Application of shRNA (short hairpin ribonucleic acid) in preparation of medicine for relieving and / or treating diabetic nephropathy

Abstract

The invention relates to application of shRNA (short hairpin Ribonucleic Acid) in preparation of a medicine for relieving and / or treating diabetic nephropathy, and belongs to the technical field of medical treatment. The invention provides shRNA (short hairpin Ribonucleic Acid). The nucleotide sequence of the shRNA is shown as SEQ ID NO. 1. The shRNA and the recombinant virus plasmid containing the shRNA can interfere with the expression of branched ketonic acid dehydrogenase kinase, so that the damage of diabetes to normal physiological functions of kidneys is effectively relieved, and the shRNA and the recombinant virus plasmid containing the shRNA can be used for relieving and / or treating diabetic nephropathy and have the characteristics of no side effect, economy, high efficiency, short treatment time and remarkable curative effect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of shRNA in preparing a medicine for alleviating and / or treating diabetic nephropathy. Background Art

[0002] Diabetic nephropathy is a disease of kidney structure and function damage caused by diabetes. It is one of the most important microvascular complications of diabetes. Its clinical manifestations include symptoms such as massive proteinuria, hypertension, edema, etc. Renal function tests show decreased glomerular filtration rate and albuminuria.

[0003] Diabetic nephropathy has become an important cause of chronic kidney disease and renal failure. Among patients with type 2 diabetes, which accounts for more than 95% of all diabetic patients, nearly 40% suffer from diabetic nephropathy, a complication with high disability and high mortality. Even most patients already have kidney involvement symptoms such as albuminuria or renal dysfunction when they are diagnosed with diabetes. Therefore, when they develop end-stage renal disease (i.e. uremia), they have to rely on expensive renal replacement therapy to maintain their lives.

[0004] At present, there is an extreme shortage of therapeutic drugs specifically targeting renal complications, and treatment options are limited to lifestyle intervention and blood sugar and blood pressure control. Problems such as recurrence of diabetic nephropathy in kidney transplant patients and increased infection risk due to immunosuppression mean that kidney transplantation cannot cure diabetic patients once and for all. Although some drugs that can delay diabetic kidney damage have been discovered, their mechanism of action has not yet been elucidated. Summary of the invention

[0005] The purpose of the present invention is to provide an application of shRNA in the preparation of a drug for alleviating and / or treating diabetic nephropathy, so as to solve the problems in the prior art of extremely scarce therapeutic drugs specifically targeting diabetic nephropathy, single treatment means and unsatisfactory treatment effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a shRNA, the nucleotide sequence of the shRNA is shown as SEQ ID NO.1.

[0008] The present invention also provides the use of the shRNA in preparing a preparation for interfering with or silencing the expression of branched-chain ketoacid dehydrogenase kinase.

[0009] The present invention also provides the use of the shRNA in preparing a preparation for alleviating and / or treating diabetic nephropathy.

[0010] The present invention also provides a recombinant viral plasmid, wherein the recombinant viral plasmid comprises the shRNA and an empty vector;

[0011] The empty vector is an adeno-associated virus vector AAV.

[0012] The present invention also provides the use of the recombinant virus plasmid in preparing a preparation for interfering with or silencing the expression of branched-chain ketoacid dehydrogenase kinase.

[0013] The present invention also provides the use of the recombinant virus plasmid in preparing a preparation for alleviating and / or treating diabetic nephropathy.

[0014] The present invention also provides a medicine for alleviating and / or treating diabetic nephropathy, wherein the medicine comprises the shRNA or the recombinant virus plasmid.

[0015] The present invention also provides the use of interfering with or silencing the expression of branched-chain ketoacid dehydrogenase kinase in preparing a preparation for alleviating and / or treating diabetic nephropathy.

[0016] Preferably, the preparation for alleviating and / or treating diabetic nephropathy comprises the shRNA or the recombinant viral plasmid.

[0017] The present invention has the following technical effects and advantages:

[0018] The shRNA of the present invention and the recombinant viral plasmid containing shRNA can interfere with the expression of branched-chain ketoacid dehydrogenase kinase, thereby effectively alleviating the damage caused by diabetes to the normal physiological function of the kidney, as well as the hypertrophic lesions of the glomeruli and renal tubules caused by diabetes, and can be used to alleviate and / or treat diabetic nephropathy, and has the characteristics of no side effects, economical and high efficiency, short treatment time and significant therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of mouse urinary albumin excretion test, in which Con+NC is the negative control blank group, Con+shBCKDK is the shRNA blank group, DM+NC is the negative control model group, and DM+shBCKDK is the shRNA model group;

[0020] Figure 2 HE pathological staining of mouse kidney tissue, in which Con+NC is the negative control treatment blank group, Con+shBCKDK is the shRNA treatment blank group, DM+NC is the negative control treatment model group, and DM+shBCKDK is the shRNA treatment model group;

[0021] Figure 3The results of the glomerular capillary loop area of ​​mice, in which Con+NC is the negative control blank group, Con+shBCKDK is the shRNA blank group, DM+NC is the negative control model group, and DM+shBCKDK is the shRNA model group;

[0022] Figure 4 The results are the percentage of mesangial area in mice. In the figure, Con+NC is the negative control blank group, Con+shBCKDK is the shRNA blank group, DM+NC is the negative control model group, and DM+shBCKDK is the shRNA model group.

