Application of regulating cAMP / Wnt / beta-catenin axis to inhibit thoracic aorta EndoMT process in kidney yin deficiency type diabetes

Abstract

The invention discloses application of regulating and controlling a cAMP / Wnt / beta-catenin axis to inhibit a thoracic aorta EndoMT process in kidney yin deficiency type diabetes, and belongs to the field of molecular biological detection of kidney yin deficiency type diabetes. The invention provides an application of regulating and controlling a cAMP / Wnt / beta-catenin axis to inhibit a thoracic aorta EndoMT process in kidney yin deficiency type diabetes mellitus through experimental research. Specifically, a preparation for regulating and controlling the cAMP / Wnt / beta-catenin axis to inhibit the thoracic aorta EndoMT process can be used for preparing a medicine for treating the kidney yin deficiency type diabetes mellitus. According to the invention, the close relation between kidney yin deficiency and diabetic great vasculopathy is proved through experiments, and a scientific basis is provided for developing a novel treatment strategy for kidney yin deficiency type diabetes and vascular complications thereof.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection of kidney yin deficiency type diabetes, and specifically relates to an application of regulating cAMP / Wnt / β-catenin axis to inhibit EndoMT process of thoracic aorta in kidney yin deficiency type diabetes. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar, and its vascular complications are one of the main causes of death and disability in patients. In traditional Chinese medicine theory, diabetes is classified as "dryness", and kidney yin deficiency is one of its main syndromes. Traditional Chinese medicine believes that the kidney is the innate foundation, which stores essence and contains the original yin and yang, and is the root of life activities. Kidney yin deficiency causes virtual fire to disturb the heart and lungs, which manifests as thirst, irritability and heat. Yin deficiency and strong fire burn the spleen and stomach, which makes people hungry. The kidney is the gate of the stomach. If it is not nourished, the opening and closing of the kidney will be lost, resulting in frequent urination and sweet urine. Kidney qi deficiency cannot protect semen, which will lead to qi and yin deficiency for a long time. Yin deficiency and strong fire will cause virtual fire to burn body fluid and damage essence, which will eventually lead to yin and yang deficiency and kidney exhaustion. Kidney yin deficiency may be one of the important factors leading to diabetic macrovascular complications. Yin deficiency develops into qi and yin deficiency over time, and yin damages yang, which eventually affects yin and yang, and deficiency can cause blood stasis, causing diabetic macrovascular lesions. And as the disease progresses, blood stasis becomes an important factor throughout the course of the disease.

[0003] Vascular disease is one of the most common complications of diabetes, involving large, medium and small blood vessels, arteries, capillaries and veins, and can cause damage to important organs such as the cardiovascular system, brain, kidneys, and fundus. In patients with kidney yin deficiency type diabetes, due to insufficient kidney yin and loss of body fluid, the blood becomes thick and the fluidity decreases, which can easily lead to blood stasis and the formation of blood stasis. This state of blood stasis further aggravates the damage to the blood vessels, promotes pathological changes such as atherosclerosis, calcification of the middle layer of the arterial wall, and intimal fibrosis, and ultimately leads to stenosis of the lumen, affecting the blood supply to the organs. Inflammatory damage plays an important role in diabetic macrovascular disease. The method of nourishing yin and activating blood circulation can play an important role in preventing and treating diabetic macrovascular damage by promoting angiogenesis.

[0004] Endothelial-mesenchymal transition (EndoMT) is a biological process that refers to the process in which endothelial cells gradually lose their original characteristics (such as the expression of markers such as VE-cadherin and CD31) under specific stimulation and transform into mesenchymal cells (such as fibroblasts and smooth muscle cells). Its abnormal activation can promote a variety of cardiovascular diseases, such as atherosclerosis, fibrosis, and pulmonary hypertension. Long-term hyperglycemia can activate the transforming growth factor-β (TGF-β) signaling pathway, which is one of the main driving factors of EndoMT. At the same time, the oxidative stress and inflammatory response caused by hyperglycemia can aggravate endothelial cell damage, promote its transformation to mesenchyme, and accelerate vascular fibrosis and sclerosis.

