Application of anti-dredging and bone-strengthening granules in preparation of medicine for treating osteoporosis and non-alcoholic fatty liver disease

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to application of anti-dredging and bone-strengthening granules in preparation of medicine for treating osteoporosis and non-alcoholic fatty liver disease and a combined action mechanism of treating different diseases at the same time, and the method comprises the following steps: establishing an osteoporosis case group and a model group, and measuring bone mineral density, blood fat and liver function indexes of the two groups; in-vivo experiments are carried out, and related indexes of osteoporosis and non-alcoholic fatty liver diseases of the model group are detected; carrying out HE staining on femur and liver tissues of the experimental rat; acting on the screened clinical biomarker and autophagy pathway protein by adopting anti-dredging and bone-strengthening particles; in-vitro experiments are carried out, a free fatty acid solution is used for inducing lipid accumulation in human liver LO2 cells, a high-fat cell model is established, and an AMPK inhibitor, anti-dredging and bone-strengthening granule drug-containing serum and fenofibrate drug-containing serum are respectively used for intervention.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and particularly to application of Kangshu Jiangu granules in preparing medicines for treating osteoporosis and non-alcoholic fatty liver disease. Background Art

[0002] Osteoporosis (OP) is a systemic metabolic disorder characterized by increased bone fragility caused by decreased bone mass, bone matrix and trabecular number, and destruction of bone tissue microstructure. It has a high incidence rate, a long course of disease, and is often accompanied by serious complications. According to statistics, more than 70% of pathological fractures are caused by osteoporosis, which in turn increases the mortality rate (the mortality rate in the first year after hip fracture can reach 20-24%) and the disability rate (40% cannot walk), which seriously threatens human health. At present, the incidence rate of osteoporosis in my country is as high as 7%, and it is estimated that by 2050, the number of osteoporosis and low bone mass patients in my country will increase to 300 million. Clinical treatment of osteoporosis drugs mainly inhibit bone resorption, promote bone synthesis and regulate hormone levels in the body, but side effects such as carcinogenicity, teratogenicity, and osteonecrosis have greatly limited the promotion and use of these drugs. So far, there is no effective drug with small toxic side effects suitable for long-term use.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic disease characterized by hepatic steatosis. Its pathogenesis is closely related to the imbalance of lipid metabolism homeostasis in hepatocytes. Its incidence (nearly 30%) ranks first among chronic liver diseases and is a global public health issue. In the past, insulin resistance and obesity were considered to be the main risk factors for NAFLD. The latest studies show that the liver may be the center of multiple organs and systems, with multiple effects on various systems, including the skeletal system. Recently, studies have found that patients with NAFLD have lower bone density and bone mass than their normal peers. However, the correlation between the two is still controversial, and the mechanism of mutual influence is unclear.

[0004] Traditional Chinese medicine has unique advantages in treating osteoporosis and non-alcoholic fatty liver disease, such as multiple targets, multiple pathways, and multiple levels. It has a significant effect on improving patients' clinical symptoms and bone and liver metabolism. In addition, traditional Chinese medicine treatment options are diverse and safe, and are easily accepted by the majority of patients in clinical practice. Based on the nearly 50 years of clinical experience summarized by Professor Yang Yuhua, a famous orthopedic expert, and Professor Chu Xiuzhen, an expert in prescriptions, this application has developed a Chinese medicine composition in combination with pharmaceutical experiments. The Chinese medicine composition is consistent with the TCM syndrome differentiation of osteoporosis and non-alcoholic fatty liver disease, and is an empirical prescription that has been continuously optimized and screened. The whole prescription is guided by the TCM theory of "liver and kidney homology" and is legislated on "tonifying the kidney, strengthening the spleen, and activating blood circulation". It is mainly used for osteoporosis caused by spleen and kidney deficiency, qi and blood stasis, and non-alcoholic fatty liver disease with liver depression and spleen deficiency syndrome. Preliminary clinical studies have confirmed that Kangshu Jiangu Granules have good clinical efficacy in increasing bone mass, relieving pain, and reducing the incidence of fractures, and no obvious toxic and side effects have been found. At the same time, animal experiments have confirmed that this prescription can inhibit bone resorption, increase bone mass, improve bone microstructure and improve liver lipid metabolism indicators in ovariectomized rats, but its specific mechanism of action is still unclear. Therefore, clarifying the targets and molecular mechanisms of Kangshu Jiangu Granules in treating osteoporosis and non-alcoholic fatty liver disease and ensuring its stable and reliable intervention effect will provide a new strategy for the effective treatment of osteoporosis and non-alcoholic fatty liver disease. Summary of the invention

