Application of bear bile heart-saving preparation in the preparation of medicines for treating diseases related to hyperlipidemia

Abstract

The present application proposes the use of bear bile heart-saving preparation in the preparation of drugs for treating hyperlipidemia-related diseases, which relates to the field of drug use. Hyperlipidemia-related diseases include hyperlipidemia, fatty liver, cognitive dysfunction, cardiovascular disease and heart failure.

Description

Technical Field

[0001] The present application relates to the field of drug use, and specifically, to the use of bear bile heart-saving preparations in the preparation of drugs for treating hyperlipidemia-related diseases. Background Art

[0002] Hyperlipidemia (HLP) is a major risk factor for the development of cardiovascular and cerebrovascular diseases and fatty liver. Studies have found that high total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are the main causes of hyperlipidemia. When the levels of TC, triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) in the blood increase, a large amount of lipids are deposited on the blood vessel wall, causing damage to vascular endothelial cells, inducing the expression of multiple adhesion factors, promoting the formation of foam cells, and leading to cardiovascular diseases. On the other hand, the liver's uptake of free fatty acids increases, leading to TG transport disorders and accumulation in the liver to form fatty liver.

[0003] Vascular dementia (VD) is a cerebrovascular disease caused by ischemic stroke, hemorrhagic stroke, and hypoperfusion of the brain area, which causes severe cognitive dysfunction such as memory, thinking and behavior. Its clinical manifestations are generally reflected in memory loss, cognitive dysfunction, lack of knowledge and skills, leading to loss of self-care ability, and its process is generally chronic, progressive and continuous. With the aging of my country's population, dementia has become one of the common diseases in the middle-aged and elderly. Vascular dementia is the most common one, and its incidence rate is second only to Alzheimer's disease (AD).

[0004] At present, statins and fibrates are commonly used drugs for the clinical treatment of hyperlipidemia. Statins can significantly reduce TC and have a good lipid-lowering effect, but they have side effects such as liver toxicity and rhabdomyolysis; fibrates can reduce TG and increase high-density lipoprotein cholesterol (HDL-C), but they have side effects such as liver and kidney toxicity. Therefore, it is necessary to develop highly effective and low-toxic lipid-lowering drugs.

[0005] In recent years, Chinese medicine has obvious advantages and characteristics in treating hyperlipidemia, which are mainly reflected in multiple pathways, multiple targets and fewer side effects. Chinese medicine mainly reduces blood lipids by inhibiting lipid absorption, affecting bile acid circulation, inhibiting lipid synthesis, resisting peroxidation, and increasing HDL.

[0006] Bear Bile Heart-Rescuing Pill is composed of bear bile powder, toad venom, borneol, artificial musk, ginseng, pearl, artificial bezoar, pig bile powder, and buffalo horn concentrated powder. It has the effects of strengthening the heart and replenishing qi, and aromatically invigorating the orifices. It is mainly used for chest pain caused by insufficient heart qi, with symptoms of chest tightness, heart pain, shortness of breath, and palpitations. Summary of the invention

[0007] The purpose of this application is to provide the use of bear bile heart-saving preparation in the preparation of medicines for treating hyperlipidemia-related diseases.

[0008] The present application solves the technical problem by adopting the following technical solution.

[0009] On the one hand, the embodiments of the present application provide the use of bear bile heart-saving preparations in the preparation of drugs for treating hyperlipidemia-related diseases.

[0010] In some embodiments of the present application, the above-mentioned bear bile heart-saving preparation includes pills, pills and tablets.

[0011] In some embodiments of the present application, the above-mentioned hyperlipidemia-related diseases include hyperlipidemia, fatty liver, cognitive dysfunction, cardiovascular disease and heart failure.

[0012] In some embodiments of the present application, the above-mentioned cognitive dysfunction includes Alzheimer's disease and vascular dementia.

[0013] In some embodiments of the present application, the above-mentioned hyperlipidemia includes hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia and low high-density lipoprotein cholesterol.

[0014] Compared with the prior art, the present invention has at least the following advantages or beneficial effects:

[0015] (1) In this application, the experimental results show that Xiongdan Jiuxin Pills can reduce the serum TC, TG, and LDL-C levels and increase the HDL-C level in hyperlipidemia rats.