[0005] The treatment principles for kidney yin deficiency type diabetes are mainly based on clearing away heat and moistening dryness, nourishing yin and promoting body fluid, and promoting blood circulation and removing blood stasis. Huangdi Anxiao Capsules are a specialty hospital preparation of the First Affiliated Hospital of Anhui University of Chinese Medicine. It uses coptis chinensis and raw rehmannia as the main medicines, panax notoginseng as the secondary medicine, and ophiopogon japonicus, kudzu root, and loquat leaves as adjuvant medicines. The whole formula has the effects of clearing away heat and moistening dryness, nourishing yin and promoting body fluid, and promoting blood circulation and removing blood stasis.

[0006] The present invention replicates the model of diabetic macroangiopathy in kidney yin deficiency type, explores the vascular protective effect of Huangdi Anxiao Capsule (HDAXC) on diabetic rats with kidney yin deficiency type, and clarifies the mechanism of action of HDAXC in treating diabetic macroangiopathy at the molecular level. Summary of the invention

[0007] The present invention proposes an application of regulating the cAMP / Wnt / β-catenin axis to inhibit the EndoMT process of the thoracic aorta in kidney yin deficiency type diabetes through experimental research, specifically: the preparation that regulates the cAMP / Wnt / β-catenin axis to inhibit the endothelial-mesenchymal transition (EndoMT) process of the thoracic aorta can be used for the preparation of therapeutic drugs for kidney yin deficiency type diabetes.

[0008] As a preferred technical solution of the present invention, the preparation in the application proposed by the present invention can inhibit the increase of cAMP level in the body, regulate the Wnt / β-catenin pathway, and reverse the aggravation of thoracic aorta endothelial-mesenchymal transition (EndoMT).

[0009] First, the present invention uses a high-fat diet combined with a small dose of STZ to induce SD rats to replicate a diabetic macroangiopathy model. STZ can damage pancreatic islet cells to produce high blood sugar. The high blood lipids caused by a high-fat diet are important factors that cause endothelial cell damage. Therefore, a high-fat diet combined with a small dose of STZ to create a type 2 diabetic macroangiopathy model is a more commonly used modeling method today. Kidney yin deficiency is induced in rats by injecting thyroid hormones.

[0010] Secondly, the present invention replicated the model of kidney-yin deficiency type diabetes mellitus macroangiopathy to explore the vascular protective effect of Huangdi Anxiao Capsule (HDAXC) on kidney-yin deficiency type diabetic rats.

[0011] The results showed that HDAXC can effectively improve the manifestations of kidney yin deficiency and vascular pathological damage in diabetic rats, reduce the expression of inflammation-related genes, and inhibit the process of EndoMT in the thoracic aorta, thereby protecting vascular structure and function. These findings not only confirm the close connection between kidney yin deficiency and diabetic macroangiopathy, but also provide a scientific basis for the development of new treatment strategies for kidney yin deficiency diabetes and its vascular complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1Effect of HDAXC on pathological changes of rat thoracic aorta tissue (×200, 50μm).

[0013] Figure 2 Effect of HDAXC on the expression of genes related to Wnt / β-catenin signaling pathway in rat thoracic aorta tissue.

[0014] Figure 3 Effect of HDAXC on the protein expression of TNF-α and IL-1β in rat thoracic aorta tissue.

[0015] Figure 4 Effect of HDAXC on the expression of EndoMT markers in rat thoracic aorta tissue.

[0016] Figure 1-4 Among them: A normal group, B model group, C low-dose HDAXC group, D medium-dose HDAXC group, E high-dose HDAXC group, F metformin group, G Shenqi Jiangtang Granule group. Specific implementation mode

[0017] In this example, a model of macrovascular lesions in diabetic rats with kidney yin deficiency was replicated to explore the vascular protective effect of Huangdi Anxiao Capsule (HDAXC) on diabetic rats with kidney yin deficiency, and to clarify the mechanism of action of HDAXC in the treatment of macrovascular lesions in diabetic rats with kidney yin deficiency at the molecular level.

[0018] 1 Materials

[0019] 1.1 Animals

[0020] The animal experiments in this study were reviewed by the Experimental Animal Ethics Committee of Anhui University of Chinese Medicine, and the ethics number is: 2022035.

[0021] 60 SPF-grade male SD rats, SPF grade. Animal certificate number: SCXK(Yu)2019-0002, provided by Huaxing Experimental Animal Farm in Huiji District, Zhengzhou; weighing 180-220g. They were housed in the animal house of the First Affiliated Hospital of Anhui University of Chinese Medicine.