[0005] The present invention aims to provide an application of Kangshu Jiangu granules in the preparation of drugs for treating osteoporosis and non-alcoholic fatty liver disease, solves the technical problem of large side effects caused by treatment with traditional Chinese medicine in the prior art, and clarifies the mechanism of action of Kangshu Jiangu granules in treating osteoporosis and non-alcoholic fatty liver disease with different diseases.

[0006] To solve the above problems, the technical solution adopted by the present invention is:

[0007] Application of Kangshujiangu granules in the preparation of drugs for treating osteoporosis and non-alcoholic fatty liver disease.

[0008] Kangshujian Bone Granules achieve the treatment of osteoporosis and non-alcoholic fatty liver disease by reducing the expression of p-Akt, p-mTOR and p-ULK1 Ser757, and increasing the expression of p-AMPK and p-ULK1 Ser555.

[0009] The Kangshu Jiangu granules include the following components in parts by weight: 15-25 parts of epimedium, 10-20 parts of eucommia bark, 10-20 parts of achyranthes bidentata, 10-15 parts of atractylodes macrocephala and 10-20 parts of salvia miltiorrhiza.

[0010] The Kangshu Jiangu granules include the following components in parts by weight: 20 parts of epimedium, 15 parts of eucommia bark, 15 parts of achyranthes bidentata, 12 parts of atractylodes macrocephala and 15 parts of salvia miltiorrhiza.

[0011] The research method of using Kangshujiangu granules in preparing a drug for treating osteoporosis and non-alcoholic fatty liver disease comprises the following steps:

[0012] Step 1: Detect biochemical indicators of patients with osteoporosis and non-alcoholic fatty liver disease to screen clinical biomarkers;

[0013] Step 2: Establish osteoporosis and non-alcoholic fatty liver disease animal models, compare indicators, and screen out autophagy pathway proteins;

[0014] Step 3: Use Kangshujiangu Granules to act on the screened clinical biomarkers and autophagy pathway proteins, and preliminarily evaluate the effects of Kangshujiangu Granules on autophagy proteins and related regulatory proteins in combating osteoporosis and non-alcoholic fatty liver disease;

[0015] Step 4: In vitro experiments were conducted to verify the effects of Kangshujian Bone Granules on autophagy proteins and related regulatory proteins in combating osteoporosis and non-alcoholic fatty liver disease.

[0016] The pharmacology of each raw material is as follows:

[0017] Epimedium: Spicy, sweet and warm. Enters the liver and kidney meridians. Has the functions of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism.

[0018] Eucommia: Sweet, warm. Enters the liver and kidney meridians. Sweet, warm and tonic, enters the liver and kidney meridians, and has a strong medicinal effect. Good at warming and tonic liver and kidney, strengthening tendons and bones, stabilizing pregnancy, and lowering blood pressure.

[0019] Achyranthes bidentata: bitter, sweet, sour, neutral. Enters the liver and kidney meridians. Bitterness is used to drain and descend, sourness enters the liver, sweetness is used to nourish and penetrate, good at descending, and enters the liver and kidney meridians. Raw use is mostly bitter, neutral and slightly cool, and can promote the flow of blood and descend. It can remove blood stasis and promote menstruation, and treat blood stasis during menstruation and childbirth, as well as arthralgia and cramps; it can also promote diuresis and relieve stranguria, and is often used for damp-heat in the downward flow; it can also lead blood and fire downward, and is often used for blood heat, fire reversal, and hyperactivity of liver yang. Processed use is mostly sweet, neutral and slightly warm, good at replenishing deficiency, good at replenishing the liver and kidney, and strengthening tendons and bones. It is an important medicine for treating soreness of the waist and knees, and weakness of tendons and bones.

[0020] Atractylodes macrocephala: bitter, sweet, warm. Enters the spleen and stomach meridians. Sweet tonic, penetrating, bitter, warm and dry, enters the spleen and stomach meridians, mainly warms and strengthens the body, and can also remove dampness. Good at replenishing qi and strengthening the spleen, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. It treats spleen and stomach qi deficiency, spleen deficiency edema, phlegm and fluid, spontaneous sweating due to superficial deficiency, and fetal movement disorder.

