divided into four groups: Sham group, high-dose AA group (AA 10 mg / kg), CLP group, CLP+AA (2.5 mg / kg), CLP+AA (5 mg / kg), and CLP+AA (10 mg / kg), denoted as groups a, A, b, c, d, and e, respectively, with 10 mice in each group. Mice in the Sham group underwent sham surgery only, mice in the high-dose AA group (AA 10 mg / kg) received only gavage, mice in the CLP group underwent cecal ligation and puncture surgery, and mice in the CLP+AA group underwent cecal ligation and puncture surgery and received three doses of AA at the corresponding concentrations preoperatively for prophylactic treatment. Real-time PCR was used to determine the effects of AA on the mRNA expression levels of the kidney injury factor KIM1, the chemokine MCP-1, and the inflammatory factor TNF-α in the CLP mouse model. The results are as follows: Figure 9 As shown in the figure, AA reduced the level of acute kidney injury and inflammation induced by sepsis. The expression levels of β-actin and KIM1 proteins in the above animal groups were measured using Western blot, and the results are shown below. Figure 10 As shown, consistent with Real-time PCR results, AA reduced protein levels in sepsis-induced acute kidney injury and inflammation.

[0071] IR model construction: Mice were randomly divided into four groups: Sham group, high-dose AA group (AA 10 mg / kg), IR group, IR+AA (2.5 mg / kg), IR+AA (5 mg / kg), and IR+AA (10 mg / kg), denoted as f, A, g, h, i, and j groups, with 10 mice in each group. Mice in the Sham group underwent sham surgery only, mice in the high-dose AA group (AA 10 mg / kg) received only gavage treatment, mice in the IR group underwent ischemia-reperfusion surgery with hemostat ischemia for 40 minutes, and mice in the IR+AA group underwent surgery and received three doses of the corresponding concentration of AA preoperatively for prophylactic treatment.

[0072] Twenty-four hours after the above operations were performed on the mice, the mice were anesthetized and killed. The kidney tissue of the mice was taken for embedding and sectioning, protein extraction and RNA extraction. At the same time, blood was taken from the eyeballs of the mice for serum index determination.

[0073] Example 6: Study on the effects of AA on serum creatinine and blood urea nitrogen in CLP and IR model mice

[0074] ① The serum creatinine level (CRE) in the kidney tissue of CLP and IR model mice was measured. The specific procedure was as follows: according to Table 1, the corresponding amount of sample, standard (concentration of 442 μmol / L), double-distilled water, and enzyme solution A were added to the test tubes of each experimental group. After incubation at 37℃ for 5 min, the absorbance A1 was measured at a wavelength of 546 nm. After the measurement, an equal amount of enzyme solution B was added to the above test tubes, and after incubation at 37℃ for 5 min, the absorbance A2 was measured at a wavelength of 546 nm.

[0075] The formula for calculating creatinine content is as follows:

[0076] Creatinine content (μmol / L) = [(Determination A2 - K * Determination A1) - (Blank A2 - K * Blank A1)] / [(Standard A2 - K * Standard A1) - (Blank A2 - K * Blank A1)] * Standard concentration (442 μmol / L)

[0077] Wherein, the dilution factor K = (sample volume + enzyme solution A volume) / (sample volume + enzyme solution A volume + enzyme solution B volume) = 186 / 246.

[0078] The results of serum creatinine (CRE) levels in kidney tissues of CLP and IR model mice are as follows: Figure 14 As shown in (a) and 20(a), AA significantly reduced serum creatinine levels in the kidney tissues of CLP and IR model mice, with the best reduction effect observed at an AA concentration of 10 mg / kg.

[0079] Table 1: Components added to the test tubes of each experimental group and their content

[0080]

[0081] ② The blood urea nitrogen (BUN) level in the serum of mouse kidney tissue in the CLP and IR models was measured. The specific operation procedure was as follows: according to Table 2, the corresponding components were added to the test tubes of each experimental group, mixed well, and placed in a water bath at 37℃ for 10 min. Then, phenol colorimetric reagent and alkaline sodium hypochlorite were added in sequence and placed in a water bath at 37℃ for 10 min. The OD value of each tube was measured at a wavelength of 640 nm, a light path of 1 cm, and zeroed with double distilled water.

