Application of CLIC5 gene or CLIC5 protein in preparing medicine for treating metabolic syndrome

Abstract

The present invention relates to the field of bioengineering technology, and in particular to the use of CLIC5 gene or CLIC5 protein in the preparation of a drug for treating metabolic syndrome. The present invention finds that the CLIC5 gene of skeletal muscle is involved in the pathogenesis of metabolic syndrome, and overexpression of the CLIC5 gene in skeletal muscle can reduce the body weight and adipose tissue weight of mice with metabolic syndrome, significantly improve the glucose tolerance and insulin sensitivity of mice with metabolic syndrome, and significantly reduce the liver weight and fat deposition in the liver of mice with metabolic syndrome. The present invention uses skeletal muscle as a target tissue and provides a new idea and method for treating human metabolic syndrome.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to application of CLIC5 gene or CLIC5 protein in preparing medicine for treating metabolic syndrome. Background Art

[0002] Over the past two decades, with the increase in energy intake and the decrease in activity, the proportion of people suffering from metabolic syndrome has continued to rise. Metabolic syndrome includes a group of diseases such as obesity, non-alcoholic fatty liver disease, glucose intolerance, insulin resistance, and hypertension. Obesity is not only one of the main manifestations of metabolic syndrome, but also plays an important role in the formation of metabolic syndrome. When these diseases occur together, they will greatly increase the morbidity and mortality of cardiovascular diseases.

[0003] Skeletal muscle is the largest tissue in the human body, accounting for 30% to 40% of the body weight of a healthy adult. In addition to its role in exercise, skeletal muscle plays an important role in the body's glucose homeostasis. As the body's largest insulin-sensitive tissue, about 1 / 3 of glucose is taken up by muscle after a meal, and muscle insensitivity to insulin is a typical feature of type 2 diabetes. In contrast, specific genetic intervention in skeletal muscle can improve the metabolic homeostasis of obese mice, including improving insulin sensitivity, reducing fatty degeneration, and alleviating non-alcoholic fatty liver disease. Skeletal muscle can also produce myokines, which affect the functions of organs such as fat, liver, pancreas, and intestines through autocrine and paracrine effects.

[0004] Chloride intracellular channel (CLIC) exists in the cytoplasm, cell membrane and membranes of various organelles, and plays an important role in the occurrence of cardiovascular diseases, cancer and neurodegenerative diseases. Previous studies have reported that systemic mutations in CLIC5 can alleviate diet-induced obesity and fat deposition in the liver, but studies have confirmed that the same gene in the whole body and in different tissues has different effects on metabolic syndrome. Studies have found that knocking out IP in skeletal muscle can reduce the incidence of metabolic syndrome. 3 R1 reduces muscle weight and worsens insulin resistance induced by a high-fat diet; however, knocking out IP in adipocytes 3 R1 can resist obesity caused by a high-fat diet and enhance glucose tolerance. In addition, knocking out P2Y in skeletal muscle 6 R reduces the muscle's ability to take up glucose, leading to glucose metabolism disorders; knocking out P2Y in adipocytes 6 R can resist diet-induced obesity, improve glucose tolerance and insulin sensitivity, and reduce the body's inflammatory response. Therefore, CLIC5 in skeletal muscle may have a different effect than other tissues in affecting metabolic syndrome, but there is no report on the association between skeletal muscle CLIC5 and metabolic syndrome. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of CLIC5 gene or CLIC5 protein in the preparation of a drug for treating metabolic syndrome. The present invention finds that overexpressing CLIC5 gene in skeletal muscle can alleviate obesity caused by a high-fat diet, significantly improve the body's glucose tolerance and insulin sensitivity, and significantly inhibit fat deposition in the liver.

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

[0007] The present invention provides use of a CLIC5 gene or a CLIC5 protein in preparing a drug for treating metabolic syndrome. The amino acid sequence encoded by the CLIC5 gene or the amino acid sequence of the CLIC5 protein is shown in SEQ ID NO.1.

[0008] The present invention provides use of a reagent for overexpressing a CLIC5 gene or a reagent for increasing the expression amount of a CLIC5 protein in preparing a drug for treating metabolic syndrome. The amino acid sequence encoded by the CLIC5 gene or the amino acid sequence of the CLIC5 protein is shown in SEQ ID NO.1.

[0009] Preferably, the CLIC5 gene or CLIC5 protein is the CLIC5 gene or CLIC5 protein in skeletal muscle.

[0010] Preferably, the treatment of metabolic syndrome includes: improving obesity, improving glucose tolerance, enhancing insulin sensitivity and reducing liver fat deposition or more.

[0011] Preferably, the nucleotide sequence of the CLIC5 gene is shown as SEQ ID NO.2.