[0023] Figure 5 This is a PAS pathological staining image of mouse kidney tissue, in which Con+NC is the negative control treatment blank group, Con+shBCKDK is the shRNA treatment blank group, DM+NC is the negative control treatment model group, and DM+shBCKDK is the shRNA treatment model group;

[0024] Figure 6 The results are for the number of renal tubules per unit area in mice. In the figure, Con+NC is the negative control treatment blank group, Con+shBCKDK is the shRNA treatment blank group, DM+NC is the negative control treatment model group, and DM+shBCKDK is the shRNA treatment model group;

[0025] Figure 7 The results are the average renal tubular area of ​​mice. In the figure, Con+NC is the negative control treatment blank group, Con+shBCKDK is the shRNA treatment blank group, DM+NC is the negative control treatment model group, and DM+shBCKDK is the shRNA treatment model group. DETAILED DESCRIPTION

[0026] The present invention provides a shRNA, the nucleotide sequence of the shRNA is shown as SEQ ID NO.1.

[0027] The present invention also provides the use of the shRNA in preparing a preparation for interfering with or silencing the expression of branched-chain ketoacid dehydrogenase kinase.

[0028] The present invention also provides the use of the shRNA in preparing a preparation for alleviating and / or treating diabetic nephropathy.

[0029] The present invention also provides a recombinant viral plasmid, wherein the recombinant viral plasmid comprises the shRNA and an empty vector;

[0030] The empty vector is an adeno-associated virus vector AAV.

[0031] The present invention also provides the use of the recombinant virus plasmid in preparing a preparation for interfering with or silencing the expression of branched-chain ketoacid dehydrogenase kinase.

[0032] The present invention also provides the use of the recombinant virus plasmid in preparing a preparation for alleviating and / or treating diabetic nephropathy.

[0033] The present invention also provides a medicine for alleviating and / or treating diabetic nephropathy, wherein the medicine comprises the shRNA or the recombinant virus plasmid.

[0034] The present invention also provides the use of interfering with or silencing the expression of branched-chain ketoacid dehydrogenase kinase in preparing a preparation for alleviating and / or treating diabetic nephropathy.

[0035] In the present invention, the preparation for alleviating and / or treating diabetic nephropathy comprises the shRNA or the recombinant virus plasmid.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] The test mice used in the present application were C57BL / 6J male mice purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0038] The empty vector used in the present application is an adeno-associated virus vector AAV, which was purchased from Yike Biotechnology Co., Ltd.

[0039] Example 1: Nucleotide sequence synthesis

[0040] Using the branched-chain keto-acid dehydrogenase kinase gene (BCKDK, see NCBI ID: 12041) as a template, the gene sequence of sites 687 to 706 was selected as the target interference sequence, and the nucleotide sequence of shRNA was designed (as shown in SEQ ID NO.1). At the same time, a scrambled nucleotide sequence that was consistent with the length and modification of the nucleotide sequence of shRNA but did not target any target gene was designed as a negative control (as shown in SEQ ID NO.2), and was commissioned to be synthesized by Yike Biotechnology Co., Ltd. The nucleotide sequences are shown in Table 1.

[0041] Table 1 Nucleotide sequences of shRNA and negative control

[0042] name Sequence (5'~3') SEQ ID NO. shRNA CGAUCAAGGACCAGGCAGATTUCUGCCUGGUCCUUGAUCGTT 1 Negative control UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT 2

[0043] Example 2: Construction of recombinant viral vector

[0044] Referring to the operating instructions of the adeno-associated virus vector AAV, the shRNA described in Example 1 was combined with the adeno-associated virus vector AAV to construct a recombinant virus vector to obtain the recombinant virus plasmid shBCKDK. At the same time, the negative control described in Example 1 was combined with the adeno-associated virus vector AAV to construct a recombinant virus vector to obtain the recombinant virus plasmid NC.

[0045] Example 3: Mouse modeling and treatment

[0046] Twenty-four 8-week-old C57BL / 6J male mice were divided into 4 groups, namely shRNA treatment model group (DM+shBCKDK), negative control treatment model group (DM+NC), shRNA treatment blank group (Con+shBCKDK) and negative control treatment blank group (Con+NC); DM+shBCKDK and DM+NC mice were injected with 120 mg / kg mouse body weight of streptozotocin by tail vein injection, and the venous blood of DM+shBCKDK and DM+NC mice was collected the next day for blood glucose detection. A blood glucose level greater than 16.7 mmol / L indicated that the diabetic model mice were successfully established; and Con+shBCKDK and Con+NC mice were injected with 10 mL / kg mouse body weight of Hank's balanced salt solution by tail vein injection.