[0021] Danshen: bitter, slightly cold, enters the heart and liver meridians. Bitterness can dissipate, and slightly cold can clear, entering the heart and liver meridians. It can activate blood circulation, remove blood stasis, relieve pain, and cool blood to eliminate restlessness and eliminate carbuncle. It is mainly used to treat blood stasis, blood heat, and heat disturbing the mind, and also treats heat toxicity, carbuncle, swelling and pain.

[0022] The beneficial effects of the present invention are:

[0023] 1. The Chinese medicine composition of the present invention is used to treat osteoporosis and non-alcoholic fatty liver disease, can significantly increase bone mass, relieve pain, reduce the incidence of fractures, and reduce the lipid accumulation index of liver tissue, promote the cure of the two diseases, and has reliable efficacy, achieving the effect of treating different diseases with the same medicine. The raw materials used are easy to obtain, low in price, convenient to make, and have a wide range of applications.

[0024] 2. The application of the Chinese medicine composition of the present invention in the treatment of osteoporosis and non-alcoholic fatty liver disease has been confirmed by animal experiments and studies, showing that the efficacy is obvious and no obvious side effects are found. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram showing the improvement of bone tissue morphology by the Kangshujiangu particles in this application;

[0026] Figure 2 The correlation analysis between BMD values ​​and age, BMI, TC, TG and ALT in osteoporosis and non-osteoporosis groups was performed;

[0027] Figure 3 The effect of Kangshujiangu granules on the body weight of rats in each group in this application;

[0028] Figure 4 This is a comparison chart of femoral trabecular changes under HE staining;

[0029] Figure 5 This is a comparison chart of the changes in the serum Ca and P content of rats in the control group, model group and intervention group;

[0030] Figure 6 This is a comparison chart of the changes in PINP and ALP levels in the serum of rats in the control group, model group, and intervention group;

[0031] Figure 7 This is a comparison chart of the changes in CTX-1 and TRACP5b levels in the serum of rats in the control group, model group, and intervention group;

[0032] Figure 8 Femoral calcium nodules were stained in the control group, model group, and intervention group;

[0033] Fig. 9 Micro-CT results of femur, tibia and lumbar spine (L4);

[0034] Fig.10 Comparison of Tb.N, Tb.Th, and Tp.Sp values ​​in rat femur;

[0035] Fig.11 The effect of Kangshujianbone granules on autophagy-related proteins in this application;

[0036] Fig.12The role of Kangshujianbone granules in regulating autophagy-related proteins in this application;

[0037] Fig.13 HE staining of rat liver tissue (400X);

[0038] Fig.14 Comparison of TG, TC, HDL-C and LDL-C levels in rat liver tissue;

[0039] Fig.15 The effect of Kangshujiangu granules on autophagy proteins in liver tissue in this application (A. Western Blot band diagram; B, C, D and E are quantification diagrams of LC3Ⅱ / LC3Ⅰ, p62, Atg7 and Beclin1 proteins, respectively, *P<0.05vs Model);

[0040] Fig.16 The effect of Kangshujiangu granules on autophagy pathway proteins in liver tissue in this application (A. Western Blot band diagram; B, C, D, E and F are quantification diagrams of p-Akt, p-mTOR, p-AMPK, p-ULK1 (Ser555) and p-ULK1 (Ser757) proteins, respectively, *P<0.05vs Model);

[0041] Fig.17 The effect of the anti-suspension bone-enhancing granule-containing serum on LO2 cell TG in this application;

[0042] Fig.18 The effect of the anti-suspension bone granule-containing serum on ALT and AST in LO2 cells in this application (A. ALT level; B. AST level, *P<0.05vs FFA);

[0043] Fig.19 The effect of the Kangshujiangu granule-containing serum in the present application on lipid accumulation in LO2 cells after AMPK inhibition (A. Oil Red O staining, 200×; B. BODIPY staining, 200×; C. BODIPY staining quantification graph, *P<0.05vs FFA, #P<0.05vs FFA+KSJG);

[0044] Fig. 20 The effect of the anti-suspension bone granule-containing serum in the present application on the autophagy proteins of LO2 cells after AMPK inhibition (A. Western Blot band diagram; B, C, D and E are quantification diagrams of LC3Ⅱ / LC3Ⅰ, p62, Atg7 and Beclin1 proteins, respectively, *P<0.05vs FFA, #P<0.05vs FFA+KSJG). DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below through embodiments.

