Application of rehmannia leaf total glycosides in preparation of medicine for down-regulating expression level of pro-inflammatory cytokines

Abstract

The invention provides application of rehmannia leaf total glycosides in preparation of a medicine for lowering the expression level of a pro-inflammatory cytokine, and relates to the technical field of inhibition of pro-inflammatory cytokine expression. The inventor finds that the rehmannia leaf total glycosides can play a role in lowering the expression level of pro-inflammatory cytokines through research; meanwhile, compared with the existing common immunosuppressant, such as tripterygium glycosides; the adverse reaction of the rehmannia leaf total glycosides is less.

Description

Technical Field

[0001] The invention relates to the technical field of inhibiting the expression of pro-inflammatory cytokines, and in particular to the application of total glycosides of Rehmannia glutinosa leaves in preparing a medicine for down-regulating the expression level of pro-inflammatory cytokines. Background Art

[0002] Pro-inflammatory cytokines are a class of endogenous polypeptides that are mainly produced by immune system cells and have many powerful biological effects. They can mediate a variety of immune responses. When pro-inflammatory cytokines are expressed at high levels in the body, they can cause inflammatory responses in the body and, in severe cases, damage to the body's organs.

[0003] Among them, IL-1β and IL-17 in proinflammatory cytokines activate extracellular regulated protein kinase and MAPK kinase pathways, inducing the production of a variety of cytokines, chemokines, and inflammatory effector substances to participate in immune inflammatory responses. When IL-1β and IL-17 are expressed at high levels in the body, they can cause inflammatory cell infiltration and tissue destruction, leading to severe inflammatory responses in the body. Summary of the invention

[0004] The purpose of the present invention is to provide the use of total glycosides of Rehmannia glutinosa leaves in the preparation of drugs for down-regulating the expression level of pro-inflammatory cytokines, so as to solve the problems mentioned in the background technology.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of drugs for downregulating the expression level of pro-inflammatory cytokines.

[0007] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of drugs for downregulating the expression level of IL-1β or IL-17 in proinflammatory cytokines.

[0008] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of a drug for treating mesangial cell hyperplasia by downregulating the expression level of IL-1β or IL-17 in proinflammatory cytokines.

[0009] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of drugs for treating increased membrane matrix by downregulating the expression level of IL-1β or IL-17 in proinflammatory cytokines.

[0010] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of drugs for treating thickening of mesangial area by downregulating the expression level of IL-1β or IL-17 in proinflammatory cytokines.

[0011] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of a drug for treating mesangial proliferative glomerulonephritis by downregulating the expression level of IL-1β or IL-17 in proinflammatory cytokines.

[0012] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of a drug for treating elevated urine protein by downregulating the expression level of IL-1β or IL-17 in proinflammatory cytokines.

[0013] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of drugs for treating elevated cystatin C by downregulating the expression levels of IL-1β or IL-17 in proinflammatory cytokines.

[0014] Application of total glycosides of Rehmannia glutinosa leaves in the preparation of a drug for treating elevated β-N-acetylglucosaminidase by downregulating the expression level of IL-1β or IL-17 in proinflammatory cytokines.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly embodied in:

[0016] The inventors have found that total glycosides from Rehmannia glutinosa can downregulate the expression level of proinflammatory cytokines, especially the expression level of IL-1β or IL-17 in proinflammatory cytokines. On this basis, the inventors have further studied and found that total glycosides from Rehmannia glutinosa can effectively treat various inflammatory reactions and diseases caused by high levels of IL-1β or IL-17 expression. For example: mesangial cell hyperplasia, increased membrane matrix, thickening of mesangial area width, mesangial proliferative glomerulonephritis, increased cystatin C, and increased β-N-acetylglucosaminidase. The advantage of using total glycosides from Rehmannia glutinosa is that compared with existing commonly used immunosuppressants, such as Tripterygium wilfordii glycosides; total glycosides from Rehmannia glutinosa can not only achieve better results, but more importantly, the toxic and side effects of total glycosides from Rehmannia glutinosa are much lower than those of Tripterygium wilfordii glycosides. The known adverse reactions of Tripterygium wilfordii glycosides include: dry mouth, nausea, vomiting, fatigue, loss of appetite, abdominal distension, diarrhea, jaundice, elevated transaminase, acute toxic liver damage, gastric bleeding, granulocytopenia, pancytopenia, edema, abnormal renal function, palpitations, chest tightness, arrhythmia, etc. The known adverse reaction of Rehmannia glutinosa glycosides is only mild abdominal distension. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0018] Figure 1 The results of observation of kidney tissue sections of mice in each group under a microscope; A is the control group, B is the model group, C is the tripterygium wilfordii polyglycosides group, and D is the total glycosides of Rehmannia glutinosa group. DETAILED DESCRIPTION

