Application of eight-ingredient travertine as RhoA / ROCK signal channel inhibitor in preparation of anti-renal fibrosis drugs

Abstract

The invention discloses an application of Tibetan medicine eight-ingredient travertine as a RhoA / ROCK signal channel inhibitor in preparation of an anti-renal fibrosis medicine. The effects of the eight-ingredient travertine pills on RhoA / ROCK signal channels and renal fibrosis are researched by establishing a mouse renal fibrosis model, and results show that after the eight-ingredient travertine pills are subjected to administration intervention, the protein expression levels of RhoA, ROCK1 and p-MYPT1 / MYPT1 in renal tissues are remarkably reduced, and the eight-ingredient travertine pills can inhibit the RhoA / ROCK1 signal channels. The eight-ingredient travertine pill can also relieve renal fibrosis by inhibiting expression of alpha-SMA, Collagen I and FN mRNA and protein expression level in renal tissues. The invention provides fundamental research for relieving renal fibrosis by the Tibetan medicine eight-ingredient travertine pill, and provides experimental basis for clinical application.

Description

Technical Field

[0001] The present invention belongs to the field of drug applications, and particularly relates to the application of Tibetan medicine Bawei Shihuahua as a RhoA / ROCK signaling pathway inhibitor in the preparation of anti-renal fibrosis drugs. Background Art

[0002] RhoA is a member of the Ras protein superfamily and plays a key role as a molecular switch. It is activated during the process of binding to chemokines, cytokines, and growth factors, and then regulates the activity states of cytoskeletal proteins and other related factors through the ROCK signaling cascade. As a widely existing and highly conserved serine / threonine kinase, ROCK is the main downstream effector of RhoA and includes two subtypes, ROCK1 and ROCK2. ROCK is widely involved in a variety of biological processes, covering multiple aspects such as cell contraction, adhesion, migration, proliferation, inflammatory response, survival, and even apoptosis.

[0003] The RhoA / ROCK signaling pathway is related to various kidney diseases such as renal fibrosis, diabetic nephropathy, and renal cell carcinoma. Some studies have shown that the activation of the RhoA / ROCK1 pathway accelerates the progression of renal fibrosis. The RhoA / ROCK signal transduction pathway plays an important role in the biological behavior of malignant tumors. Its abnormal activation can not only promote the invasion and metastasis of tumor cells but also accelerate the malignant progression of tumors by regulating the cell microenvironment. The RhoA / ROCK signaling pathway can participate in the development of kidney diseases by regulating biological processes such as actin cytoskeleton remodeling, cell migration ability, and cell proliferation. In view of these important biological functions, further clarifying the molecular mechanism of the RhoA / ROCK signaling pathway in kidney diseases has important theoretical value. Actively exploring more drugs that can inhibit or block this pathway is expected to open up a new way for the treatment strategy of kidney diseases.

[0004] Tibetan medicine Bawei Shihuahua Pills were first recorded in the "Four Medical Canons" and are included in the "Tibetan Medicine Formula Compendium" of the Tibetan Hospital of Tibet Autonomous Region. It is a traditional Tibetan medicine raw medicine water pill. The prescription of Bawei Shihuahua Pills is: Shihuahua (106.4 g), Gansu Oxytropis Extract (42.6 g), Clove (21.3 g), Safflower (69.1 g), Piper longum (21.3 g), Meconopsis (79.8 g), Pomegranate Seeds (106.4 g), Cinnamon (53.2 g). Its functions and indications are diuretic and detumescent, used for various edema diseases, cough and asthma, fatigue, leg swelling, oliguria, loss of appetite, especially for heat-induced edema with very good effects. Finding effective prevention and treatment strategies is of great clinical significance for delaying the progression of kidney diseases and improving renal function. Summary of the Invention

[0005] The present invention aims to provide the application of Tibetan medicine Bawei Shihuahua as an inhibitor of the RhoA / ROCK signaling pathway in the preparation of anti-renal fibrosis drugs.

[0006] The first aspect of the present invention provides the application of Bawei Shihuahua in the preparation of an inhibitor of the RhoA / ROCK signaling pathway.