[0046] The invention provides an anti-sparse bone strengthening granule, which comprises the following components in parts by weight: 15-25 parts of epimedium, 10-20 parts of eucommia bark, 10-20 parts of achyranthes bidentata, 10-15 parts of atractylodes macrocephala and 10-20 parts of salvia miltiorrhiza.

[0047] Preferably, the epimedium is selected from wild medicinal materials produced in Ankang City, Shaanxi Province, the Eucommia ulmoides is selected from the artificially cultivated product produced in the GAP base of Lueyang County, Hanzhong, the Achyranthes bidentata is selected from the Achyranthes bidentata produced in Henan Province, the Atractylodes macrocephala is selected from the artificially cultivated product in Yaozhou District, Tongchuan City, and the Salvia miltiorrhiza is selected from the artificially cultivated product in the GAP base of Shangluo Tasly Botanical Pharmaceutical Co., Ltd.

[0048] Preferably, an anti-sparse bone-strengthening granule comprises the following components in parts by weight: 20 parts of Epimedium, 15 parts of Eucommia, 15 parts of Achyranthes, 12 parts of Atractylodes and 15 parts of Salvia miltiorrhiza.

[0049] Furthermore, the dosage form of the medicine is granules or decoction.

[0050] The anti-osteoporosis and bone-strengthening granules of the present invention can be used in drugs for treating osteoporosis, and can also be used in drugs for treating non-alcoholic fatty liver disease.

[0051] Embodiment 1

[0052] The anti-osteoporosis bone-strengthening particles of the present invention are used in the treatment of osteoporosis.

[0053] The specific experimental process includes the following steps:

[0054] Step 1: Select 20 parts of Epimedium, 15 parts of Eucommia, 15 parts of Achyranthes, 12 parts of Atractylodes and 15 parts of Salvia miltiorrhiza.

[0055] Step 2: A case-control study was conducted, that is, randomly selected research subjects were divided into an osteoporosis case group and a control group according to their osteoporosis T scores, and the bone density, blood lipids, liver function and other indicators of the two groups were measured, and a correlation analysis of osteoporosis and lipid-related indicators was performed.

[0056] Step 3, in vivo experiment, 60 3-4 months old female SD rats were randomly divided into 6 groups, namely control group (normal saline / d), model group (normal saline / d), low (0.287g / kg / d), medium (0.573g / kg / d), high (1.146g / kg / d), Kangshu Jiangu granule intervention group and icariin group (0.573g / kg / d). Except for the control group, bilateral ovariectomy was performed in each group, and the relevant indicators of osteoporosis model were detected.

[0057] Step 4: Verify the changes in autophagy-related proteins and AMPK / ULK1 (Ser555) pathways. That is, all experimental animals were killed after blood was drawn from the abdominal aorta, and femur and liver tissues were taken for HE staining; Micro-CT was used to detect rat bone density, trabecular structure and parameters; biochemical tests were performed on serum calcium and phosphorus content and lipid metabolism indicators, and Elisa was used to detect alkaline phosphatase (ALP), type I procollagen N-terminal peptide (PINP), tartrate-resistant acid phosphatase (TRACP-5b) and type I collagen C-terminal peptide (CTX-I); Western Blot was used to detect autophagy and related pathway proteins.

[0058] Step 5: Use the anti-osteoporosis granules to act on the screened clinical biomarkers and autophagy pathway proteins, and preliminarily evaluate the effects of the anti-osteoporosis granules on autophagy proteins and related regulatory proteins in combating osteoporosis.

[0059] Step 6. In vitro experiments, free fatty acid (FFA) solution (oleic acid: palmitic acid = 2:1) was used to induce lipid accumulation in human liver LO2 cells to establish a high-fat cell model. AMPK inhibitor (Compound C), the anti-sparse bone granules containing serum and fenofibrate (FF) containing serum were used for intervention. The cells were divided into 6 groups, namely BSA control group, FFA model group, FFA+Compound C group, FFA+KSJG group, FFA+FF positive control group and FFA+KSJG+Compound C group. Cell lipid accumulation was detected by Oil Red O staining and BODIPY 493 / 503 fluorescence staining; cell lipid metabolism and liver function indicators were detected by biochemical detection; Western Blot, immunofluorescence co-localization and immunoprecipitation methods were used to deeply detect the co-expression relationship of various targets in autophagy and related pathway proteins in the AMPK / ULK1 pathway, proving that the anti-sparse bone granules have a multi-target overall regulatory effect on the autophagy pathway.