[0016] (2) In the present application, based on the analysis of the two indicators of aspartate aminotransferase (glutamic oxaloacetic transaminase, AST) and alanine aminotransferase (glutamic pyruvic transaminase, ALT) and the results of HE staining of the liver, it was found that Xiongdan Jiuxin Pills can improve fatty degeneration of the liver.

[0017] (3) In the present application, the model group rats had endothelial damage, intimal thickening, and disordered smooth muscle cell arrangement. The experimental results showed that Xiongdan Jiuxin Pills could alleviate endothelial damage by improving the blood lipid levels of hyperlipidemia rats.

[0018] (4) In this application, the experimental results show that Xiongdan Jiuxin Pills can reduce cholesterol synthesis and accelerate metabolism by reducing the cholesterol ester transfer protein (CETP) content in rats with hyperlipidemia, the activity of rat 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and the activity of rat acyl transferase (ACAT) in liver tissue. Xiongdan Jiuxin Pills downregulates the expression of HMGCR to inhibit cholesterol biosynthesis, and regulates the levels of ACAT and CETP to increase lipid metabolism and transport, thereby improving blood lipid levels and playing a certain therapeutic role in hyperlipidemia.

[0019] (5) In this application, the experimental results show that Xiongdan Jiuxin Pills can significantly reduce the damage of brain neurons in hyperlipidemia rats and improve cognitive dysfunction. Its molecular mechanism is related to anti-inflammatory mechanisms such as upregulating brain tissue PPARγ protein expression, inhibiting NF-κB signaling pathway, reducing the production of proinflammatory factors (IL6, TNFa), and anti-apoptotic mechanisms such as inhibiting Caspase3 activation. Xiongdan Jiuxin Pills can exert neuroprotective effects by intervening in multiple targets and pathways related to neuronal inflammatory damage and apoptosis caused by hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The effect of XP on liver histopathology of hyperlipidemic rats (HE staining, ×400);

[0022] Figure 2 The figure shows the pathological changes of aorta in hyperlipidemia rats (HE staining);

[0023] Figure 3 The results of the water maze experiment;

[0024] Figure 4 The results of the Y-maze experiment;

[0025] Figure 5 Results of the novel object discrimination experiment;

[0026] Figure 6 It is a representative picture of NeuN immunofluorescence staining in the CA1 region of rat hippocampus;

[0027] Figure 7 This is the quantitative analysis result of NeuN positive expression in the CA1 region of the rat hippocampus;

[0028] Figure 8 This is a representative WB image of cleaved-caspase-3 and pro-caspase-3 proteins in rat brain tissue protein;

[0029] Fig. 9 It is a statistical graph of the ratio of cleaved-caspase-3 / pro-caspase-3;

[0030] Fig.10 The relative content of IL-6 in the pro-inflammatory factor level in the brain tissue of the rats in the Xiongdan Jiuxin Pills inhibition model control;

[0031] Fig.11 The relative content of TNF-α in the pro-inflammatory factor level in the brain tissue of the rats in the model control was inhibited by Xiongdan Jiuxin Pills;

[0032] Fig.12 This is a representative WB image of PPARγ and β-actin proteins in rat brain tissue;

[0033] Fig.13 is a statistical graph of PPARγ protein levels;

[0034] Fig.14 This is a representative WB image of NF-κB p65 and Histone H3 nuclear protein in rat brain tissue nuclear protein;

[0035] Fig.15 is a statistical graph of NF-κB p65 nuclear translocation protein levels;

[0036] Fig.16 The effect of XP on cardiac tissue pathology in hyperlipidemic rats (HE staining). DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0038] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0039] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0040] Example 1

[0041] Experimental animals: SPF grade SD male rats, 200 ± 20 g, were purchased from Chengdu Enswell Biotechnology Co., Ltd. [License No.: SCXK (Yu) 2018-0003] and routinely kept in an SPF animal room with a constant temperature of (24 ± 2) °C and a relative humidity of (60 ± 5)%. During the experiment, each rat was given 25 g of maintenance feed or high-fat feed (Beijing Boaigang Biotechnology Co., Ltd.) per day and had free access to water.