[0007] The Bawei Shihuahua described in the above technical solution is composed of Shihuahua, Gansu Oxytropis paste, cloves, safflower, piper longum, meconopsis, pomegranate seeds, and cinnamon.

[0008] Furthermore, the prescription of the Bawei Shihuahua is composed as follows by weight: Shihuahua (106.4 parts), Gansu Oxytropis paste (42.6 parts), cloves (21.3 parts), safflower (69.1 parts), piper longum (21.3 parts), meconopsis (79.8 parts), pomegranate seeds (106.4 parts), and cinnamon (53.2 parts).

[0009] Furthermore, the Bawei Shihuahua is a pharmaceutical preparation prepared according to the above prescription. Preferably, the Bawei Shihuahua of the present invention is Bawei Shihuahua Pills (National Medicine Approval Number Z20083038) produced by Tibet Shenhou Pharmaceutical Co., Ltd.

[0010] In some embodiments of the present invention, a mouse renal fibrosis model was established to study the effect of Bawei Shihuahua Pills on the RhoA / ROCK signaling pathway.

[0011] During renal fibrosis, the activation of the RhoA / ROCK signaling pathway leads to the excessive accumulation of extracellular matrix and promotes the progression of renal fibrosis. RhoA has two forms, among which it binds to GTP to form the activated form GTP-RhoA, and GTP-RhoA translocates to the cell membrane, mediating the downstream molecule ROCK1. The activation of ROCK1 causes phosphorylation modification of MYPT1, thereby inhibiting the activity of myosin light chain phosphatase. This process hinders the phosphorylation process of myosin light chain and ultimately promotes the differentiation of fibroblasts into myofibroblasts. In some specific embodiments of the present invention, a mouse renal fibrosis model was established by a single intravenous injection of adriamycin. The protein expression levels of RhoA, ROCK1, and p-MYPT1 / MYPT1 in the renal tissues of the model group mice increased, indicating that the RhoA / ROCK signaling pathway was activated. Compared with the model group, after the administration of Bawei Shihuahua Pills, the protein expression levels of RhoA, ROCK1, and p-MYPT1 / MYPT1 in the renal tissues decreased significantly, indicating that Bawei Shihuahua Pills can be used as an inhibitor of the RhoA / ROCK1 signaling pathway.

[0012] The RhoA / ROCK signaling pathway inhibitor described in the present invention can be used to prepare drugs for the treatment of diseases related to the activation of the RhoA / ROCK signaling pathway.

[0013] Modern pharmacological studies have shown that the activation of the RhoA / ROCK signaling pathway is associated with a variety of diseases, including but not limited to cardiovascular diseases such as hypertension, coronary heart disease, and heart failure; neurological diseases such as Alzheimer's disease, Parkinson's disease, and spinal cord injury; metabolic diseases such as diabetes and its complications including diabetic nephropathy, diabetic retinopathy, and diabetic peripheral neuropathy; liver fibrosis caused by various etiologies; renal fibrosis caused by various etiologies; pulmonary fibrosis caused by various etiologies; inflammatory diseases such as rheumatoid arthritis; and tumor diseases including but not limited to renal cell carcinoma and other kidney cancers, breast cancer, lung cancer, and colorectal cancer.

[0014] The second aspect of the present invention provides the use of the above-mentioned eight-flavor tufa in the preparation of a drug for anti-renal fibrosis.

[0015] In some embodiments of the present invention, the effect of the eight-flavor tufa pill on renal fibrosis was studied by establishing a mouse renal fibrosis model.

[0016] Serum creatinine (Scr) and blood urea nitrogen (BUN) are commonly used indicators for clinically evaluating renal function. The research results of the present invention show that after the administration of the eight-flavor tufa pill, the levels of the renal function indicators BUN and Scr decreased significantly, indicating that it can improve renal function. Compared with the pomegranate seed group, the tufa group, and the pomegranate seed + tufa group, the levels of Scr and BUN in the serum of the mice in the eight-flavor tufa pill administration group were significantly reduced.