[0060] 2. Experimental Results

[0061] 1. Comparison of biochemical indicators between the two groups of clinical data

[0062] The prevalence of osteoporosis increased with age (P<0.01). The weight and BMI of the osteoporosis group were higher than those of the non-osteoporosis group, while the height was lower than that of the non-osteoporosis group, and the difference was statistically significant (P<0.01). The TC and TG of the osteoporosis group were higher than those of the non-osteoporosis group (P<0.01), and there was no statistically significant difference in HDL-C, LDL-C, ALT, AST, total bilirubin and direct bilirubin.

[0063] 2. Correlation between BMD value, T-socre and age, BMI and biochemical indexes

[0064] Pearson correlation analysis was used to analyze whether the BMD values ​​and T-score of the included patients were correlated with age, BMI, TG, TC, HDL-C, LDL-C, ALT, AST, total bilirubin, and direct bilirubin. The results showed that BMD and T-score were correlated with age, BMI, TC, TG, and ALT. BMD was significantly negatively correlated with age, weight, TG, and TC, and significantly positively correlated with ALT (P<0.05). For details, please refer to Figure 2 In addition, T-score was negatively correlated with LDL-C, and the correlation was statistically significant.

[0065] 3. Multifactorial analysis of osteoporosis

[0066] The results of binary logistic regression analysis showed that age (β=-0.103, OR=0.902, P=0.001), BMI (β=-0.535, OR=0.585, P=0.000), TC (β=-0.617, OR=0.539, P=0.033) and TG (β=-0.623, OR=0.536, P=0.031) were risk factors for osteoporosis.

[0067] 3. Clinical study on improving osteoporosis with Kangshujian bone granules in this application

[0068] 1. Through two clinical studies, it was found that the Kangsuojian bone granules can significantly improve the clinical efficacy of osteoporosis. Compared with Gusongbao capsules, the therapeutic efficacy and total efficacy increased by 22.3% (48.1%, 25.8%) and 22% (83.3%, 61.3%) respectively; compared with Calcitrate, the total efficacy increased by 12.5% ​​(87.5%, 75%), indicating that the therapeutic efficacy and total efficacy of the Chinese medicine composition are higher than those of traditional Chinese and Western medicines. At the same time, it can also increase the BMD value of the lumbar spine and femoral neck (as shown in the table below). Animal experimental studies have found that in the osteoporosis rat model with bilateral ovarian resection, the Chinese medicine composition can improve the morphology of bone tissue such as trabeculae (such as Figure 1 And no obvious side effects were found in long-term toxicity tests.

[0069]

[0070] Note: *P<0.05 compared with before treatment

[0071] 2. In vivo study of Kangshujiangu granules on osteoporosis in ovariectomized rats

[0072] 1) General conditions:

[0073] After 12 weeks of free feeding, the body weight of rats in each group increased. Compared with the control group, the model group had the largest increase, and a statistically significant difference was observed in the second week (p<0.05). Compared with the control group, the hair of the rats in the model group became dry, yellow, dull, and prone to hunching and slow reaction from the sixth week. The three doses of intervention groups and icariin improved these phenomena and effectively controlled the weight of the animals. For details, refer to Figure 3 shown.

[0074] 2) Bone histopathology

[0075] HE staining was used to evaluate the changes in femoral trabeculae. The green part in the figure is trabeculae. 12 weeks after the rats were ovariectomized, compared with the control group, the trabeculae in the model group were disordered in arrangement, uneven in thickness, widened in distance, and had more trabecular breakpoints; compared with the model group, the number of trabeculae in the low, medium, and high doses of the Chinese medicine combination intervention groups increased significantly, and the disordered arrangement, thinning, and sparseness of the trabeculae were significantly alleviated. The structure was generally clear, and trabecular fractures were occasionally seen. The effect was particularly significant in the medium dose group. For details, please refer to Figure 4 shown.