[0042] Reagents and instruments: Xiongdan Jiuxin Pills (XP, Shenyang Qinggong Pharmaceutical Group Co., Ltd.); simvastatin (Hangzhou Merck Pharmaceutical Co., Ltd.); TC, TG, LDL-C, HDL-C, AST, ALT, nitric oxide (NO) were purchased from Nanjing Jiancheng Bioengineering Institute; endothelin (ET-1), CETP, ACAT, HMGCR were all purchased from Quanzhou Ruixin Biotechnology Co., Ltd.; multifunctional microplate reader VarioskanTM LUX (Thermo Fisher); upright fluorescence microscope (Nikon).

[0043] Modeling and grouping: 48 SD male rats were randomly divided into a normal control group (Normal control group) and a model control group according to body weight after 3 days of adaptive feeding with ordinary diet. The normal control group was given ordinary diet, and the model control group was given a high-fat diet for 6 consecutive weeks. After 6 weeks of modeling, the high-fat rats with successful modeling were randomly divided into a model control group (Modelcontrol group), a simvastatin group (Simvastatin group), and an XP dose group (XP group), with 12 rats in each group.

[0044] The preparation of simvastatin solution (4 mg / kg) includes: each simvastatin tablet is 20 mg, one simvastatin tablet is taken, 50 ml of 0.5% CMCNa is added to dissolve it, and a 0.4 mg / ml simvastatin solution is prepared;

[0045] The preparation of XP solution (15.75 mg / kg) includes: each Bear Bile Heart-saving Pill is 25 mg, take an appropriate amount of Bear Bile Heart-saving Pill and grind it into powder with a mortar, add 0.5% CMCNa, and prepare it into 1.575 mg / ml Bear Bile Heart-saving Pill solution.

[0046] During the administration period, each administration group was given the corresponding drug (1ml / 100g) by gavage, and the model control group and the normal control group were given the same volume of saline once a day for 9 consecutive weeks. The administration group and the model group continued to be fed with high-fat diet. After 9 weeks of administration, the rats were killed and the samples were collected and stored in a -80℃ refrigerator for testing.

[0047] Experimental methods: During the experiment, the body weight of rats was recorded every week. After the administration, the rats were anesthetized, blood was collected from the abdominal aorta, and serum was separated. The TC, TG, LDL-C, HDL-C, NO, ET-1, CETP content and AST, ALT, HMGCR activity were determined according to the instructions of the kit; liver tissue was taken and TC, TG content and ACAT activity were determined according to the instructions of the kit; the aorta, heart and liver were fixed in 4% paraformaldehyde for 24 hours, and then dehydrated with 70%, 85%, 95% and 100% ethanol solutions for 1 hour each, embedded in paraffin, cut into 4μm thick sections, and then stained with HE.

[0048] Statistical analysis: All data were analyzed using SPSS 26.0 software, and the results were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison among multiple groups, and P < 0.05 was considered statistically significant.

[0049] Experimental results:

[0050] (1) Effect of Xiongdan Jiuxin Pills on blood lipids in hyperlipidemic rats

[0051] The experimental results are shown in Table 1:

[0052] Table 1 Effect of XP on blood lipid levels in hyperlipidemic rats ( n=12)

[0053]

[0054] Compared with the model control group, * P<0.05, ** P<0.01

[0055] The results in Table 1 show that compared with the normal control group, the TC, TG, and LDL-C levels in the model control group were significantly increased, and the HDL-C level was significantly decreased (P<0.01), indicating that the high-fat diet feeding method can successfully replicate the hyperlipidemia model of rats. Compared with the model control group, after 9 weeks of administration, the TC, TG, and LDL-C levels in the XP group and the Simvastatin group were decreased, and the HDL-C level was increased (P<0.01, P<0.05), indicating that the XP group had the most significant lipid-lowering effect, which was similar to the Simvastatin group.