[0019] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. In the description of the present invention, it should be understood that the term "or" means "either this, that, or both"; "NAG" means the abbreviation of "β-N-acetylglucosaminidase"; "CysC" means the abbreviation of "cystatin C"; "ELISA" means the abbreviation of "enzyme-linked immunosorbent assay"; "24hUP" stands for "24-hour urine protein". If no specific conditions are specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0020] Pro-inflammatory cytokines are a class of endogenous polypeptides that are mainly produced by immune system cells and have many powerful biological effects. They can mediate a variety of immune responses. When pro-inflammatory cytokines are expressed at high levels in the body, they can cause inflammatory responses in the body, and in severe cases, they can also cause damage to the body's organs. For example, when IL-1β and IL-17, which are pro-inflammatory cytokines, are expressed at high levels in the body, they can cause inflammatory cell infiltration and tissue destruction, leading to severe inflammatory responses in the body.

[0021] The immunosuppressants used in clinical practice, such as Tripterygium wilfordii polyglycosides, often have various adverse reactions due to individual differences among patients, such as dry mouth, nausea, vomiting, fatigue, loss of appetite, abdominal distension, diarrhea, jaundice, elevated transaminase, acute toxic liver damage, gastric bleeding, granulocytopenia, pancytopenia, edema, abnormal renal function, palpitations, chest tightness, arrhythmia, etc.

[0022] Based on this, this embodiment provides a new way to down-regulate the expression level of pro-inflammatory cytokines, that is, a new use of total glycosides from the leaves of Rehmannia glutinosa; its greatest advantage is that it greatly reduces adverse reactions. The details are as follows:

[0023] This embodiment provides the use of total glycosides of Rehmannia glutinosa leaves in the preparation of a drug for down-regulating the expression level of pro-inflammatory cytokines, especially in the preparation of a drug for down-regulating the expression level of IL-1β or IL-17 in pro-inflammatory cytokines.

[0024] The inventors have found that total glycosides from Rehmannia glutinosa can effectively downregulate the expression levels of IL-1β or IL-17 in the blood and kidneys.

[0025] Furthermore, the inventors have found that when high-level expression of IL-1β or IL-17 is accompanied by mesangial cell hyperplasia, total glucosides of Rehmannia glutinosa can effectively improve the symptoms of mesangial cell hyperplasia; therefore, total glucosides of Rehmannia glutinosa can be used to prepare drugs for treating mesangial cell hyperplasia caused by high-level expression of IL-1β or IL-17.

[0026] Furthermore, the inventors have found that when high-level expression of IL-1β or IL-17 is accompanied by increased membrane matrix, total glycosides of Rehmannia glutinosa can effectively improve the symptoms of increased membrane matrix; therefore, total glycosides of Rehmannia glutinosa can be used to prepare drugs for treating increased membrane matrix caused by high-level expression of IL-1β or IL-17.

[0027] Furthermore, the inventors have found that when high-level expression of IL-1β or IL-17 is accompanied by thickening of the mesangial width, total glucosides of Rehmannia glutinosa can effectively improve the symptoms of thickening of the mesangial width; therefore, total glucosides of Rehmannia glutinosa can be used to prepare drugs for treating thickening of the mesangial width caused by high-level expression of IL-1β or IL-17.