[0047] The recombinant viral plasmid shBCKDK constructed in Example 2 was injected into DM+shBCKDK and Con+shBCKDK mice by tail vein injection, and the recombinant viral plasmid NC constructed in Example 2 was injected into DM+NC and Con+NC mice by tail vein injection. The injections were performed twice, the first injection time was at the 12th week after the mouse model was established, and the second injection time was at the 14th week after the mouse model was established. The injection volume was 1.29×10 11 All mice were killed at 16 weeks after mouse modeling.

[0048] Example 4: Detection of urinary albumin excretion in mice

[0049] The urine albumin-creatinine ratio (ACR) of the DM+shBCKDK, DM+NC, Con+shBCKDK and Con+NC mice sacrificed in Example 3 was measured to analyze the renal filtration and reabsorption function lesions caused by diabetes. The results are as follows: Figure 1 shown.

[0050] The results showed that the ACR of DM+NC mice was significantly higher than that of Con+NC mice, indicating that the renal filtration and reabsorption functions of diabetic model mice were impaired; the ACR of DM+shBCKDK mice was significantly lower than that of DM+NC mice, indicating that shRNA interference with the expression of branched-chain ketoacid dehydrogenase kinase can effectively alleviate the damage caused by diabetes to the normal physiological function of the kidney.

[0051] Example 5: Mouse glomerular pathological morphology staining

[0052] The renal tissues of the DM+shBCKDK, DM+NC, Con+shBCKDK and Con+NC mice sacrificed in Example 3 were respectively subjected to HE pathological staining, and the percentage of the glomerular capillary loop area and the mesangial area of ​​each mouse was calculated to analyze the glomerular lesions caused by diabetes. The results are shown in FIG. Figure 2-4 shown.

[0053] The results showed that the glomerular capillary loop area and the percentage of mesangial area of ​​DM+NC mice were significantly increased compared with those of Con+NC mice, indicating that the glomeruli of diabetic model mice showed hypertrophic lesions; the glomerular capillary loop area and the percentage of mesangial area of ​​DM+shBCKDK mice were significantly decreased compared with those of DM+NC mice, indicating that shRNA interference with the expression of branched-chain ketoacid dehydrogenase kinase can effectively alleviate the glomerular hypertrophic lesions caused by diabetes.

[0054] Example 6: Mouse renal tubular pathological morphology staining

[0055] The renal tissues of the DM+shBCKDK, DM+NC, Con+shBCKDK and Con+NC mice sacrificed in Example 3 were subjected to PAS pathological staining, and the number of renal tubules per unit area and the average renal tubular area of ​​each mouse were calculated to analyze the renal tubular lesions caused by diabetes. The results are shown in FIG. Figure 5-7 shown.

[0056] The results showed that the number of renal tubules per unit area in DM+NC mice was significantly reduced compared with that in Con+NC mice, and the average renal tubular area was significantly increased compared with that in Con+NC mice, indicating that the renal tubules of diabetic model mice showed hypertrophic lesions; the number of renal tubules per unit area in DM+shBCKDK mice was significantly increased compared with that in DM+NC mice, and the average renal tubular area was significantly decreased compared with that in DM+NC mice, indicating that shRNA interference with the expression of branched-chain ketoacid dehydrogenase kinase can effectively alleviate renal tubular hypertrophy caused by diabetes.

[0057] As can be seen from the above examples, the present invention provides an application of shRNA in the preparation of a drug for alleviating and / or treating diabetic nephropathy. The shRNA of the present invention and the recombinant viral plasmid comprising shRNA can interfere with the expression of branched-chain ketoacid dehydrogenase kinase, thereby effectively alleviating the damage caused by diabetes to the normal physiological function of the kidney, as well as the hypertrophic lesions of the glomeruli and renal tubules caused by diabetes, and can be used to alleviate and / or treat diabetic nephropathy, with the advantages of no side effects, economical and efficient, short treatment time, and significant efficacy.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A shRNA, characterized in that The nucleotide sequence of the shRNA is shown in SEQ ID NO.

1.

2. Use of the shRNA according to claim 1 in the preparation of a preparation for interfering with or silencing the expression of branched-chain ketoacid dehydrogenase kinase.

3. Use of the shRNA according to claim 1 in preparing a preparation for alleviating and / or treating diabetic nephropathy.

4. A recombinant viral plasmid, characterized in that: The recombinant viral plasmid comprises the shRNA according to claim 1 and an empty vector; The empty vector is an adeno-associated virus vector AAV.

5. Use of the recombinant viral plasmid according to claim 4 in the preparation of a preparation for interfering with or silencing the expression of branched-chain ketoacid dehydrogenase kinase.

6. Use of the recombinant viral plasmid according to claim 4 in the preparation of a preparation for alleviating and / or treating diabetic nephropathy.

7. A drug for alleviating and / or treating diabetic nephropathy, characterized in that: The drug comprises the shRNA according to claim 1 or the recombinant viral plasmid according to claim 4.

8. Use of interference or silencing of the expression of branched-chain ketoacid dehydrogenase kinase in the preparation of a preparation for alleviating and / or treating diabetic nephropathy.

9. The use according to claim 8, characterized in that: The preparation for alleviating and / or treating diabetic nephropathy comprises the shRNA according to claim 1 or the recombinant virus plasmid according to claim 4.

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