[0017] The results of HE staining and Masson staining showed that after the administration of the eight-flavor tufa pill, the pathological damage of the renal tissue was alleviated and the collagen deposition and fibrosis were reduced, indicating that the eight-flavor tufa pill can reduce renal tissue fibrosis.

[0018] Myofibroblasts are a heterogeneous cell population with multiple origins. During tissue fibrosis, they produce a large amount of extracellular matrix and promote the development of tissue fibrosis by expressing extracellular matrix components such as collagen I (CollagenⅠ), α-smooth muscle actin (α-SMA), and fibronectin (FN). α-SMA is a specific marker for myofibroblast activation, and CollagenⅠ and FN are classic extracellular matrices secreted by myofibroblasts, which are closely related to renal fibrosis. The research results of the present invention show that the eight-flavor tufa pill may reduce renal fibrosis by inhibiting the mRNA and protein expression levels of α-SMA, Collagen I, and FN in the renal tissue.

[0019] Furthermore, the renal fibrosis described in the above technical solution may include: renal fibrosis caused by chronic glomerulonephritis, chronic pyelonephritis, obstructive nephropathy, systemic lupus erythematosus nephropathy, hereditary nephropathy such as Alport syndrome, diabetic nephropathy, hypertensive nephropathy, drug-induced nephropathy, nephropathy caused by hepatitis B or HIV, and kidney transplantation, etc.

[0020] The present invention establishes a renal fibrosis model in mice to explore the effect of Tibetan medicine Bawei Shihuahua Pills on inhibiting the RhoA / ROCK1 signaling pathway and alleviating renal fibrosis, providing basic research for Tibetan medicine Bawei Shihuahua Pills to alleviate renal fibrosis and providing experimental basis for clinical application. Brief Description of the Drawings

[0021] Figure 1 It is the HE staining diagram (×400) of mice in each group.

[0022] Figure 2 It is the Masson staining diagram (×400) of mice in each group.

[0023] Figure 3 It is the effect of Bawei Shihuahua Pills on the expression of Collagen I, α-SMA, and FN mRNA in the renal tissue of mice with renal fibrosis ( n = 5); A - C are the relative expression levels of Collagen I, α-SMA, and FN mRNA respectively. Compared with the normal group, ** P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01; compared with the low-dose group of Bawei Shihuahua Pills, ▲ P < 0.05, ▲▲ P < 0.01.

[0024] Figure 4 It is the effect of Bawei Shihuahua Pills on the expression of Collagen I, α-SMA, and FN proteins in the renal tissue of mice with renal fibrosis ( n = 5); A is the protein band diagram, and B - D are the expression levels of Collagen I, α-SMA, and FN proteins respectively. Compared with the normal group, ** P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01; compared with the low-dose group of Bawei Shihuahua Pills, ▲ P < 0.05, ▲▲ P < 0.01.

[0025] Figure 5 It is the effect of Bawei Shihuahua Pills on the expression of proteins related to the RhoA / ROCK1 signaling pathway in the renal tissue of mice with renal fibrosis ( n = 5); A and B are protein band diagrams, and C - E are the protein expression levels of RhoA, ROCK1, and p - MYPT1 / MYPT1 respectively. Compared with the normal group, ** P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01; compared with the low - dose of Bawei Shihuahua Pills, ▲ P < 0.05, ▲▲ P < 0.01. Specific Embodiments

[0026] The present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] The Tibetan medicine Bawei Shihuahua Pills used in the following specific embodiments of the present invention are provided by Tibet Shenhou Pharmaceutical Co., Ltd.

[0028] Example 1, Effect of Bawei Shihuahua Pills on Renal Fibrosis in Mice

[0029] 1. Experimental animals and grouping: SPF - grade male BALB / c mice, weighing (18 ± 2) g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., with the license number: SCXK(Xiang)2021 - 0002. After 7 days of adaptive feeding, during which they had free access to food and water. This experiment was approved by the Experimental Animal Ethics Committee (Ethical batch number: TOP - IACUC - 2024 - 0252). After 7 days of adaptive feeding, 45 mice were randomly divided into 9 groups: 5 mice in the normal group, 5 mice in the model group, 5 mice in the low - dose group of Bawei Shihuahua Pills, 5 mice in the middle - dose group of Bawei Shihuahua Pills, 5 mice in the high - dose group of Bawei Shihuahua Pills, 5 mice in the imidapril hydrochloride group, 5 mice in the pomegranate seed group, 5 mice in the travertine group, and 5 mice in the pomegranate seed + travertine group.