[0012] The present invention provides a drug for treating metabolic syndrome, comprising an agent for overexpressing the CLIC5 gene; the amino acid sequence encoded by the CLIC5 gene is shown in SEQ ID NO.1.

[0013] Preferably, the reagent for overexpressing the CLIC5 gene comprises a vector for overexpressing the CLIC5 gene.

[0014] Preferably, the vector for overexpressing the CLIC5 gene is a vector for overexpressing the CLIC5 gene in skeletal muscle.

[0015] Preferably, the vector overexpressing the CLIC5 gene comprises an adeno-associated virus vector overexpressing the CLIC5 gene.

[0016] Preferably, the drug further comprises pharmaceutically acceptable excipients; the excipients comprise physiological saline and / or phosphate buffer.

[0017] Beneficial effects:

[0018] The present invention found that the CLIC5 gene of skeletal muscle is involved in the pathogenesis of metabolic syndrome. Overexpression of the CLIC5 gene in skeletal muscle can reduce the body weight and adipose tissue weight of metabolic syndrome mice, significantly improve the glucose tolerance and insulin sensitivity of metabolic syndrome mice, and significantly reduce the liver weight and fat deposition in the liver of metabolic syndrome mice. The present invention uses skeletal muscle as the target tissue and provides a new idea and method for treating human metabolic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 These are the results of the effect of overexpression of CLIC5 gene in skeletal muscle on the liver tissue morphology of mice with metabolic syndrome. DETAILED DESCRIPTION

[0021] The present invention provides the use of CLIC5 gene or CLIC5 protein in preparing a drug for treating metabolic syndrome. The amino acid sequence encoded by the CLIC5 gene or the amino acid sequence of the CLIC5 protein is shown in SEQ ID NO.1, which is as follows:

[0022] MTDSATTNGDDRDPEIELFVKAGIDGESIGNCPFSQRLFMILWLKGVVFNVTTVDLKRKPADLHNLAPGTHPPFLTFNGDVKTDVNKIEEFLEETLTPEKYPKLAAKHRESNTAGIDIFSKFSAYI KNTKQQNNAALERGLTKALRKLDDYLNSPLPEEIDTNTHGDEKGSQRKFLDGDELTLADCNLLPKLHVVKIVAKKYRNYDIPAEMTGLWRYLKNAYARDEFTNTCAADSEIELAYADVARRLSRS.

[0023] The present invention finds that overexpression of CLIC5 gene in skeletal muscle can significantly improve metabolic syndrome caused by a high-fat diet, and CLIC5 gene or CLIC5 protein can be combined with a drug delivery system for targeted drug delivery in skeletal muscle to treat metabolic syndrome.

[0024] The present invention provides an application of an agent for overexpressing the CLIC5 gene or an agent for increasing the expression of the CLIC5 protein in the preparation of a drug for treating metabolic syndrome, wherein the amino acid sequence encoded by the CLIC5 gene or the amino acid sequence of the CLIC5 protein is shown in SEQ ID NO.1. In the present invention, the CLIC5 gene or CLIC5 protein is preferably the CLIC5 gene or CLIC5 protein in skeletal muscle, that is, the agent for overexpressing the CLIC5 gene in skeletal muscle or the agent for increasing the expression of the CLIC5 protein in skeletal muscle can be used to prepare a drug for treating metabolic syndrome; the treatment of metabolic syndrome preferably includes: improving obesity, improving glucose tolerance, enhancing insulin sensitivity and reducing one or more of liver fat deposition; the improvement of obesity preferably includes reducing body weight and / or reducing adipose tissue weight; the nucleotide sequence of the CLIC5 gene is preferably shown in SEQ ID NO.2, specifically as follows:

[0025] 5'-atgacggactcagcgacaactaatggggacgacagggaccccgagatcgagctcttcgtgaaggctgggat cgacggggaaagcattggcaactgtcccttctctcagcgtctcttcatgatcctctggctgaaaggagtcgtgttcaatgtcaccaccgtggatctgaaaagaaagccagccgatctacacaacctggctcctggcacgcacccgcccttcctgaccttcaatggggatgtgaagacagatgtcaacaagattgaagagttcctggaggagaccctaacccccgagaagtaccccaaactggctgcaaaacaccgggaatctaacaccgcgggcatcgacatcttctccaagttctcagcctacatcaaaaacaccaaacaacagaacaatgctgcccttgagagaggcttgacaaaggcgctgaggaagctggatgactacctaaacagccctctgccagaggagattgacaccaacacccacggggacgagaaggggtcccagcgcaagttcctggatggggatgagctgaccctggccgactgcaatctgctgcccaagctgcatgtggtcaagattgtggctaagaagtaccgaaactatgacatcccagctgagatgacaggcttgtggcgatacctcaagaatgcctatgcacgggacgagttcaccaacacctgtgcagctgacagtgagatcgagttggcctacgcagatgtcgccaggcgcctcagccgatcgtga-3'。