[0082] [Note]: The standard concentration is 10 mmol / L urea nitrogen, which is equivalent to 280.1 mg / L.

[0083] The formula for calculating urea nitrogen content is as follows:

[0084] Urea nitrogen content (mmol / L) = [(Determination A - Blank A) - (Standard A - Blank A)] * Standard concentration (10 mmol / L) * Sample dilution factor

[0085] The results of serum urea nitrogen (BUN) levels in kidney tissues of CLP and IR model mice are as follows: Figure 14 As shown in (b) and 20(b), it can be seen that AA significantly reduced the serum urea nitrogen level in the kidney tissue of CLP model mice and IR model mice, with the best reduction effect observed at an AA concentration of 10 mg / kg.

[0086] Table 2: Components added to the test tubes of each experimental group and their content

[0087]

[0088] ③ The mRNA expression levels of kidney injury factors, inflammatory factors, and chemokines in kidney tissue were measured using real-time quantitative PCR. The results of the CLP model are shown below. Figure 9 As shown in the figure, the mRNA expression levels of kidney injury factor KIM1, chemokine MCP-1, and inflammatory factor TNF-α were significantly increased in the kidney tissue of mice in the CLP group, leading to sepsis infection. Conversely, the expression levels of these kidney injury factors, inflammatory factors, and chemokines were significantly decreased in mice in the CLP+AA group. This indicates that AA can significantly inhibit kidney injury and inflammation in the CLP model, thereby improving kidney function in mice infected with sepsis and reducing serum creatinine and urea nitrogen levels in the kidney tissue. Similarly, from... Figure 15 It can be seen that the mRNA expression levels of kidney injury factor KIM1, chemokine MCP-1, and inflammatory factor IL-6 in the kidney tissue of IR group mice were significantly increased, consistent with the CLP model. The above indicators in IR+AA group mice were significantly reduced in a dose-dependent manner, indicating that AA can significantly inhibit the expression of the above factors, thereby improving kidney function damage in ischemia-reperfusion model mice and reducing the levels of serum creatinine and urea nitrogen in mouse kidney tissue.

[0089] Example 7: Study on the effects of AA on kidney histopathology in CLP and IR model mice

[0090] Example 5: After establishing CLP and IR models in mice for 24 hours, the mice were anesthetized, and kidney tissue was collected. The CLP group tissue samples were stained using a hematoxylin and eosin (H&E) staining kit to observe renal tubular damage in the CLP model mice. The results are as follows: Figure 11 As shown, the level of renal tubular damage in mice in the CLP group was significantly increased, manifested as dilation of renal tubules and cytoplasmic vacuoles, while the level of renal tubular damage in mice in the CLP+AA (10mg / kg) group was significantly reduced, indicating that AA has a significant ameliorative effect on renal tissue pathology in septic mice.

[0091] Example 5: Twenty-four hours after establishing an IR model in mice, the mice were anesthetized, and kidney tissue was collected. The mouse kidney tissue was stained using a glycogen (PAS) staining kit. The staining results are as follows: Figure 17 As shown in the figure, the renal tubular injury of IR model mice was observed. It can be seen that the level of renal tubular injury in the IR group mice was significantly increased, mainly manifested as renal tubular dilation and glycogen deposition, while the level of renal tubular injury in the IR+AA (10mg / kg) group mice was significantly reduced, indicating that AA has a significant ameliorative effect on the renal tissue pathology of ischemia-reperfusion mice.