[0030] 2. Dose design:

[0031] The Tibetan medicine Bawei Shihuahua Pills, with the specification of: 5 g for every 10 pills. According to the clinical medication guiding principles, the dose is 4 - 5 pills once and 2 - 3 times a day. After calculation, the maximum daily dose for humans is 7.5 g·person -1 ·day -1 , based on the standard adult body weight of 70 kg, the clinical equivalent dose is converted to 107 mg·kg -1 ·d -1 . The inter - species dose conversion was carried out using the body surface area conversion coefficient method, and the low, middle, and high doses of Bawei Shihuahua Pills administered to mice were determined to be 250 mg·kg -1 , 500 mg·kg -1 , 1000 mg·kg -1Similarly, referring to the above calculation method, the dosage of the positive drug imidapril hydrochloride in the mouse group was 1.3 mg·kg -1 , the pomegranate seed group was 210 mg·kg -1 , the travertine group was 210 mg·kg -1 , the pomegranate seed + travertine group was 420 mg·kg -1 (210 mg·kg each of pomegranate seeds and travertine -1 ).

[0032] 3. Model establishment and drug administration:

[0033] Mice in the normal group were intravenously injected with an equal volume of normal saline once through the tail vein; mice in each experimental group were intravenously injected with doxorubicin at 10 mg·kg -1 . The treatment groups were intragastrically administered with the prepared dosage at a volume of 0.1 mL / 10 g, and the normal group and the model group were intragastrically administered with an equal volume of normal saline, once a day for 6 consecutive weeks. After the last drug administration, the mice were subjected to a 24-hour fasting period, during which only free drinking water was allowed. Pentobarbital sodium was administered by intraperitoneal injection for anesthesia. After the anesthesia took effect, blood was collected from the mouse orbital cavity. The collected whole blood was centrifuged at 5000 r·min -1 at 4°C for 15 min, and the supernatant was stored in a -80°C refrigerator for biochemical detection. After blood collection, the kidney tissue was quickly separated on an ice table. The left kidney tissue was immediately transferred to a -80°C ultra-low temperature refrigerator for storage for subsequent RT-qPCR and Western Blot experiments; the right kidney tissue was placed in a pre-prepared fixing solution for subsequent HE staining to evaluate kidney pathological changes and Masson staining to analyze the distribution of collagen fibers.

[0034] 4. Observation indicators:

[0035] Serum was taken from mice in each group, and the expression levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were detected using an automatic biochemical analyzer. HE staining was used to observe the pathological changes of rat kidney tissue; RT-qPCR was used to detect the expression of Collagen I, α-SMA, and FN mRNA; Western Blot was used to detect the expression of Collagen I, α-SMA, and FN proteins.

[0036] 5. Data analysis:

[0037] Data statistical processing was completed using SPSS 25.0 statistical software. The experimental data were expressed as mean ± standard deviation In the formal representation, after the normality test and the homogeneity of variance test, if the data meet the conditions for parametric tests, one-way analysis of variance is used for comparison among multiple groups, and P < 0.05 indicates statistical significance.

[0038] 6. Results:

[0039] (1) Effects of Bawei Shihuahua Pills on the levels of BUN and Scr in mouse serum

[0040] Compared with the normal group, the levels of BUN and Scr in the serum of mice in the model group increased (P < 0.01); compared with the model group, the levels of BUN and Scr in the serum of mice in the low, medium, and high-dose groups of Bawei Shihuahua Pills and the imidapril hydrochloride group decreased (P < 0.05, P < 0.01); compared with the low-dose group of Bawei Shihuahua Pills, the levels of BUN and Scr in the serum of mice in the medium and high-dose groups of Bawei Shihuahua Pills decreased (P < 0.01), and the levels of BUN and Scr in the serum of mice in the pomegranate seed group, the travertine group, and the pomegranate seed + travertine group increased significantly (P < 0.05), as shown in Table 1.