[0026] The present invention provides a drug for treating metabolic syndrome, comprising an agent for overexpressing the CLIC5 gene; the amino acid sequence encoded by the CLIC5 gene is shown in SEQ ID NO. 1. In the present invention, the agent for overexpressing the CLIC5 gene comprises a vector that preferably overexpresses the CLIC5 gene; the vector that overexpresses the CLIC5 gene is preferably a vector that overexpresses the CLIC5 gene in skeletal muscle; the vector that overexpresses the CLIC5 gene preferably comprises an adeno-associated virus vector that overexpresses the CLIC5 gene; the drug preferably further comprises a pharmaceutically acceptable excipient; the excipient comprises physiological saline and / or phosphate buffer.

[0027] The present invention finds that overexpressing the CLIC5 gene in skeletal muscle can reduce the body weight and adipose tissue weight of mice with metabolic syndrome, significantly improve the glucose tolerance and insulin sensitivity of mice with metabolic syndrome, and significantly reduce the liver weight and fat deposition in the liver of mice with metabolic syndrome. This characteristic is utilized to prepare a reagent that can overexpress the CLIC5 gene in skeletal muscle into a drug that can be used to treat metabolic syndrome.

[0028] To further illustrate the present invention, the application of the CLIC5 gene or CLIC5 protein provided by the present invention in preparing a drug for treating metabolic syndrome is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] The therapeutic effect of overexpression of CLIC5 gene in skeletal muscle on metabolic syndrome

[0031] 1. Animal modeling, grouping and treatment

[0032] A total of 36 mice with overexpressed CLIC5 in skeletal muscle and mice injected with control AAV vectors in the tibialis anterior muscle were used. The preparation method of the 36 mice refers to Example 2 in Chinese patent CN202311139530.X, wherein the control AAV vector is AAV9:CMV-MSC-3×Flag-EF1-ZsGreen-T2A-Puro plasmid [purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.], and the 36 mice were divided into 3 groups, and the specific groups are as follows:

[0033] ① Control group: 12 mice injected with control AAV vector into the tibialis anterior muscle, fed with control feed (10% fat calories, purchased from Beijing Huafukang Biotechnology Co., Ltd., product number H10010), and distilled water for daily drinking;

[0034] ② High-fat model group: 12 mice injected with control AAV vector into the tibialis anterior muscle, fed with high-fat diet (60% fat calories, purchased from Beijing Huafukang Biotechnology Co., Ltd., product number H10060), and distilled water for daily drinking;

[0035] ③CLIC5 overexpression group: 12 mice with overexpression of CLIC5 in skeletal muscle were fed with high-fat diet (60% of calories from fat, purchased from Beijing Huafukang Biotechnology Co., Ltd., product number H10060), and distilled water was used for daily drinking.

[0036] 2. Index detection

[0037] The total duration of the experiment was 10 weeks. In the 8th week of the experiment, the mice were fasted for 6 hours in the morning and injected with 2g / kg body weight of glucose solution for glucose tolerance test. Blood was collected from the tail vein, and the blood glucose of the mice was measured with a blood glucose meter at fasting and 15, 30, 60, 90 and 120 minutes after glucose injection. In the 9th week of the experiment, the mice were fasted for 6 hours in the morning and injected with 0.75U / kg body weight of insulin for insulin tolerance test. Blood was collected from the tail vein, and the blood glucose of the mice was measured with a blood glucose meter at fasting and 15, 30, 60, 90 and 120 minutes after insulin injection. The area under the blood glucose curve (AUC) was calculated based on the blood glucose values ​​at each time point in the glucose tolerance test and insulin tolerance test. The glucose tolerance and insulin sensitivity of the mice were judged based on the AUC. The lower the AUC, the stronger the glucose tolerance and insulin sensitivity of the mice.

[0038] After the end of the 10th week experiment, the mice were fasted for 12 hours, anesthetized and killed, and then bled. The epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), tibialis anterior muscle (TA), gastrocnemius muscle (GAS) and liver were separated and weighed. A part of the liver was removed and fixed in 4% paraformaldehyde for more than 48 hours. The preparation of liver tissue sections includes dehydration, transparency, preparation of wax blocks, sectioning, baking, hydration, hematoxylin staining, eosin staining, sealing and image collection. The results are shown in Figure 1 (10×).