[0076] 3) Serological indicators

[0077] Serum Ca and P levels: Compared with the control group, the Ca and P contents in the model group decreased (P<0.05); compared with the model group, the Ca and P contents in the medium-dose intervention group increased (P<0.05), and the P content in the high-dose intervention group increased (P<0.05). There was no significant difference in Ca content between the low-dose and high-dose intervention groups (P>0.05), and there was no significant difference in P content between the low-dose intervention group (P>0.05). For details, please refer to Figure 5 shown.

[0078] Serum PINP and ALP levels: Compared with the control group, the PINP and ALP levels in the model group increased (P<0.05); compared with the model group, the three dose intervention groups could reduce the PINP and ALP levels (P<0.05); the medium dose group was the most significant, see Figure 6 shown.

[0079] Serum CTX-I and TRACP5b levels: Compared with the control group, the CTX-I and TRACP5b levels in the model group increased (P<0.05); compared with the model group, the medium and high dose intervention groups could reduce the CTX-I level (P<0.05), and the three dose intervention groups could reduce the TRACP5b level (P<0.05), with the medium dose group being the most significant. There was no significant difference in CTX-I level in the low dose intervention group (P>0.05). For details, please refer to Figure 7 shown.

[0080] 4) Calcium salt deposition detection

[0081] Von Kossa staining was performed on the distal end of the femur to evaluate the amount of calcium salt deposition in the femur. Compared with the control group, the amount of calcium salt deposition in the model group decreased. Compared with the model group, the amount of calcium salt deposition in the low, medium, and high dose intervention groups increased. Among them, the increase in calcium salt deposition in the medium dose intervention group was the most obvious, and the difference was statistically significant (P<0.05). For details, refer to Figure 8 shown.

[0082] 5) Micro-CT test results

[0083] Micro-CT scans of the femur, tibia, and lumbar spine (L4) showed Fig. 9 Compared with the control group, the model group had decreased bone mass, sparse and loose trabeculae, and a "rod-shaped" structure; compared with the model group, the bone mass of the three dose intervention groups increased.

[0084] 6) Evaluation of trabecular bone parameters

[0085] Compared with the control group, the Tb.N and Tb.Th values ​​of the model group decreased (P<0.05), and the Tp.Sp value increased (P<0.05); compared with the model group, the Tb.N and Tb.Th values ​​of the three dose intervention groups increased (P<0.05), and the Tp.Sp value decreased (P<0.05), with the middle dose group being the most significant. Fig.10 shown.

[0086] 7) Effect of Kangshujianbone Granules on Autophagy Level in Osteoporosis

[0087] Effects on autophagy marker proteins: Compared with the control group, the expressions of LC3Ⅱ / LC3Ⅰ, Atg7 and Beclin1 in the model group were decreased, and the expression of p62 was increased; compared with the model group, the low, medium and high dose intervention groups could increase the expressions of LC3Ⅱ / LC3Ⅰ and Beclin1; the medium dose intervention group could increase the expression of Atg7 and decrease the expression of p62; the low, medium and high dose intervention groups could promote the autophagy level to different degrees, and the effect of the medium dose intervention group was the most significant, with significant difference (P<0.05), please refer to Fig.11 shown.

[0088] Effect on autophagy pathway: Compared with the control group, the model group induced p-Akt expression, p-mTOR expression and p-ULK1 (Ser757) expression increased, while p-AMPK and p-ULK1 (Ser555) expression decreased; compared with the model group, the medium and high dose intervention groups reduced p-Akt, p-mTOR and p-ULK1 (Ser757) expression to varying degrees, and increased p-AMPK and p-ULK1 (Ser555) expression, and the differences were significant. For details, please refer to Fig.12 shown.

[0089] Embodiment 2

[0090] 1. Example 2 The Kangshu Jiangu granules of the present invention are applied to the treatment of non-alcoholic fatty liver disease, and the specific experimental steps are the same as those in Example 1.

