Application of combination of bicyclol and tetrandrine in preparation of medicine for treating silicosis fibrosis

Abstract

The invention belongs to the technical field of medicines, and discloses application of combination of bicyclol and tetrandrine in preparation of a medicine for treating silicosis.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a new use of bicyclol in combination with tetrandrine as a drug for treating silicosis fibrosis. Background Art

[0002] Silicosis is a systemic disease caused by long-term occupational exposure to dust containing high levels of free silica (SiO2). It is characterized by inflammatory infiltration and diffuse fibrosis of the lung tissue, and is common among workers in industries such as mining, jeans production, and jewelry polishing. In the early stages of silicosis, inflammatory cells infiltrate and upregulate proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, leading to pulmonary inflammatory lesions. As the disease progresses, profibrotic markers such as TGF-β, α-SMA, and fibronectin are expressed in high concentrations, promoting fibroblast activation and pathological collagen deposition. Currently, the only FDA-approved drugs for the treatment of idiopathic pulmonary fibrosis are pirfenidone and nintedanib. However, the mechanisms of action of these two drugs in treating silicosis remain unclear, and long-term treatment cycles can lead to drug resistance, reducing therapeutic efficacy. Therefore, finding a cost-effective, safe, and effective treatment option for silicosis is crucial.

[0003] Tetrandrine (TET), a traditional Chinese medicine extract used clinically in my country, has a good anti-silicosis fibrosis effect, but the drug is slowly metabolized and easily accumulated in the body. The toxic side effects caused by long-term use inhibit the cardiovascular system and reduce liver and kidney function, which has great clinical application limitations. Bicyclol (BIC) is a class of innovative drugs with independent intellectual property rights in China. It has been widely used in the clinical treatment of chronic hepatitis. Studies have shown that bicyclol has a significant alleviating effect on fibrosis in multiple organs, and its mechanism of action involves anti-inflammatory, anti-oxidation, and inhibition of collagen deposition. In addition, clinical trials have shown that bicyclol tablets have the characteristics of protecting the liver and reducing enzymes, high safety, and convenient oral administration. Therefore, the inventors hope to combine bicyclol with tetrandrine to improve the treatment effect of silicosis. By utilizing the hepatoprotective and anti-fibrotic properties of bicyclol, the dosage and liver toxicity of tetrandrine can be reduced, thereby improving the safety of clinical drug use.

[0004] The present invention aims to target the fibrosis and inflammatory reactions that occur during the progression of silicosis. Currently, there are no reports on the combined use of bicyclol and tetrandrine in alleviating or treating diseases such as lung damage or pulmonary fibrosis caused by industrial dust. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of bicyclol combined with tetrandrine in the treatment of silicotic pneumonia and fibrosis and to provide an optimal combined dosage to solve the problems in the prior art.

[0006] There have been no studies reporting the combined use of bicyclol and tetrandrine for the treatment of silicotic fibrosis. The inventors have demonstrated through in vivo experiments that the combination of bicyclol and tetrandrine has a significant interventional effect on silica-induced pulmonary fibrosis in rats. They have also identified the optimal dosage of bicyclol and tetrandrine for this combination, suggesting that their combination could be used to prepare a therapeutic drug for pulmonary fibrosis.

[0007] The present invention provides the following technical solutions:

[0008] The first aspect of the technical solution of the present invention is to provide the use of a bicyclic alcohol represented by formula I in combination with tetrandrine represented by formula II in the preparation of a silicosis fibrosis drug;

[0009]

[0010] When the drugs are used in combination, the mass ratio of the bicyclol to tetrandrine is 3-15:1, preferably 6-15:1, and more preferably 13-14:1.

[0011] When treating silicosis rats, the dosage of the bicyclol suspension is 100-200 mg / kg, and the dosage of the tetrandrine is 15-30 mg / kg.

[0012] The optimal combined drug dose is bicyclol 200 mg / kg and tetrandrine 15 mg / kg.