[0028] Furthermore, mesangial cell proliferation, increased membrane matrix, and thickening of the mesangial area are typical symptoms of mesangial proliferative glomerulonephritis; therefore, total glycosides of Rehmannia glutinosa can be used to prepare drugs for treating mesangial proliferative glomerulonephritis caused by high levels of IL-1β or IL-17 expression.

[0029] Furthermore, mesangial proliferative glomerulonephritis is usually accompanied by symptoms of increased urinary protein; the inventors have found that total glycosides from Rehmannia glutinosa leaves can effectively improve the symptoms of increased urinary protein; therefore, total glycosides from Rehmannia glutinosa leaves can be used to prepare drugs for treating increased urinary protein caused by high levels of IL-1β or IL-17 expression.

[0030] Furthermore, mesangial proliferative glomerulonephritis is usually accompanied by symptoms of elevated cystatin C; the inventors have found that total glycosides from Rehmannia glutinosa leaves can effectively improve the symptoms of elevated cystatin C; therefore, total glycosides from Rehmannia glutinosa leaves can be used to prepare drugs for treating elevated cystatin C caused by high levels of IL-1β or IL-17 expression.

[0031] Furthermore, mesangial proliferative glomerulonephritis is usually accompanied by symptoms of elevated β-N-acetylglucosaminidase; the inventors have found that total glycosides from Rehmannia glutinosa leaves can effectively improve the symptoms of elevated β-N-acetylglucosaminidase; therefore, total glycosides from Rehmannia glutinosa leaves can be used to prepare drugs for treating elevated β-N-acetylglucosaminidase caused by high levels of IL-1β or IL-17 expression.

[0032] Herein, the term "drug" may refer to tablets, capsules, granules, aerosols, syrups or emulsions.

[0033] The application effect of the total glycosides of Rehmannia glutinosa provided by the embodiment of the present invention is evaluated in combination with the test data.

[0034] This animal experiment was reviewed and approved by the Animal Ethics Committee with the resolution number: DWLL-20230103. Two hundred ICR clean-grade male mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd., weighing 20 to 40 g and aged 8 to 10 weeks.

[0035] The mice were first fed for 5 days to adapt to the environment, and then randomly divided into four groups: control group, model group, tripterygium wilfordii polyglycosides group, and rehmannia glutinosa total glycosides group.

[0036] Construction of MsPGN model: For the model group, tripterygium wilfordii polyglycosides group, and rehmannia glutinosa total glycosides group, anti-thymocyte serum was injected through the tail vein, 1 ml / 100 g, once a week, for a total of four injections, to construct the MsPGN mouse model.

[0037] Preparation of medicine: Dissolve tripterygium wilfordii polyglycoside tablets in distilled water to prepare a 10 mg / L tripterygium wilfordii polyglycoside solution; dissolve the contents of Rehmannia glutinosa total glycoside capsules in distilled water to prepare a 20 mg / L Rehmannia glutinosa total glycoside solution. Among them, tripterygium wilfordii polyglycoside tablets are produced by Yuanda Pharmaceutical Huangshi Feiyun Pharmaceutical Co., Ltd.; Rehmannia glutinosa total glycoside capsules are produced by Sichuan Meidakang Pharmaceutical Co., Ltd.

[0038] Drug treatment: The tripterygium wilfordii polyglycosides group was given tripterygium wilfordii polyglycosides solution by gavage, 10 ml / kg, once a day, for 12 consecutive weeks; the total glycosides of Rehmannia glutinosa group was given total glycosides of Rehmannia glutinosa solution by gavage, 20 ml / kg, once a day, for 12 consecutive weeks; the control group and the model group were given 0.9% sodium chloride solution by gavage during these 12 weeks.

[0039] Among them, the test data were analyzed using SPSS24.0 statistical software; all experimental data were normally distributed, and the results were One-way analysis of variance was used to perform statistical analysis on the results of multi-sample experiments.