[0056] (2) Effects of Xiongdan Jiuxin Pills on lipid content and pathology in liver tissue of hyperlipidemia rats

[0057] The experimental results are shown in Table 2:

[0058] Table 2 Effects of XP on lipid content in liver tissue of hyperlipidemia rats ( n=12)

[0059] Group Dose / (mg / kg) TC / (mmol / g prot) TG / (mmol / g prot) Normal control / 0.045±0.014** 0.127±0.035** Model control / 0.142±0.042 0.434±0.142 Simvastatin 4 0.077±0.026* 0.264±0.062** XP 15.75 0.084±0.036* 0.258±0.106*

[0060] Compared with the model control group, * P<0.05, ** P<0.01

[0061] From the analysis in Table 2, compared with the normal control group, the TG and TC contents in the liver tissue of the rats in the model control group were significantly increased (P<0.01); the TG and TC contents in the liver tissue of the XP group and the Simvastatin group were reduced (P<0.05), indicating that XP can alleviate the degree of fatty liver lesions in hyperlipidemia rats to a certain extent.

[0062] And from Figure 1 HE staining of the liver showed that the hepatocyte structure of the rats in the normal control group was intact, and no fat vacuoles were formed; the hepatocytes of the rats in the model control group were disordered, and a large number of fat vacuoles were seen in the cells, indicating the formation of fatty liver. Compared with the model control group, the pathological changes of the liver tissue in the XP group were significantly improved, the hepatocytes were more neatly distributed, and the macrovesicular lipid droplets were reduced, which was similar to the Simvastatin group. In summary, XP can improve the lipid product of the liver in rats with hyperlipidemia.

[0063] (3) Effects of Xiongdan Jiuxin Pills on NO and ET-1 in serum and aortic morphology in hyperlipidemic rats

[0064] The experimental results are shown in Table 3 and Figure 2 As shown:

[0065] Table 3 Effect of XP on the levels of NO and ET-1 in serum of hyperlipidemic rats ( n=12)

[0066] Group Dose / (mg / kg) NO / (μmol / L) ET-1 / (ng / L) Normal control 4.4±0.7** 20.9±3.5** Model control 2.5±0.7 38.5±3.8 Simvastatin 4 3.9±0.3** 25.6±5.4** XP 15.75 3.9±1.1* 25.7±3.5**

[0067] Compared with the model control group, * P<0.05, ** P<0.01

[0068] ET-1 and NO are important vasodilator and vasoconstrictor, respectively. They interact with each other to maintain the normal function of blood vessels. When hyperlipidemia occurs, abnormal metabolism of NO and ET-1 in the vascular endothelium leads to impaired endothelial function.

[0069] From the analysis in Table 3, compared with the normal control group, the serum ET-1 content of the model control group rats was significantly increased (P<0.01), and the NO content was significantly decreased (P<0.01); compared with the model control group, the XP dose group could significantly reduce the serum ET-1 content and increase the serum NO content, which was similar to the SIM group. The above results show that the XP group can reduce vascular endothelial damage by improving the blood lipid level of hyperlipidemia rats.

[0070] Figure 2 The results showed that the aortic intima of rats in the normal control group was intact, smooth muscle cells were arranged in an orderly manner, and no foam cells were found; the aortic intima of rats in the model control group was damaged, the intima-media thickened, the smooth muscle cells were arranged in a disordered manner, and the lipid vacuoles in the media increased significantly; the aortic intima structure of rats in the XP group and simvastatin group was relatively complete, with fewer fat vacuoles, and the smooth muscle in the middle layer was arranged more neatly, indicating that the XP group had a certain protective effect on the vascular endothelial structure damage in rats with hyperlipidemia.

[0071] (4) Effects of Xiongdan Jiuxin Pills on serum AST and ALT levels and liver tissue structure in hyperlipidemia rats

[0072] The experimental results are shown in Table 4:

[0073] Table 4 Effects of XP on liver function in hyperlipidemic rats ( n=12)

[0074] Group Dose / (mg / kg) ALT / (U / L) AST / (U / L) Normal control 6.4±1.0** 10.5±1.8** Model control 13.1±2.6 17.4±3.8 Simvastatin 4 9.2±1.9** 11.9±1.7** XP 15.75 9.2±1.9** 11.9±2.4**

[0075] Compared with the model control group, * P<0.05, ** P<0.01

[0076] From the analysis in Table 4, compared with the normal control group, the AST and ALT activities in the serum of rats in the model control group were significantly increased (P<0.01); compared with the model control group, the AST and ALT activities in the XP group were significantly reduced, which was similar to the Simvastatin group.