[0013] The second aspect of the technical solution of the present invention is to provide a use of a pharmaceutical composition in the preparation of a silicosis fibrosis drug, characterized in that the pharmaceutical composition comprises a bicyclic alcohol represented by formula I in combination with tetrandrine represented by formula II and a pharmaceutically acceptable carrier or excipient;

[0014]

[0015] Specifically: The present invention is based on in vivo pharmacological experimental results that prove that bicyclol combined with tetrandrine can improve silica-induced inflammation and pulmonary fibrosis in rats, observe lung tissue pathological sections by HE and Masson staining, measure hydroxyproline representing collagen content in lung tissue, detect changes in indicators such as inflammatory cells representing inflammation levels, measure changes in lung function of rats after administration, and compare changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), and creatinine (CRE) in rats before and after administration.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] 1. There is no report on the application of the bicyclic alcohol combined with tetrandrine in the present invention in the treatment of silicosis, and its application can be expanded.

[0018] 2. The present invention reduces the therapeutic dosage of tetrandrine, improves the therapeutic effect, and reduces toxic and side effects.

[0019] 3. The raw materials of the present invention are easily available, suitable for mass use, and have good application prospects.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 , The body weight changes of rats in each group during the whole administration process.

[0022] Figure 2 After 8 weeks of administration, the body weight changes of rats in each group were expressed as mean ± standard error of the mean (SD). Compared with the model group, *** P < 0.001, ** P < 0.01, * P<0.05; compared with the tetrandrine group, ### P < 0.001, ## P < 0.01, # P<0.05; compared with the bicyclol group, Compared with the low-dose BT group ※※※ P < 0.001, ※※ P < 0.01, ※ P<0.05.

[0023] Figure 3 , Effects of silica-induced lung tissue inflammation in rat pulmonary fibrosis model (HE staining).

[0024] Figure 4 , HE staining Szapiel inflammation score, the values ​​of each group are expressed as mean ± standard error of the mean (SD), compared with the model group, *** P < 0.001, ** P < 0.01, * P<0.05; compared with the tetrandrine group, ### P < 0.001, ## P < 0.01, # P<0.05; compared with the bicyclol group, Compared with the low-dose BT group ※※※ P < 0.001, ※※ P < 0.01, ※ P<0.05.

[0025] Figure 5 , Effects of silica on lung tissue fibrosis in each group in the rat pulmonary fibrosis model (Masson staining).

[0026] Figure 6 , Masson staining collagen fiber deposition ratio, the values ​​of each group are expressed as mean ± standard error of the mean (SD), compared with the model group, *** P < 0.001, ** P < 0.01, * P<0.05; compared with the tetrandrine group, ### P < 0.001, ## P < 0.01, # P<0.05; compared with the bicyclol group, Compared with the low-dose BT group ※※※ P < 0.001, ※※ P < 0.01, ※ P<0.05.

[0027] Figure 7 , Masson staining Ascroft fibrosis score, the values ​​of each group are expressed as mean ± standard error of the mean (SD), compared with the model group, *** P < 0.001, ** P < 0.01, * P<0.05; compared with the tetrandrine group, ### P < 0.001, ## P < 0.01, # P<0.05; compared with the bicyclol group, Compared with the low-dose BT group ※※※ P < 0.001, ※※ P < 0.01, ※ P<0.05.

[0028] Figure 8 , Comparison of hydroxyproline content in lung tissue of rats in each group, the values ​​of each group are expressed as mean ± standard error of the mean (SD), compared with the model group, *** P < 0.001, ** P < 0.01, * P<0.05; compared with the tetrandrine group, ### P < 0.001, ## P < 0.01, # P<0.05; compared with the bicyclol group, Compared with the low-dose BT group ※※※ P < 0.001, ※※ P < 0.01, ※ P<0.05. DETAILED DESCRIPTION

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] The technical solution of the present invention will be further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0032] The present invention proposes a potential anti-pulmonary fibrosis drug use scheme for the treatment of silicosis fibrosis, which is further described in detail below in conjunction with specific embodiments of the present invention:

[0033] 1. Application of bicyclol combined with tetrandrine in the treatment of silicosis fibrosis

[0034] 1 Materials and Instruments

[0035] Male Wistar rats, weighing 170–200 g, were provided by Spectrum Biotechnology Co., Ltd. (Beijing). A hydroxyproline assay kit was purchased from Nanjing Jiancheng Bioengineering Institute. ALT, AST, UREA, and CRE assays were purchased from Biosino Biotechnology Co., Ltd. Silica particles were purchased from Sigma-Aldrich.