[0040] 1. Comparison of the expression levels of pro-inflammatory cytokines in each group of mice

[0041] Peripheral blood was collected from mice one day before treatment and after 4, 8, and 12 weeks of treatment, and centrifuged at 2500 r / min for 15 min. The supernatant was tested by ELISA to obtain the expression levels of pro-inflammatory cytokines IL-1β and IL-17 in the serum of mice in each group. The results are shown in Table 1.

[0042] After 12 weeks of treatment, the mice were anesthetized and killed, and the kidneys were separated and divided into two equal parts. One of the kidney tissues was placed in a glass homogenizer, put into a 0.9% sodium chloride solution at a mass ratio of 1:9, and fully ground under ice bath conditions to make a 10% kidney homogenate; then centrifuged at 5000r / min for 20min, and the supernatant was taken for detection by ELISA to obtain the expression levels of proinflammatory cytokines IL-1β and IL-17 in the kidney homogenate of each group of mice. The results are shown in Table 2.

[0043] Table 1. Expression levels of IL-1β and IL-17 in the serum of mice in each group ( n=8)

[0044]

[0045]

[0046] * P < 0.05 compared with the control group; # P<0.05 compared with the model group.

[0047] Table 2. Expression levels of IL-1β and IL-17 in kidney homogenates of mice in each group ( n=8)

[0048]

[0049] * P < 0.05 compared with the control group; # P<0.05 compared with the model group.

[0050] As can be seen from Tables 1 and 2, the expression levels of IL-1β and IL-17 in the kidney homogenate and serum of the mice in the model group were significantly higher than those in the control group (P < 0.05); after treatment, the expression levels of IL-1β and IL-17 in the kidney homogenate and serum of the mice in the Tripterygium wilfordii polyglycosides group and the total glycosides of Rehmannia glutinosa group were significantly lower than those in the model group (P < 0.05). In addition, the expression levels of IL-1β and IL-17 in the kidney homogenate of mice were consistent with the change trend in serum.

[0051] 2. Comparison of renal tissue pathological morphology of mice in each group

[0052] Another kidney tissue obtained after the mice were killed was rinsed with 0.9% sodium chloride solution; then dried with filter paper, and then placed in 10% neutral formaldehyde solution for 48 hours; then dehydrated, paraffin embedded, and sliced. The thickness of the pathological slices was 4μm; after slicing, it was stained with hematoxylin and alcohol-eosin, and sealed with neutral resin; then the tissue morphology and pathological changes were observed under a microscope. The results are detailed in Figure 1 .

[0053] Depend on Figure 1 It can be seen that the glomerular structure of the control group mice was normal, and there was no abnormality in the capillary wall and the width of the mesangial area; the glomerular capillary wall and the width of the mesangial area of ​​the model group mice were thickened, and the glomerular mesangial cells proliferated, and the mesangial matrix increased; after treatment, the width of the mesangial area of ​​the mice in the tripterygium wilfordii polyglycoside group and the total glycosides of Rehmannia glutinosa group was smaller than that of the model group mice, and the proliferation of glomerular mesangial cells and the increase of mesangial matrix were significantly alleviated.

[0054] 3. General situation and comparison of 24hUP

[0055] None of the mice in each group died during the experiment. The mice in the control group had shiny fur, normal food intake, and sensitive reactions. After the model was established, the mice in the model group, the tripterygium wilfordii polyglycosides group, and the total glycosides of Rehmannia glutinosa group all developed systemic edema and ascites, and had slow reactions, dull fur, and reduced food intake. After treatment, the mice in the tripterygium wilfordii polyglycosides group had dull fur, slightly increased activity, and significantly increased food intake; the mice in the total glycosides of Rehmannia glutinosa group performed slightly better than the model group and the tripterygium wilfordii polyglycosides group; the systemic edema and ascites of the mice in the model group worsened, and other conditions did not improve.

[0056] The 24-hour urine of mice in each group was collected using metabolic cages one day before treatment and after 4, 8, and 12 weeks of treatment, and the 24-hour UP was detected using conventional biochemical methods. The results are shown in Table 3.