[0077] (5) Effect of Xiongdan Jiuxin Pills on CETP and HMGCR in the serum of hyperlipidemic rats

[0078] The experimental results are shown in Table 5:

[0079] Table 5 Effect of XP on HMGCR and ACAT activity in hyperlipidemia rats ( n=12)

[0080] Group Dose / (mg / kg) HMGCR / (U / L) CETP / (μg / L) Normal control <![CDATA[87.1±42.7 ** ]]> <![CDATA[1510.7±133.5 ** ]]> Model control 264.8±109.2 2641.5±465.9 Simvastatin 4 <![CDATA[128.2±77.8 * ]]> <![CDATA[1869.7±387.5 ** ]]> XP 15.75 <![CDATA[134.1±70.3 * ]]> <![CDATA[2004.1±283.3 * ]]>

[0081] Compared with the model control group, *P<0.05, ** P<0.01

[0082] HMGCR is the rate-limiting enzyme for cholesterol synthesis in the liver. When HMGCR expression is upregulated, cholesterol synthesis in the body increases. Currently, statins in clinical practice lower blood lipids by inhibiting the activity of HMGCR. ACAT is the only enzyme in cells that synthesizes cholesterol esters (CE). Lowering ACAT can reduce the production of cholesterol esters and the secretion of very low-density lipoprotein (VLDL). CETP promotes the transport of CE and TG in the blood between HDL-C, LDL-C and VLDL. By regulating the expression level of CETP, lipid metabolism balance can be maintained.

[0083] The results in Table 5 showed that compared with the normal control group, the levels of CETP and HMGCR in the serum of the rats in the model control group were increased (P < 0.01); compared with the model group, the levels of CETP and HMGCR in the serum of the rats in the XP group and the simvastatin group were decreased.

[0084] (6) Effect of Xiongdan Jiuxin Pills on ACAT in the Liver Tissue of Hyperlipidemic Rats

[0085] The experimental results are shown in Table 6:

[0086] Table 6 Effects of XP on HMGCR and ACAT activity in hyperlipidemia rats ( n=12)

[0087] Group Dose / (mg / kg) ACAT / (U / L) Normal control <![CDATA[5.5±1.1 ** ]]> Model control 13.4±1.2 Simvastatin 4 <![CDATA[8.1±0.8 ** ]]> XP 15.75 <![CDATA[9.1±1.5 ** ]]>

[0088] Compared with the model control group, * P<0.05, ** P<0.01

[0089] The results in Table 6 showed that compared with the normal control group, the ACAT activity in the liver tissue of rats in the model control group increased (P<0.01); compared with the model group, the XP group had a significant decrease.

[0090] The results of (5) and (6) indicate that Xiongdan Jiuxin Pills can reduce the serum CETP content, HMGCR activity and ACAT activity in liver tissue of hyperlipidemia rats, thereby reducing cholesterol synthesis and accelerating metabolism, thereby improving blood lipid levels and playing a certain therapeutic role in hyperlipidemia.

[0091] (7) Effect of Xiongdan Jiuxin Pills on cognitive dysfunction in hyperlipidemic rats

[0092] 7.1 At the end of the experiment, we used water maze, Y maze, and novel object discrimination tests to evaluate the learning and memory abilities of rats.

[0093] like Figure 3As shown in the figure, analysis of the results of the water maze experiment showed that in the hidden platform experiment, as the training time of the rats increased, the latency of the three groups of rats to find the platform decreased accordingly, and the latency of each group of rats was the shortest on the fifth day, indicating that the three groups of rats had acquired a certain learning and memory ability during training.