[0022] 1.2 Drugs

[0023] HDAXC is composed of Coptis chinensis Franch., Puerariae Lobatae (Wild.) Ohwi, Ophiopogon japonicus (Linn.f.) Ker-Gawl., Rehmannia Glutinosa Libosch., Panax notoginseng (Burk.) FHChen, and Eriobotrya Japonica (Thunb.) Lindl. All the medicinal materials were purchased from the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine.

[0024] 1.3 Experimental instruments

[0025] KD-TS3A biological tissue automatic dehydrator (Jinhua Kedi Medical Instrument Co., Ltd.); Tissue-TEKRTECTM biological tissue embedding machine (SAKURA); Shandon Finesse325 Leica slicer (Thermo Fisher Scientific); Nikon eclipse 50i microscope (Nikon); PTC-200 ordinary PCR instrument (Bio-Rad); S-1-150S desktop high-speed refrigerated centrifuge (Zhengzhou Honghua Instrument Co., Ltd.); EPS300 electrophoresis instrument (Shanghai Tianneng Technology Co., Ltd.).

[0026] 1.4 Experimental reagents

[0027] Xylene (Sinopharm Group Co., Ltd.); hematoxylin staining solution (Zhuhai Besuo Biotechnology Co., Ltd.); eosin staining solution (alcohol soluble, Zhuhai Besuo Biotechnology Co., Ltd.); DEPC-H2O (Beyotime); 2×SYBR GreenqPCR Master Mix (High ROX, Servicebio); RIPA lysis buffer (Biosharp); PMSF (Biosharp); SDS (Sokarbio); PAGE gel accelerator (Solarbio); Tris (Solarbio); APS (BBI Life Sciences); prestained protein marker (Thermo); Western primary and secondary antibody removal solution (Beyotime); ECL ultrasensitive luminescence kit (Thermo).

[0028] 2 Methods

[0029] 2.1 Preparation of Huangdi Anxiao Capsule Samples

[0030] According to the prescription ratio: loquat leaf 12g, coptis root 6g, raw earth 10g, ophiopogon 8g, kudzu root 10g, panax notoginseng 5g. Add water to coptis root, kudzu root, raw earth, ophiopogon, loquat leaf and 5 herbs and boil them 3 times. Add 10 times the water for the first time and boil.

[0031] 1.5 hours; add 8 times the amount of water for the second time, boil for 1 hour, combine the extracts, filter, and let stand for 24 hours. Take the supernatant, concentrate under reduced pressure, add Panax notoginseng powder, collect the paste, and vacuum dry it to obtain a dry extract.

[0032] 2.2 Modeling, grouping and drug administration

[0033] Sixty male SD rats were randomly divided into groups after one week of adaptive feeding considering the influence of the rat breeding environment. After the normal group was selected, the remaining rats were equally and randomly divided into the model group, HDAXC high-, medium-, and low-dose groups, the positive drug metformin group, and the Shenqi Jiangtang Granule group after modeling.

[0034] A STZ-induced diabetic rat model was established according to the literature method (Gorelova A, Berman M, Al Ghouleh I. Endothelial-to-Mesenchymal Transition in Pulmonary Arterial Hypertension. Antioxid Redox Signal. 2021; 34(12): 891-914. doi: 10.1089 / ars.2020.8169): except for the normal group, the rats were fed with a high-sugar and high-fat diet for 4 weeks, fasted but not deprived of water for 12 hours, and then received a single intraperitoneal injection of 40 mg / kg of streptozotocin (STZ) dissolved in 0.1 mmol / L, pH 4.5 sodium citrate buffer, followed by injection of thyroid hormone for 7 consecutive days. Three days later, the model rats were fasted but not watered for 12 hours, and blood glucose was measured from the tail vein. The random blood glucose was >11.1mmol / L. After continuing to be fed with a high-sugar and high-fat diet for 2 weeks, the thoracic aorta of the model rats was randomly taken. HE staining results showed that the structure and morphology of the rat thoracic aorta were disordered, the intima was thickened, and the smooth muscle proliferated, indicating that the model was successfully established.