[0036] 2 Experimental methods

[0037] Wistar male rats were divided into control group, model group, tetrandrine group, bicyclol group, BT combined with low-dose group, BT combined with medium-dose group, and BT combined with high-dose group, with 9 rats in each group. After isoflurane inhalation anesthesia, the rats were fixed on the operating table and instilled with 1 mL of SiO2 suspended particles (100 mg / mL) using a non-exposed tracheal instillation method. The rats were then quickly stood upright and their breathing was observed. After waking up, they were fed normally. Four weeks after modeling, blood was collected from the canthal vein and medication was started at the same time. The control group and the model group were given 0.5% CMC-Na by gavage every day, and the tetrandrine group, bicyclol group, and each dose combination group were given the corresponding drugs by gavage for 8 consecutive weeks. Considering the greater hepato-renal toxicity of tetrandrine, the tetrandrine group and all combination groups in this study adopted a 6-day medication and 1-day rest medication method. The body weight of the rats was monitored every week during the medication period. Table 1 shows the modeling method and medication dosage of each group of rats.

[0038] Table 1. Rat modeling method and dosage

[0039]

[0040] After administration, the rats were deeply anesthetized with sodium pentobarbital, and their lung function was measured. Blood was then collected from the heart, the right side of the lung was ligated, and the left lung was lavaged with normal saline to obtain bronchoalveolar lavage fluid. The right lung tissue was then dissected and removed for staining and subsequent analysis. All data are expressed as mean ± standard error of the mean (SD) and analyzed using one-way ANOVA. P < 0.05 was considered statistically significant.

[0041] 3 Experimental results

[0042] 3.1 Effects of drugs on rat body weight and lung function

[0043] like Figure 1 As shown in Figure 2, one week after silica instillation, the weight gain of rats in the model group slowed down. Figure 2 As shown, after 8 weeks of administration (i.e., 12 weeks of modeling), the body weight of the medium-dose BT combination group was significantly higher than that of the model group, and the degree of weight increase was significantly better than that of the tetrandrine monotherapy group, indicating that at this time, the rats in the medium-dose BT combination group were in better physical condition and the degree of morbidity was most significantly reduced.

[0044] After 8 weeks of administration, the rats were deeply anesthetized and their lung function was tested using the FlexiVent forced respiratory oscillation method. Table 2 shows the respiratory parameters of the rats in each group. It is obvious that the expiratory and inspiratory abilities of the rats were improved after administration. The degree of improvement in lung function in the medium and high-dose BT combination groups was better than that in the single-dose and low-dose BT combination groups.

[0045] Table 2. Pulmonary function test parameters of rats

[0046]

[0047]

[0048] The values ​​of each group are expressed as mean ± standard error of the mean (SD). Compared with the model group, *** P < 0.001, ** P < 0.01, * P<0.05; compared with the tetrandrine group, ### P < 0.001, ## P < 0.01, # P<0.05; compared with the bicyclol group, Compared with the low-dose BT group ※※※ P < 0.001, ※※ P < 0.01, ※ P<0.05.