[0094] Compared with the normal control group, the latency of the model control group rats to find the hidden platform in the last three days of training was significantly prolonged (P<0.01, P<0.05); the latency of the XP group to find the hidden platform on the last day of training was significantly shortened (P<0.05), compared with the model control group rats. In the probe experiment, the number of times the normal control group rats crossed the platform and the percentage of platform quadrant time were 3.2±1.0 times and 31.08±6.1%, respectively, while the number of times the model control group rats crossed the platform was reduced to 2.0±0.9 times, and the percentage of platform quadrant time was shortened to 24.58±5.18%, and compared with the normal control group, the data of the two groups showed significant differences (P<0.01), which shows that the model control group rats have a decreased memory of the safe platform location; the XP dose group rats have different degrees of recovery of the memory of the safe platform location (P<0.05), which shows that XP can improve the learning and memory ability of hyperlipidemia rats; the typical path diagram of the probe experiment further verifies this point.

[0095] The results of the Y-maze experiment are as follows Figure 4 As shown, we found that the alternation rate of the rats in the model control group was higher than that in the normal control group (P<0.01), indicating that the short-term working memory of the rats in the model control group decreased; while the short-term working memory of the rats in the XP dose group recovered (P<0.05).

[0096] The results of the novel object discrimination experiment are as follows Figure 5 As shown, compared with the rats in the normal control group, the discrimination index of the rats in the model control group was significantly reduced (P<0.01), indicating that the learning and memory ability of the rats in the model control group was weakened; the discrimination index of the rats in the XP dose group increased, which was significantly different from that of the rats in the model control group (P<0.05).

[0097] 7.2 Immunofluorescence staining of hippocampal neurons

[0098] Neuronal nucleus (NeuN) is a specific neuronal protein. In this experiment, NeuN immunofluorescence staining was used to detect neuronal loss in the CA1 region of the rat hippocampus.

[0099] Experimental method: Rats were killed 24 hours after the last administration, the chest cavity was quickly opened, the right atrial appendage was cut, and pre-cooled sterile saline was perfused from the left ventricle. Then the rat brain was removed, the left hemisphere was fixed with 4% paraformaldehyde, dehydrated and embedded in OCT embedding medium, frozen sections (20μm), antigen repaired and blocked in sequence, incubated with NeuN antibody (1:200) at 4℃ overnight, and then incubated with FITC-labeled donkey anti-rabbit secondary antibody at 37℃ for 1h, and anti-fluorescence quenching sealing agent was added and sealed. Images were collected using a fluorescence microscope, and the percentage of positive fluorescence area was calculated using Image-pro plus 6.0.0 software.

[0100] The experimental results are shown in Figure 6 and Figure 7 (Data are expressed as mean ± SEM (n = 6 / group), ** P<0.01, compared with the model control group; ns P>0.05, compared with the normal control group). Figure 6 and Figure 7 The results showed that compared with the normal control group, the number of neurons in the CA1 region of the hippocampus of the model control group was significantly reduced, indicating that long-term high-fat diet can cause serious neuronal damage in the CA1 region; compared with the model control group, the number of neurons in the CA1 region of the hippocampus of the XP group was significantly increased, and there was no statistical difference with the normal control group. The results suggest that Xiongdan Jiuxin Pills can significantly reduce neuronal damage in the CA1 region of the hippocampus, thereby improving cognitive dysfunction in the brain of rats caused by a high-fat diet.

[0101] 7.3 Western Blot Analysis of Caspase 3 Activation in Brain Tissue

[0102] Experimental methods: Western Blot was used to analyze the expression levels of pro-Caspase 3 (antibody to pro-caspase 3) and cleaved-Caspase 3 (activated caspase 3) in brain tissue to detect the degree of Caspase 3 activation. Brain tissue samples frozen at -80°C were taken as before, and total brain tissue protein was extracted and the protein concentration was determined. Equal amounts of protein were taken from each group of samples, separated by SDS-PAGE, and transferred to PVDF membranes. After blocking with 5% skim milk for 2 hours, the membranes were incubated with Caspase 3 antibody (1:800) at 4°C overnight, and then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 hour. Gel imaging and data statistics were performed using the BIO-RAD gel imaging system.