[0035] The normal group was fed with ordinary feed, and the same amount of sodium citrate buffer was injected intraperitoneally as the modeling group. The normal group and the model group were given an equal amount of normal saline by gavage every day; according to the conversion ratio of human and mouse body surface area, the daily required amount of crude drug for rats was calculated to be 4.59g / kg, the yield of the prescription extract was 33%, and the daily required amount of extract for rats was calculated to be 1.51g / kg. According to 8, 4, and 2 times of the clinical equivalent dose, HDAXC high (12.0g / kg), medium (6.0g / kg) and low (3.0g / kg) dose groups were set up respectively. The metformin group was given 0.72g / kg metformin by gavage every day. The Shenqijiangtang granule group was given 1g / kg Shenqijiangtang granule aqueous solution (concentration of 0.25g / mL) by gavage every day. The operations of each group were carried out simultaneously, and the drug was administered continuously for 6 weeks.

[0036] 2.3 Thoracic aorta specimen collection

[0037] After 6 weeks of administration, the rats were fasted but not watered for 12 h. After anesthesia, the thoracic aorta was removed and placed in 4% paraformaldehyde and stored at -80°C for later use.

[0038] 2.4 Observation of thoracic aorta pathological morphology

[0039] After routine tissue processing and sectioning, HE staining was performed. Hematoxylin staining solution was used for 3-5 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol solution for a few seconds, washed with water, blued with dilute lithium carbonate aqueous solution for 30 seconds, and then washed with water several times. The sections were dehydrated with 80% ethanol and then stained with eosin staining solution for 15 seconds, washed with water, dehydrated with gradient ethanol, sealed, and examined under a microscope.

[0040] 2.5 Western blot detection of thoracic aorta-related protein expression

[0041] Take 100 mg of tissue, add RIPA cell lysis buffer at a ratio of 1:100, place on ice for lysis for 30 minutes, centrifuge at 12,000 rpm for 15 minutes, collect the supernatant, heat at 100°C for 10 minutes, load the protein sample directly into the SDS-PAGE gel loading hole, transfer to PVDF membrane, add Western blocking solution (5% skim milk powder) for 2 hours, add primary antibody, incubate overnight at 4°C, rinse 3 times with PBST. Add HRP-labeled secondary antibody diluted at 1:10000, incubate at room temperature for 2 hours. Add PBST and rinse 3 times. Perform ECL color reaction and adjust the exposure conditions according to different luminescence intensities.

[0042] 2.6 RT-qPCR detection of thoracic aorta-related mRNA expression

[0043] Take 100 mg of tissue into a mortar, grind it into powder with liquid nitrogen, and add 1 ml of TRIzol to lyse it. Extract total RNA and store it at -80℃ for later use. Then perform reverse transcription cDNA synthesis, and store it at -20℃ for later use. Take 3uL of cDNA, add 10uL of 2×SYBR Green qPCR Master Mix, 0.4uL of upstream and downstream primers, 6.2uL of RNase Free water, and a total system of 20μL, and perform real-time quantitative PCR. The results were obtained using 2 -ΔΔCt Calculate the relative expression of the target gene. The primer sequences are shown in Table 1.

[0044] Table 1 Primer sequences

[0045]

[0046] 2.7 Statistical analysis

[0048] The experimental data were analyzed using SPSS25.0 statistical analysis program. The calculated data were expressed as x±s. One-way analysis of variance (ANOVA) was used to compare the groups, and then LSD was used to compare the groups pairwise. P<0.05 was used to indicate different statistical values.

[0049] 3 Results

[0050] 3.1 Effects of HDAXC on pathological changes of rat thoracic aorta

[0051] like Figure 1 As shown in the figure, the thoracic aorta of rats in the normal group had normal morphology and structure, clear cell boundaries, and no obvious cell shedding. The thoracic aorta of rats in the model group had disordered structure and thickened intima, indicating that the model was successfully established. Compared with the normal group, the thoracic aorta of rats in the model group had severe vascular pathological damage. Compared with the model group, the pathological damage of the thoracic aorta tissue structure of rats in the low, medium and high dose groups of HDAXC and metformin groups was alleviated to varying degrees.