[0092] Example 8: Study on the effect of AA on renal tissue damage in in vivo CLP and IR model mice

[0093] Example 5: After establishing CLP and IR models in mice for 24 hours, the mice were anesthetized, and kidney tissue was collected. KIM1 fluorescence staining was performed on the kidneys to observe the effect on kidney injury in mice. The staining results were... Figure 12 Observation of renal tubular injury in CLP model mice showed that the level of renal tubular injury was significantly increased in the CLP group, while it was significantly decreased in the CLP+AA (10 mg / kg) group, indicating that AA can improve acute kidney injury in septic mice. The staining results of the renal injury factor KIM1 in the IR model are as follows: Figure 18 As shown, the level of renal tubular injury in mice in the IR+AA (10 mg / kg) group was significantly reduced, indicating that AA can improve ischemia-reperfusion induced acute kidney injury in mice.

[0094] Example 9: Study on the effect of AA on renal tissue inflammation in CLP and IR model mice in vivo.

[0095] Example 5: After establishing CLP and IR models in mice for 24 hours, the mice were anesthetized, and kidney tissue was collected. F4 / 80 histochemical staining of the kidneys was performed to observe the effect on acute kidney injury and inflammation in mice. The staining results of the CLP model are as follows: Figure 13 As shown, the CLP group mice showed a significant increase in renal inflammation, indicating successful model establishment. Conversely, the CLP+AA (10 mg / kg) group mice showed a significant decrease in renal inflammation, indicating that AA can reduce the number of macrophages infiltrating the renal tissue of septic mice and has a significant anti-inflammatory effect. The F4 / 80 histochemical staining results of the IR model are shown below. Figure 19 As shown in the figure (IR+AA in the figure refers to IR+AA (10mg / kg)), the results are consistent with the CLP model.

[0096] Example 10: Study on the effects of AA on the histopathology of heart, liver, spleen, lung and kidney tissues in wild-type mice.

[0097] In Example 5, while establishing CLP and IR models in mice, six mice in the WT group were subjected to three gavage administrations of AA (10 mg / kg). After 24 hours, the mice were anesthetized, and tissues of the heart, liver, spleen, lungs, and kidneys were collected, along with blood samples from the eyeballs. The hematoxylin and eosin (H&E) staining kit was used to stain the mouse tissues. The staining results are as follows: Figure 21 As shown, no tissue damage was observed in the mice in the drug-only group. Serum ALT and AST levels were measured, and the results are as follows: Figure 22 As shown in the above, this dose of AA is not toxic to normal mouse tissues.

[0098] Example 5: After establishing CLP and IR models in mice for 24 hours, the mice were anesthetized, and kidney tissue was collected. The KIM1 protein level in the kidney tissue of both model mice was measured using Western blot. The results are as follows: Figure 10 , Figure 16 As shown in the figure. The results showed that AA could inhibit the expression of KIM1 protein in mouse kidney tissue in CLP and IR models.

[0099] In conclusion, experiments have demonstrated that AA plays a crucial role in acute kidney injury and exhibits significant protective effects against kidney damage. Therefore, this drug holds promise as a key agent in the prevention and treatment of acute kidney injury.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of orange-yellow piperamide in the preparation of drugs for treating acute kidney injury, characterized in that, Its structural formula is .

2. The application according to claim 1, characterized in that, The concentration of the orange-yellow piperidine is 0.1-100 μM.

3. The application according to claim 2, characterized in that, The concentration of the orange-yellow piperidine is 6-100 μM.

4. The application according to claim 2, characterized in that, The concentration of the orange-yellow piperidine is 12.5-50 μM.

5. The application according to claim 2, characterized in that, The concentration of the orange-yellow piperidine was 6.25 μM.

6. The application according to claim 2, characterized in that, The concentration of the orange-yellow piperidine was 12.5 μM.

7. The application according to claim 2, characterized in that, The concentration of the orange-yellow piperidine was 25 μM.

8. The application according to claim 2, characterized in that, The concentration of the orange-yellow piperidine was 50 μM.

9. The application according to claim 2, characterized in that, The concentration of the orange-yellow piperidine was 100 μM.

10. The application according to any one of claims 1-9, characterized in that, The medications for treating acute kidney injury also include pharmaceutically acceptable carriers or excipients.

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