[0103] Figure 8 and Fig. 9 (Data are expressed as mean ± SEM (n = 4 / group), * P<0.05, **P<0.01, compared with the model control group; # P<0.05, compared with the normal control group) showed that compared with the normal control group, the cleaved-caspase-3 / pro-caspase-3 ratio in the brain of the model control group rats was significantly increased (P<0.01); while the cleaved-caspase-3 / pro-caspase-3 ratio of the XP group rats was significantly lower than that of the model control group (P<0.05), but significantly higher than that of the normal control group (P<0.05). The results showed that Xiongdan Jiuxin Pills can significantly reduce caspase-3 activation, reduce apoptosis and loss of brain neurons in hyperlipidemic rats, play a neuroprotective role, and alleviate cognitive dysfunction.

[0104] According to the results of 7.1-7.3, after 9 weeks of administration, the results of Y-maze, novel object discrimination test, and water maze behavioral tests showed that Xiongdan Jiuxin Pills can significantly improve the learning and memory ability of hyperlipidemia rats. Further immunofluorescence staining results showed that Xiongdan Jiuxin Pills can significantly reduce the damage to brain neurons in hyperlipidemia rats.

[0105] Apoptosis is the main pathological mechanism of brain neuron loss due to damage. Caspase-3 is a key enzyme that induces apoptosis. Under physiological conditions, it mainly exists in the form of pro-caspase-3. Under pathological conditions, it is activated and cleaved into cleaved-caspase-3. The increase in the level of its cleaved protein indicates the initiation of apoptosis. This experiment found that the level of cleaved-caspase-3 protein in the XP group was significantly reduced, suggesting that Xiongdan Jiuxin Pills may reduce neuronal damage and cognitive dysfunction caused by a high-fat diet by inhibiting neuronal apoptosis.

[0106] 7.4 ELISA (enzyme linked immunosorbent assay) detection of proinflammatory factors in brain tissue

[0107] Experimental method: rats were killed 24 hours after the last administration, the chest cavity was quickly opened, the right atrial appendage was cut, and pre-cooled sterile saline was perfused from the left ventricle. The rat brain was then removed and the right hemisphere was frozen with liquid nitrogen and kept at -80°C for later use.

[0108] Extract total protein from brain tissue, prepare brain tissue homogenate, and centrifuge at 4°C to obtain supernatant. According to the instructions of the BCA protein concentration assay kit, determine the total protein concentration of brain tissue samples. A rat ELISA kit was used to detect the protein levels of proinflammatory factors (IL-6 and TNF-α) in rat brain tissue. According to the instructions of the kit, the absorbance of brain tissue samples was detected at 450nm, and the cytokine content was calculated based on the standard curve and absorbance.

[0109] Fig.10 (Data are expressed as mean ± SEM (n = 6 / group), * P<0.05, ** P<0.01, compared with the model control group; ns P>0.05, compared with the normal control group) and Fig.11 (Data are expressed as mean ± SEM (n = 6 / group), * P<0.05, ** P<0.01, compared with the model control group; ns P>0.05, compared with the normal control group) The results showed that compared with the normal control group, the content of IL-6 and TNF-α in the brain of the model control group was significantly increased (P<0.01); while the levels of IL-6 and TNF-α in the brain of the XP group rats were significantly lower than those in the model control group (P<0.01 or P<0.05), and there was no statistical difference between the normal control group and the normal control group (P>0.05). The results showed that Xiongdan Jiuxin Pills can significantly reduce the inflammatory response of the central nervous system in hyperlipidemia rats.

[0110] 7.5 Western Blot Analysis of PPARγ (Peroxisome Proliferator-Activated Receptor γ) Expression in Brain Tissue

[0111] Experimental method: rats were killed 24 hours after the last administration, the chest cavity was quickly opened, the right atrial appendage was cut, and pre-cooled sterile saline was perfused from the left ventricle. The rat brain was then removed and the right hemisphere was frozen with liquid nitrogen and kept at -80°C for later use.

[0112] Western Blot was used to analyze the expression level of PPARγ in brain tissue. Brain tissue samples frozen at -80℃ were obtained as before, and total brain tissue protein was extracted and the protein concentration was determined. Equal amounts of protein were taken from each group of samples, separated by SDS-PAGE (polyacrylamide gel electrophoresis), and transferred to PVDF (polyvinylidene fluoride) membranes. After blocking with 5% skim milk for 2 hours, the membranes were incubated with PPARγ antibody (1:1000) at 4℃ overnight, and then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at 37℃ for 1 hour. Gel imaging and data statistics were performed using the BIO-RAD gel imaging system.