[0091] 2. Experimental Results

[0092] 1. In vivo study on the improvement effect of Kangshujiangu granules on non-alcoholic fatty liver disease

[0093] HE staining results of liver tissues of rats in each group showed that the liver cell cords of rats in the control group were neatly arranged, the liver lobules were clear, and no fatty degeneration of the liver was observed; the liver cell cords of rats in the model group were disordered, the liver cells were swollen, and obvious macrovesicular fatty degeneration was observed; compared with the model group, the fatty degeneration of liver cells in the low, medium, high dose groups and icariin group was significantly improved, the cytoplasmic vacuolar changes were significantly alleviated, and the lipid droplets were significantly reduced or basically disappeared, among which the high dose group had the most significant effect, and the specific results are shown in Table 1. Fig.13 shown.

[0094] 1) Determination of TG, TC, HDL-C and LDL-C levels in liver tissue: Compared with the control group, the TG, TC and LDL-C levels in the model group increased, and the HDL-C level decreased (P<0.05); compared with the model group, the TG, TC and LDL-C levels in the low, medium, high dose and icariin groups decreased (P<0.05), among which there was no statistically significant difference in LDL-C after intervention in the low dose group, and there was no statistically significant difference in HDL-C after intervention in the low, medium, high dose and icariin groups. For details, please refer to Fig.14 shown.

[0095] 2) Effect of the Chinese medicine composition on autophagy proteins in liver tissue: Compared with the control group, the expression levels of LC3Ⅱ / LC3Ⅰ, Atg7 and Beclin1 proteins in the model group were decreased, and the level of p62 was increased (P<0.05); Compared with the model group, the medium-dose, high-dose and icariin groups could increase the expression levels of LC3Ⅱ / LC3Ⅰ, Atg7 and Beclin1 proteins to varying degrees, and reduce the level of p62, with the medium-dose group having the most significant effect (P<0.05); Among them, the low-dose group had increased LC3Ⅱ / LC3Ⅰ levels and decreased p62 levels compared with the model group, but there was no significant difference in Atg7 and Beclin1, for details, refer to Fig.15 shown.

[0096] 3) Effect of the Kangshujianbone granules on the autophagy pathway of liver tissue: Compared with the control group, the expression levels of p-Akt, p-mTOR and p-ULK1 (Ser757) proteins in the model group were increased, while the levels of p-AMPK and p-ULK1 (Ser555) were decreased (P<0.05); Compared with the model group, the low, medium, high dose groups and icariin group could reduce the expression levels of p-Akt, p-mTOR and p-ULK1 (Ser757) to varying degrees, and increase the levels of p-AMPK and p-ULK1 (Ser555) (P<0.05); Among them, the high dose group had the most significant difference in reducing the levels of p-Akt, p-mTOR and increasing the levels of p-ULK1 (Ser555), and the medium dose group had the most significant effect in increasing the levels of p-AMPK (P<0.05), please refer to Fig.16 shown.

[0097] 2. In vitro study on the mechanism of improvement of non-alcoholic fatty liver disease by Kangshujiangu Granules

[0098] 1) Establishment of high lipid cell model: The cells in the BSA group and the FFA group were fixed and stained with Oil Red O. The Oil Red dye passed through the cell membrane and combined with the LD in the cell, dyeing the LD into red plaques. The results showed that compared with the BSA group, the red plaques in the FFA group increased significantly, indicating that the lipid accumulation level in the FFA group was significantly increased; in BODIPY 493 / 503 fluorescence staining, LD can show green fluorescence. By comparing the fluorescence intensity, it was found that the fluorescence intensity of the FFA group was significantly higher than that of the BSA group. Through quantitative analysis, it was found that the lipid accumulation level in the FFA group was significantly higher than that in the BSA group.

[0099] 2) Effect of the Kangshujiangu Granule-containing serum on lipid accumulation in LO2 cells:

[0100] Effect of drug-containing serum on cell TG: Increased TG level is one of the important manifestations of non-alcoholic fatty liver disease. The results of TG level detection showed that compared with the cells in the BSA group, the TG level in the FFA group was significantly increased; the FFA+KSJG group and the FFA+FF group could reduce the TG level (P<0.05). Fig.17 This result shows that the serum containing Kangshujiangu granules can effectively improve the lipid accumulation in hyperlipidemia cells.

[0101] Effect of drug-containing serum on cell ALT and AST: ALT and AST are important indicators of acute liver cell injury and are highly sensitive to liver cell injury. The results showed that compared with the BSA group, the ALT and AST values ​​of the FFA group were significantly increased, while the FFA+KSJG group and the FFA+FF group could effectively reduce the ALT and AST levels of the FFA group (P<0.05). For details, please refer to Fig.18The above results show that the serum containing Kangshujiangu granules can effectively improve liver cell damage and has a certain protective effect on liver cells.