[0052] 3.2 Effect of Huangdi Anxiao on serum cAMP / cGMP levels in rats with kidney yin deficiency type diabetes

[0053] As shown in Table 2, compared with the normal control group, the cAMP / cGMP ratio was significantly increased; compared with the model group, the cAMP and cGMP levels were significantly increased (P<0.05, P<0.01), and the cAMP / cGMP ratio was significantly decreased (P<0.05, P<0.01). The results showed that Huangdi Anxiao Capsule can improve kidney yin deficiency in diabetic rats.

[0054] Table 2 Effect of Huangdi Anxiao on serum cAMP / cGMP levels in rats with kidney yin deficiency type diabetes

[0055]

[0056] 3.3 Effect of HDAXC on the expression of genes related to the Wnt / β-catenin signaling pathway in rat thoracic aorta tissue

[0057] As shown in Table 3, Figure 2 As shown in the results, compared with those in the normal group, the gene expression levels of Wnt, β-catenin, and GSK3β in the thoracic aorta tissue of the rats in the model group were significantly increased (P<0.01); compared with the model group, the gene expression levels of TNF-α and IL-1β in the thoracic aorta tissue of the rats in the low, medium, and high doses of HDAXC groups and metformin groups were decreased to varying degrees (P<0.05).

[0058] Table 3 Effect of Huangdi Anxiao on the expression of genes related to the Wnt / β-catenin signaling pathway in the thoracic aorta tissue of rats with kidney yin deficiency diabetes

[0059]

[0060] 3.4 Effect of HDAXC on the expression of inflammation-related genes in rat thoracic aorta tissue

[0061] As shown in Table 4, Figure 3 As shown in the data, compared with the normal group, the gene expression levels of TNF-α and IL-1β in the thoracic aorta tissue of the rats in the model group were significantly increased (P<0.01); compared with the model group, the gene expression levels of TNF-α and IL-1β in the thoracic aorta tissue of the rats in the HDAXC low, medium and high dose groups and metformin groups were decreased to varying degrees (P<0.05).

[0062] Table 4 Effect of HDAXC on the expression of TNF-α and IL-1β mRNA in rat thoracic aorta tissue Note: Compared with the model group ** P<0.01, * P<0.05.

[0063]

[0064] Effect of 3.5HDAXC on the expression of EndoMT markers in rat thoracic aorta tissue

[0065] like Figure 4 As shown, compared with the normal group, the CD31 in the thoracic aorta tissue of the rats in the model group was significantly decreased, and the FSP1 gene expression was significantly increased (P<0.01); compared with the model group, the CD31 in the thoracic aorta tissue of the rats in the low, medium and high dose groups of HDAXC and metformin groups was increased to varying degrees, and the FSP1 gene expression was decreased to varying degrees (P<0.05).

[0066] The above research results show that the gene expressions of cAMP, Wnt, GSK3β, β-catenin, TNF-α, IL-1β, and FSP1 in the thoracic aorta tissue of rats in the low, medium, and high dose groups of Huangdi Anxiao Capsules and the metformin group decreased to varying degrees, and CD31 increased to varying degrees. This indicates that Huangdi Anxiao Capsules may regulate the cAMP / Wnt / β-catenin axis to promote the EndoMT process of the thoracic aorta, thereby improving vascular function and intervening in macroangiopathy in diabetic patients with kidney yin deficiency.

[0067] In summary, the present invention explores that Huangdi Anxiao Capsule can improve kidney yin deficiency in diabetic rats, and inhibits the EndoMT process of the thoracic aorta by regulating the inhibition of the cAMP / Wnt / β-catenin axis in diabetic rats with kidney yin deficiency, thereby improving the macrovascular disease of diabetes with kidney yin deficiency, providing an experimental basis for the clinical application of Huangdi Anxiao Capsule to intervene in the macrovascular disease of diabetes with kidney yin deficiency.

[0068] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. The application of regulating cAMP / Wnt / β-catenin axis to inhibit EndoMT process of thoracic aorta in kidney yin deficiency type diabetes, characterized in that: Preparations that regulate the cAMP / Wnt / β-catenin axis and inhibit the process of thoracic aorta endothelial-mesenchymal transition (EndoMT) can be used for the preparation of therapeutic drugs for kidney yin deficiency type diabetes.

2. The use according to claim 1, characterized in that The preparation can inhibit the increase of cAMP levels in the body, regulate the Wnt / β-catenin pathway, and reverse the aggravation of thoracic aorta endothelial-mesenchymal transition (EndoMT).

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