[0057] Table 3. 24hUP of mice in each group ( mg / d, n=8)

[0058]

[0059] * P < 0.05 compared with the control group; # P<0.05 compared with the model group.

[0060] As can be seen from Table 3, before treatment, the 24hUP of mice in the model group, the tripterygium wilfordii polyglycosides group, and the total glycosides of Rehmannia glutinosa group were significantly higher than those in the control group (P < 0.05). After 4 weeks of treatment, the 24hUP of mice in the tripterygium wilfordii polyglycosides group and the total glycosides of Rehmannia glutinosa group did not decrease significantly; after 8 and 12 weeks of treatment, the 24hUP of mice in the tripterygium wilfordii polyglycosides group and the total glycosides of Rehmannia glutinosa group was significantly lower than that of mice in the model group (P < 0.05).

[0061] 4. Comparison of renal function of mice in each group

[0062] Peripheral blood was collected from mice one day before treatment and after 4, 8, and 12 weeks of treatment; the blood was centrifuged at 2500 r / min for 15 min, and the supernatant was collected; the CysC content was detected by ELISA, and the NAG content was detected by colorimetry. The results are shown in Table 4.

[0063] Table 4. CysC and NAG contents in serum of mice in each group ( n=8)

[0064]

[0065] As can be seen from Table 4, the CysC content and NAG content in the serum of the mice in the model group were significantly higher than those in the control group (P < 0.05); after 4, 8, and 12 weeks of treatment, the CysC content and NAG content in the serum of the mice in the Tripterygium wilfordii polyglycosides group and the total glycosides of Rehmannia glutinosa group were significantly lower than those in the model group (P < 0.05), and continued to decrease with the increase of treatment time.

[0066] In summary, the present invention discloses that total glycosides of Rehmannia glutinosa can down-regulate the expression level of pro-inflammatory cytokines, especially the expression level of IL-1β or IL-17 in inflammatory cytokines. Therefore, total glycosides of Rehmannia glutinosa can be used to prepare drugs for treating diseases or symptoms caused by high-level expression of IL-1β or IL-17. Such as: mesangial cell hyperplasia, increased membrane matrix, thickening of mesangial area width, mesangial proliferative glomerulonephritis, increased urine protein, increased cystatin C, and increased β-N-acetylglucosaminidase caused by high-level expression of IL-1β or IL-17.

[0067] Although specific embodiments of the present invention are described above, those skilled in the art should understand that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention, and these changes and modifications are all within the scope of protection of the present invention.

Claims

1. Application of total glycosides from Rehmannia glutinosa leaves in the preparation of drugs for downregulating the expression levels of pro-inflammatory cytokines.

2. The use according to claim 1, characterized in that: Application of total glycosides of Rehmannia glutinosa leaves in the preparation of drugs for downregulating the expression level of IL-1β or IL-17 in proinflammatory cytokines.

3. The use according to claim 2, characterized in that: Application of total glycosides of Rehmannia glutinosa leaves in preparing medicine for treating mesangial cell hyperplasia.

4. The use according to claim 2, characterized in that: Application of total glycosides of Rehmannia glutinosa leaves in the preparation of medicines for treating increased membrane matrix.

5. The use according to claim 2, characterized in that: Application of total glycosides of Rehmannia glutinosa leaves in the preparation of medicines for treating thickening of mesangial area width.

6. The use according to claim 2, characterized in that: Application of total glycosides of Rehmannia glutinosa leaves in preparing medicines for treating mesangial proliferative glomerulonephritis.

7. The use according to claim 2, characterized in that: Application of total glycosides of Rehmannia glutinosa leaves in preparing medicine for treating elevated urine protein.

8. The use according to claim 2, characterized in that: Application of total glycosides of Rehmannia glutinosa leaves in the preparation of medicines for treating elevated cystatin C.

9. The use according to claim 2, characterized in that: Application of total glycosides of Rehmannia glutinosa leaves in preparing medicine for treating elevated β-N-acetylglucosaminidase.

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