[0102] Effect of drug-containing serum on cellular lipid accumulation after AMPK inhibition: Oil red O staining and BODIPY 493 / 503 fluorescence staining results showed that compared with the BSA group, the number of LD in the FFA group increased, and the lipid accumulation level increased (P<0.05); compared with the FFA group, the lipid accumulation in the FFA+Compound C group increased further, while the cellular LD content in the FFA+KSJG group and the FFA+FF group decreased significantly (P<0.05); when Compound C was added to the FFA+KSJG group, the KSJG effect was inhibited, and the LD content increased (P<0.05), refer to Fig.19 The above results indicate that the inhibition of AMPK aggravates the accumulation of cellular lipids induced by FFA, and the serum containing Kangshujiangu granules can improve the level of cellular lipid accumulation induced by FFA induced by AMPK inhibition.

[0103] 3) Effect of drug-containing serum on autophagy proteins after AMPK inhibition: Western Blot was used to detect the levels of LC3, p62, Atg7 and Beclin1 proteins in six groups of cells: BSA, FFA, FFA+Compound C, FFA+KSJG, FFA+FF and FFA+KSJG+Compound C. The results showed that compared with the cells in the BSA group, the levels of LC3Ⅱ / LC3Ⅰ, Atg7 and Beclin1 in the FFA group were decreased, and the level of p62 was increased (P<0.05); the levels of LC3Ⅱ / LC3Ⅰ, Atg7 and Beclin1 in the FFA+Compound C group were further decreased, and the level of p62 was increased compared with the FFA group, while the levels of LC3Ⅱ / LC3Ⅰ, Atg7 and Beclin 1 in the FFA+KSJG group and the FFA+FF group were significantly increased, the level of p62 was decreased (P<0.05), and the level of autophagy was increased; in addition, the addition of Compound C to the FFA+KSJG group could reverse the effect of KSJG (P<0.05) and inhibit autophagy, but there was no significant difference in Beclin1, for details, refer to Fig. 20 shown.

[0104] In summary, the anti-osteoporosis and bone-strengthening granules can improve non-alcoholic fatty liver disease under the same mechanism as osteoporosis by improving autophagy, indicating that the Chinese medicine composition has the effect of treating osteoporosis and non-alcoholic fatty liver disease with the same treatment.

[0105] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of Kangshujiangu Granules in the preparation of drugs for the treatment of osteoporosis and non-alcoholic fatty liver disease.

2. The use according to claim 1, Features: Kangshujian Bone Granules achieve the treatment of osteoporosis and non-alcoholic fatty liver disease by reducing the expression of p-Akt, p-mTOR and p-ULK1 Ser757, and increasing the expression of p-AMPK and p-ULK1 Ser555.

3. The use according to claim 1, Features: The Kangshu Jiangu granules include the following components in parts by weight: 15-25 parts of epimedium, 10-20 parts of eucommia bark, 10-20 parts of achyranthes bidentata, 10-15 parts of atractylodes macrocephala and 10-20 parts of salvia miltiorrhiza.

4. The use according to claim 3, Features: The Kangshu Jiangu Granules include the following components in parts by weight: 20 parts of Epimedium, 15 parts of Eucommia, 15 parts of Achyranthes, 12 parts of Atractylodes and 15 parts of Salvia miltiorrhiza.

5. The research method of application as claimed in claim 1, It is characterized in that The following steps are involved: Step 1: Detect biochemical indicators of patients with osteoporosis and non-alcoholic fatty liver disease to screen clinical biomarkers; Step 2: Establish osteoporosis and non-alcoholic fatty liver disease animal models, compare indicators, and screen out autophagy pathway proteins; Step 3: Use Kangshujiangu Granules to act on the screened clinical biomarkers and autophagy pathway proteins, and preliminarily evaluate the effects of Kangshujiangu Granules on autophagy proteins and related regulatory proteins in combating osteoporosis and non-alcoholic fatty liver disease; Step 4: In vitro experiments were conducted to verify the effects of Kangshujian Bone Granules on autophagy proteins and related regulatory proteins in combating osteoporosis and non-alcoholic fatty liver disease.

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