[0039] 3. Data processing

[0040] The original data were sorted using Excel 2013, and the experimental data were analyzed and compared using SAS (SAS 9.2). The differences between the groups were tested using the independent sample t test. P < 0.05 was considered to be significantly different, P < 0.01 was considered to be extremely significantly different, and 0.05 ≤ P ≤ 0.10 was considered to have a trend of change. The data were expressed as "mean ± standard deviation".

[0041] 4. Test results

[0042] Table 1 Effect of overexpression of CLIC5 in skeletal muscle on body weight of mice with metabolic syndrome (g)

[0043]

[0044]

[0045] Note: Compared with the control group, ΔP<0.05, ΔΔP<0.01; compared with the high-fat model group, *P<0.05, **P<0.01. Table 2 Effects of skeletal muscle overexpression of CLIC5 on the weight of adipose tissue, skeletal muscle and liver in mice with metabolic syndrome

[0046]

[0047] As shown in Table 1, compared with the control group, the high-fat diet significantly increased the body weight of mice from the second week of feeding (P<0.05), leading to obesity, while overexpression of CLIC5 in skeletal muscle significantly reduced the body weight of mice at the 4th, 7th, 8th, 9th and 10th weeks of the experiment (P<0.05). At the end of the experiment, the body weight of mice in the CLIC5 overexpression group was reduced by 11.31% compared with the high-fat model group. Analysis of the body composition of mice showed that overexpression of CLIC5 in skeletal muscle significantly reduced the weight of iWAT and liver of mice and increased the weight of TA. It also significantly reduced the relative weight of iWAT and liver and increased the relative weight of TA (Table 2). Further analysis of the histomorphology of the liver showed that overexpression of CLIC5 in skeletal muscle greatly reduced liver fat deposition caused by a high-fat diet ( Figure 1 The above results indicate that overexpression of CLIC5 in skeletal muscle can significantly improve obesity induced by a high-fat diet, reduce fat deposition in adipose tissue and liver, and increase muscle weight.

[0048] Table 3 Effect of overexpression of CLIC5 in skeletal muscle on glucose tolerance in mice with metabolic syndrome (mmol / L)

[0049]

[0050]

[0051] The glucose tolerance test showed that (Table 3) after intraperitoneal injection of glucose, compared with the high-fat model group, overexpression of CLIC5 in skeletal muscle significantly reduced blood glucose levels at 15 and 120 min after injection (P<0.05), and the fasting blood glucose and blood glucose levels at 30, 60 and 90 min after injection tended to decrease (0.05≤P≤0.10), and the AUC was significantly reduced (P<0.05), indicating that overexpression of CLIC5 in skeletal muscle significantly improved the glucose tolerance of mice fed a high-fat diet.

[0052] Table 4 Effect of overexpression of CLIC5 in skeletal muscle on insulin sensitivity in mice with metabolic syndrome (mmol / L)

[0053] Group High-fat model group CLIC5 overexpression group P-value Fasting blood sugar 9.15±1.53 7.24±1.90 0.05 15min 8.27±1.37 6.93±1.75 0.06 30min 7.00±1.82 5.53±1.61 0.06 60min 6.62±1.33 4.92±1.73 0.02 90min 6.50±1.49 4.93±1.51 0.02 120min 6.22±1.09 4.93±1.36 0.02 AUC 649±122 511±127 0.02

[0054] The insulin tolerance test showed that (Table 4) after intraperitoneal injection of insulin, compared with the high-fat model group, overexpression of CLIC5 in skeletal muscle significantly reduced blood glucose levels at 60, 90 and 120 min after injection (P<0.05), and there was a trend of decreasing fasting blood glucose and blood glucose levels at 15 and 30 min after injection (0.05≤P≤0.10), and the AUC was significantly reduced (P<0.05), indicating that overexpression of CLIC5 in skeletal muscle significantly improved the insulin sensitivity of mice fed a high-fat diet.

[0055] In summary, in the metabolic syndrome model constructed using a high-fat diet, overexpression of the CLIC5 gene in skeletal muscle can improve obesity, reduce adipose tissue weight, and significantly improve other metabolic syndrome symptoms in mice, including improving glucose tolerance and insulin sensitivity, and greatly reducing fat deposition in the liver.

[0056] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Overexpression CLIC5 Use of a gene vector in the preparation of a drug for treating metabolic syndrome, the CLIC5 The nucleotide sequence of the gene is shown in SEQ ID NO.2; the treatment of metabolic syndrome is to reduce fat deposition in the mouse liver; the overexpression CLIC5 Gene vectors for overexpression in skeletal muscle CLIC5 Gene vector.

2. The use according to claim 1, characterized in that: Said CLIC5 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The overexpression CLIC5 Gene vectors include overexpression CLIC5 Adeno-associated virus vectors.

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