[0113] Fig.12 and Fig.13 (Data are expressed as mean ± SEM (n = 4 / group), * P<0.05, ** P<0.01, compared with the model control group; #P<0.05, compared with the normal control group) showed that compared with the normal control group, the PPARγ protein level in the brain tissue of the model control group was significantly reduced (P<0.05); while the PPARγ protein level of the XP group rats was significantly higher than that of the model control group and the normal control group (P<0.01 or P<0.05). The results showed that Xiongdan Jiuxin Pills can significantly increase the PPARγ protein level in the brain of hyperlipidemia rats.

[0114] 7.6 Western Blot Analysis of NF-κB (Nuclear Transcription Factor) Activation in Brain Tissue

[0115] Experimental methods: Western Blot was used to analyze the nuclear translocation of NF-κB p65 in brain tissue to detect the degree of NF-κB activation. Brain tissue frozen at -80℃ was obtained as before, and tissue nuclear protein was extracted according to the instructions of the subcellular structure nuclear and cytoplasmic protein extraction kit, and the nuclear protein concentration of the sample was determined according to the instructions of the BCA protein concentration determination kit. Equal amounts of nuclear protein were taken from each group of samples, separated by SDS-PAGE and transferred to PVDF membrane. After blocking with 5% skim milk for 2 hours, it was incubated with NF-κBp65 antibody (1:400) at 4℃ overnight, and then the membrane was incubated with horseradish peroxidase-conjugated secondary antibody at 37℃ for 1 hour, and gel imaging and data statistics were performed using the BIO-RAD gel imaging system.

[0116] Fig.14 and Fig.15 (Data are expressed as mean ± SEM (n = 4 / group), * P<0.05, compared with the model control group; # P<0.05, compared with the normal control group) showed that compared with the normal control group, the nuclear level of NF-κB p65 in the model control group was significantly increased (P<0.01); while the nuclear level of NF-κB p65 in the XP group was significantly lower than that in the model control group (P<0.05), and also higher than that in the normal control group (P<0.05). The results showed that Xiongdan Jiuxin Pills may play an anti-inflammatory role by reducing the nuclear transfer of NF-κB p65, thereby inhibiting the activation of NF-κB in the brain of hyperlipidemic rats.

[0117] Neuroinflammation is the main pathological mechanism of cognitive dysfunction. According to the results of 7.4-7.6 tests, the ELISA test results showed that Xiongdan Jiuxin Pills can significantly reduce the content of TNFa and IL6. PPARγ can inhibit the NF-κB pathway to exert anti-inflammatory effects. Western blot test results showed that Xiongdan Jiuxin Pills can significantly increase the expression level of PPARγ protein in the brain tissue of hyperlipidemic rats and inhibit the nuclear transfer and activation of NF-κBp65 protein.

[0118] The results showed that Bear Bile Heart-Saving Pills can upregulate the expression of PPARγ protein in the brain and inhibit the activation of NF-κB, thereby alleviating the neuroinflammatory response in rats with hyperlipidemia and exerting a neuroprotective effect.

[0119] (8) Effects of Xiongdan Jiuxin Pills on cardiac pathology in hyperlipidemic rats

[0120] Fig.16 From the HE staining results, it can be seen that the myocardial tissue structure of rats in the normal control group is intact, the myocardial cells are arranged neatly, and there is no inflammatory infiltration; the myocardial cells of rats in the model control group are arranged disorderly, a large number of myocardial cells are hypertrophic and deformed, and there is a small amount of inflammatory cell infiltration; each drug-treated group can alleviate the pathological changes of the heart of rats with hyperlipidemia to varying degrees, among which the pathological changes in the XP dosage group are the most obvious, the myocardial cells are arranged slightly neatly, a small number of myocardial cells are hypertrophic and degenerated, and the cell nuclei are evenly distributed.

[0121] In summary, XP can have a certain protective effect on the cardiac function of hyperlipidemic rats.

[0122] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

Claims

1. Application of Bear Bile Heart-saving Pills in the preparation of drugs for the treatment of hyperlipidemia-related cognitive dysfunction.

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