[0049] 3.2. Effects of drugs on rat lung tissue

[0050] HE staining results showed that the lung tissue structure of the control group was intact and clear, the alveolar septa were not thickened, the alveolar cavity was translucent, no obvious exudates were seen in the cavity, no inflammatory cell infiltration was seen in the alveolar cavity, and no fibroblast proliferation was seen. In the model group, the alveolar structure was destroyed, the alveolar septa were widened, a large number of inflammatory cells infiltrated and fibroblasts proliferated, a large amount of collagen was deposited, and pulmonary fibrosis was formed. The lung tissue structure of the drug-treated group was relatively intact and clear, the alveolar septa were slightly thickened, and the inflammatory cell infiltration was slightly reduced. The inflammatory infiltration of the lungs of rats in the medium and high dose BT combined groups was significantly reduced ( Figure 3 ), Szapiel inflammation score further confirmed that the BT combined with medium-dose B group had better anti-inflammatory properties than the single drug group and BT combined with low-dose group ( Figure 4 The results of Masson staining showed that a small amount of blue-dyed collagen fibers, which are the main components of the extracellular matrix, were observed in the lung tissues of the control rats. In the model group, a large amount of dense blue-dyed collagen fibers were observed, which were deposited in bundles or sheets. Compared with the model group, the collagen content in the drug-treated group was reduced ( Figure 5 ). Analysis of Masson staining collagen fiber deposition ratio and Ascroft fibrosis score showed that the fibrosis deposition in the medium and high dose BT combined group was significantly improved compared with the bicyclol and tetrandrine single use groups ( Figure 6 , Figure 7 )

[0051] 3.3 Effects of drugs on lung inflammation in model rats

[0052] Alveolar lavage fluid was collected from the rats and the levels of white blood cells, neutrophils, macrophages, and lymphocytes in the lavage fluid were measured. The results showed that the model group had severe pulmonary inflammatory cell infiltration, while the treatment group had some relief. Table 3 shows the levels of inflammatory cells in the alveolar lavage fluid of each group.

[0053] Table 3. Inflammatory cell counts

[0054]

[0055] The values ​​of each group are expressed as mean ± standard error of the mean. Compared with the model group, *** P < 0.001, ** P < 0.01, * P<0.05; compared with the bicyclol group, Compared with the tetrandrine group, there was no significant difference.

[0056] 3.4 Effects of drugs on fibrosis in model rats

[0057] The hydroxyproline content in the lungs of rats in each group was measured. Figure 8The results showed that the hydroxyproline content in each drug-treated group was significantly reduced, and the hydroxyproline content in the medium and high-dose BT combination groups was significantly lower than that in the single-dose group, further confirming the significant anti-silicotic pharmacological effect of the combination group. At the same time, the degree of hydroxyproline reduction in the medium-dose combination group was better than that in the low-dose group.

[0058] 3.5 Changes of ALT, AST, UREA, and CRE in each group before, during, and after administration

[0059] The results are shown in Tables 4 and 5. After administration of tetrandrine group and high-dose BT combined group, serum ALT, AST, UREA, and CRE levels were significantly increased compared with before administration, and were significantly higher than those in the model group, confirming the greater hepato-renal toxicity of tetrandrine. However, in the bicyclol group, ALT, AST, UREA, and CRE levels remained the same or even decreased after administration, which to a certain extent illustrates the hepatoprotective properties of bicyclol. At the same time, it can be clearly seen that after the combination of bicyclol, the medium-dose BT combined group has more obvious liver and kidney safety.

[0060] Table 4 Comparison of serum ALT, AST, UREA and CRE levels in each group before and after administration

[0061]

[0062] The values ​​of each group are expressed as mean ± standard error of the mean, and the differences in serum levels before and after administration were compared using t-test. *** P < 0.001, ** P < 0.01, * P<0.05.

[0063] Table 5 Comparison of serum ALT, AST, UREA, and CRE levels in each group after administration

[0064]

[0065] Compared with the model group, *** P < 0.001, ** P < 0.01, * P<0.05; compared with the tetrandrine group, ### P < 0.001, ## P < 0.01, # P<0.05.

[0066] Discussion

[0067] During silica-induced pulmonary fibrosis, rats in the model group showed pathological changes such as slow weight gain and reduced activity as alveolar function became more impaired, while the drug-treated group showed slight relief. Due to the diffuse fibrotic lesions in the lungs, the inspiratory and expiratory functions of silicotic rats were severely affected, eventually leading to respiratory failure. Therefore, improving lung function is also one of the important tasks in the treatment of silicosis. Compared with the normal control group, the lung function of rats in the model group was severely reduced, mainly manifested in reduced deep inspiration volume, impaired expiratory function, and decreased airway compliance. However, after drug treatment, lung function-related indicators improved, especially in the combined medium and high dose groups.