[0041] Table 1 Comparison of BUN and Scr levels in mice of each group ( n = 5)

[0042]

[0043] Note: Compared with the normal group, ** P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01; compared with the low-dose group of Bawei Shihuahua Pills, ▲▲ P < 0.01.

[0044] (2) Effects of Bawei Shihuahua Pills on the histopathology of mouse kidneys

[0045] The renal tissue structure of mice in the normal group was clear, the glomeruli and renal tubules had normal structures, and the tubules in the renal tissue were arranged tightly; compared with the normal group, the renal tissue of mice in the model group was arranged loosely, the structure was disordered, the glomeruli were atrophied, the lumens of the renal tubules were dilated, the interstitial area was enlarged, and there was infiltration of inflammatory cells; compared with the model group, the pathological damage of the renal tissue of mice in the low, medium, and high-dose groups of Bawei Shihuahua Pills and the imidapril hydrochloride group was reduced, the infiltration of inflammatory cells was decreased, and the dilation of the lumens of the renal tubules was alleviated; the degree of reduction of pathological damage of the renal tissue of mice in the medium and high-dose groups of Bawei Shihuahua Pills was better than that of the low-dose group of Bawei Shihuahua Pills, and the degree of pathological damage of the pomegranate seed group, the travertine group, and the pomegranate seed + travertine group was more severe than that of the low-dose group of Bawei Shihuahua Pills, as shown in Figure 1 .

[0046] (3) Effects of Bawei Shihuahua Pills on renal fibrosis in mice

[0047] A small amount of collagen fibers were visible in the normal group of mice; compared with the normal group, obvious collagen fiber deposition was observed in the renal interstitium of the model group of mice, and the fibrosis level increased; compared with the model group, the collagen fiber deposition in the low, medium, and high-dose groups of Bawei Shihuahua Pills and the imidapril hydrochloride group of mice decreased, and the fibrosis level decreased; compared with the low-dose group of Bawei Shihuahua Pills, the renal fibrosis level in the medium and high-dose groups of Bawei Shihuahua Pills decreased significantly, while the renal fibrosis levels in the pomegranate seed group, the travertine group, and the pomegranate seed + travertine group increased significantly; see Figure 2 。

[0048] (4) Effects of Bawei Shihuahua Pills on the mRNA expression of Collagen I, α-SMA, and FN in the renal tissue of mice

[0049] Compared with the normal group, the mRNA expression levels of Collagen I, α-SMA, and FN in the renal tissue of the model group of mice increased significantly (P < 0.01); compared with the model group, the mRNA expression levels of Collagen I, α-SMA, and FN in the renal tissue of the low, medium, and high-dose groups of Bawei Shihuahua Pills and the imidapril hydrochloride group of mice decreased significantly (P < 0.05, P < 0.01); compared with the low-dose group of Bawei Shihuahua Pills, the mRNA expression levels of Collagen I, α-SMA, and FN in the renal tissue of the medium and high-dose groups of Bawei Shihuahua Pills decreased significantly (P < 0.05, P < 0.01); see Figure 3 。

[0050] (5) Effects of Bawei Shihuahua Pills on the protein expression of Collagen I, α-SMA, and FN in the renal tissue of mice

[0051] Compared with the normal group, the protein expressions of Collagen I, α-SMA, and FN in the renal tissue of the model group of mice increased significantly (P < 0.01); compared with the model group, the protein expressions of Collagen I, α-SMA, and FN in the renal tissue of the low, medium, and high-dose groups of Bawei Shihuahua Pills and the imidapril hydrochloride group of mice decreased (P < 0.05, P < 0.01); the protein expressions of Collagen I, α-SMA, and FN in the renal tissue of the medium and high-dose groups of Bawei Shihuahua Pills were significantly lower than those in the low-dose group (P < 0.05, P < 0.01); see Figure 4 。

[0052] (6) Effects of Bawei Shihuahua Pills on the Expression of Proteins Related to the RhoA / ROCK1 Signaling Pathway in the Renal Tissues of Mice After ROCK activation, its substrate myosin light chain phosphatase targeting subunit (MYPT1) is phosphorylated to become p-MYPT1. The ratio of p-MYPT1 / MYPT1 represents the degree of phosphorylation. It was found that the total MYPT1 protein expression did not change significantly, while the p-MYPT1 protein expression changed significantly. Compared with the normal group, the protein expressions of RhoA, ROCK1, and p-MYPT1 / MYPT1 in the renal tissues of mice in the model group were significantly increased (P<0.01); compared with the model group, the protein expressions of RhoA, ROCK1, and p-MYPT1 / MYPT1 in the renal tissues of mice in the low-, medium-, and high-dose groups of Bawei Shihuahua Pills and the imidapril hydrochloride group were significantly decreased (P<0.05, P<0.01); compared with the low-dose group of Bawei Shihuahua Pills, the protein expressions of RhoA, ROCK1, and p-MYPT1 / MYPT1 in the renal tissues of mice in the medium- and high-dose groups of Bawei Shihuahua Pills were decreased (P<0.05, P<0.01); see Figure 5 .

[0053] 7. Conclusion:

[0054] After the administration of Bawei Shihuahua Pills, the levels of renal function indicators BUN and Scr decreased significantly, the pathological damage of the renal tissues was alleviated, the collagen deposition fibers decreased, and the mRNA and protein expression levels of α-SMA, Collagen I, and FN in the renal tissues decreased significantly, indicating that Bawei Shihuahua Pills can alleviate renal fibrosis and improve renal function. Mechanism studies have shown that after the administration of Bawei Shihuahua Pills, the protein expression levels of RhoA, ROCK1, and p-MYPT1 / MYPT1 in the renal tissues decreased significantly, suggesting that the effect of Tibetan medicine Bawei Shihuahua Pills in alleviating renal fibrosis is related to the RhoA / ROCK1 signaling pathway.

[0055] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Bawei Shihuahua in the preparation of RhoA / ROCK signaling pathway inhibitors, wherein the Bawei Shihuahua is composed of Shihuahua, Gansu Sophora moorcroftiana paste, cloves, safflower, piper longum, meconopsis, pomegranate seeds, and cinnamon.

2. The application according to claim 1, wherein The prescription of the Bawei Shihuahua is composed of the following parts by weight: 106.4 parts of Shihuahua, 42.6 parts of Gansu Sophora moorcroftiana paste, 21.3 parts of cloves, 69.1 parts of safflower, 21.3 parts of piper longum, 79.8 parts of meconopsis, 106.4 parts of pomegranate seeds, and 53.2 parts of cinnamon.

3. The application according to claim 1, characterized in that, The Bawei Shihuahua is Bawei Shihuahua Pills produced by Tibet Shenhou Pharmaceutical Co., Ltd.

4. The application according to claim 1, characterized in that, Use of the RhoA / ROCK signaling pathway inhibitor in the preparation of a drug for treating diseases related to activation of the RhoA / ROCK signaling pathway.

5. The application according to claim 4, wherein The diseases related to activation of the RhoA / ROCK signaling pathway include cardiovascular diseases, nervous system diseases, metabolic diseases, liver diseases, kidney diseases, lung diseases, inflammatory diseases, and tumors.

6. The application according to claim 5, characterized in that, The cardiovascular diseases include hypertension, coronary heart disease, and heart failure; the nervous system diseases include Alzheimer's disease, Parkinson's disease, and spinal cord injury; the metabolic diseases include diabetes and its complications; the liver diseases include liver fibrosis; the kidney diseases include renal fibrosis; the lung diseases include pulmonary fibrosis; the tumors include renal cancer, breast cancer, lung cancer, and colorectal cancer.

7. The application according to claim 1, wherein Use of the Bawei Shihuahua in the preparation of a drug for anti-renal fibrosis.

8. The application according to claim 7, wherein The renal fibrosis includes chronic glomerulonephritis, chronic pyelonephritis, obstructive nephropathy, lupus nephritis, hereditary nephropathy, diabetic nephropathy, hypertensive nephropathy, drug-induced nephropathy, virus-related nephropathy, and renal fibrosis caused by kidney transplantation.

Images