[0068] HE and Masson staining results showed that the drug treatment groups improved alveolar inflammatory infiltration and alveolar septal thickening, with the combined treatment group showing particularly significant effects. Furthermore, the drug treatment group also inhibited the deposition of fibrillar collagen during pulmonary fibrosis. To further quantify the collagen content in lung tissue, we also measured hydroxyproline content in rat lung tissue after administration. Hydroxyproline is an amino acid produced by the hydrolysis of connective tissue proteins, accounting for approximately 14% of collagen by weight. It plays a key role in collagen stability. Therefore, observing the level and changes in collagen content is an important indicator of the degree of pulmonary fibrosis. The results showed that the drug treatment significantly reduced tissue hydroxyproline content, with the reduction being more pronounced in the combined treatment group. Pathological findings in the combined treatment groups revealed significant improvements in lung tissue structure, with significantly reduced alveolar structural damage and alveolar septal thickening, decreased inflammatory cell infiltration, and reduced collagen fiber content in the medium- and high-dose BT treatment groups. This suggests that the combined treatment with medium- and high-dose BT has a certain inhibitory effect on the development and progression of pulmonary fibrosis, and is more effective in treating fibrosis than either drug alone. In addition, the number of inflammatory cells in the alveolar lavage fluid was detected after drug treatment. The results showed that the number of inflammatory cells in the medium and high-dose groups of BT combination decreased significantly, indicating that the degree of lung inflammation in rats was significantly reduced after administration. Combined with the above results, it proves that the combination group has excellent pharmacological effects against silicosis inflammation and fibrosis.

[0069] Studies have shown that tetrandrine has significant hepatotoxicity and renal toxicity. Therefore, it is very necessary to measure the changes in serum ALT, AST, UREA, and CRE in rats before and after administration and in each group to test the safety of the drug. The results showed that after 8 weeks of continuous administration, the tetrandrine group had greater hepatotoxicity and renal toxicity and low safety. At the same time, the high-dose BT combination group also showed significantly high ALT, AST, UREA, and CRE levels. However, the medium-dose BT combination group reduced the dosage of tetrandrine while ensuring good anti-inflammatory and anti-fibrosis therapeutic effects, reducing the accumulation of tetrandrine in the body and achieving the purpose of long-term continuous administration; at the same time, the medium-dose BT combination group exerted the excellent hepatoprotective effect of bicyclol, further reducing the toxic side effects caused by tetrandrine. The medium-dose BT combination group not only has the characteristics of low toxicity, but is even significantly better than the single-drug group to some extent, and has good clinical application prospects.

[0070] In summary, the medium and high-dose groups of BT combined with silica significantly intervened in the process of pulmonary fibrosis induced by silica in rats, reduced the degree of lung inflammation, and improved lung function. However, the high-dose group had higher liver and kidney toxicity. The medium dose not only played a good role in anti-silicosis, but also had higher safety and better therapeutic effect.

Claims

1. Use of a bicyclic alcohol of Formula I in combination with tetrandrine of Formula II in the preparation of a medicament for treating silicosis fibrosis; 2. The use according to claim 1, characterized in that When the drugs are used in combination, the mass ratio of the bicyclol to tetrandrine is 3-15:1, preferably 6-15:1, and more preferably 13-14:

1.

3. The use according to claim 2, characterized in that When treating silicosis rats, the dosage of the bicyclol suspension is 100-200 mg / kg, and the dosage of the tetrandrine is 15-30 mg / kg.

4. The use according to claim 3, characterized in that The optimal combined drug dose is bicyclol 200 mg / kg and tetrandrine 15 mg / kg.

5. Use of a pharmaceutical composition in the preparation of a silicosis fibrosis drug, characterized in that: The pharmaceutical composition comprises the bicyclic alcohol of formula I described in claim 1 in combination with tetrandrine of formula II and a pharmaceutically acceptable carrier or excipient;

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