CN116115619B

CN116115619B - Use of risperidone for treating organ fibrosis

Info

Publication number: CN116115619B
Application number: CN202310260008.0A
Authority: CN
Inventors: 李卫东; 李雪敏; 邱家丽; 杨珂; 付运; 李申
Original assignee: Tianjin Medical University
Current assignee: Tianjin Medical University
Priority date: 2023-03-17
Filing date: 2023-03-17
Publication date: 2024-03-01
Anticipated expiration: 2043-03-17
Also published as: CN116115619A

Abstract

The invention relates to application of risperidone in treating organ fibrosis, in particular to application in preparing a medicament for treating organ fibrosis; treatment of fibrotic organs is achieved by inhibition of the glycine transporter GLYT1 by risperidone, and medicaments comprising risperidone are particularly suitable for the treatment of pulmonary fibrosis. The collagen synthesis can be inhibited by inhibiting glycine transporter GLYT1 by risperidone, so that the pulmonary fibrosis caused by bleomycin can be obviously improved, and the formed pulmonary fibrosis can be obviously improved; risperidone acts on the glycine transporter GLYT1, and has little influence on other physiological functions; in addition, risperidone is a medicine which has been clinically used for many years, and the safety and side effects of risperidone are studied for many years, so that risperidone is more easily accepted by human bodies and has low side effects.

Description

Use of risperidone for treating organ fibrosis

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of risperidone in treating organ fibrosis.

Background

Organ fibrosis is a pathological change of fibrous connective tissue increase and parenchymal cytopenia in organ tissues, and is a common pathological feature of various chronic diseases. Fibrosis can affect any organ, and it is counted that up to 45% of deaths are due to fibrosis. Furthermore, common diseases associated with fibrosis include cirrhosis, hepatitis, nonalcoholic steatohepatitis, chronic kidney disease, myocardial infarction, heart failure, diabetes, idiopathic pulmonary fibrosis, scleroderma, and the like. Patients with fibrosis-related diseases account for about 1/20 of the number of important organ diseases. Thus, the medical burden of fibrosis is significant, and about 1/4 of the population worldwide is directly or indirectly affected by fibrosis of the viscera.

Fibrosis has now become an important economic burden for global healthcare. Therefore, the discovery of key therapeutic targets highly correlated with human fibrotic diseases, and the development of efficient anti-fibrotic therapies for these targets, is the focus of future research. However, despite the substantial progress currently made in understanding the pathology of fibrosis, there is no effective therapeutic approach.

One of the main means for treating organ fibrosis is to apply a large dose of glucocorticoid to reduce inflammatory response, but has serious side effects such as femoral head necrosis and the like, and has poor effect on chronic organ fibrosis caused by non-acute infection. Blocking the TGF- β pathway is an important target for the treatment of fibrotic diseases, but TGF- β signaling is widely distributed in the human body and is involved in important physiological roles; in addition, drugs acting on Wnt and Notch signaling pathways have the problem of poor selectivity. Anti-organ fibrosis drugs can also be ameliorated by modulating oxidative stress, lipid metabolism, MMP inhibitor enzymes, and the like. Pirfenidone (pirfenidone) and nintedanib (nintedanib) were approved by the FDA in the united states for the treatment of idiopathic pulmonary fibrosis. Nidamnification is a powerful inhibitor of tyrosine kinase receptors such as VEGF, FGF, PDGF, the action mechanism of pirfenidone is not well defined, and anti-fibrosis effects can be exerted by inhibiting inflammatory mediators such as TNF-alpha, IL-6, IL-12, IL-8 and the like.

At present, effective drug treatment means for organ fibrosis are not available except support treatment, anti-inflammatory treatment, stem cell treatment and the like.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of risperidone in treating organ fibrosis.

The technical scheme adopted by the invention is as follows: use of risperidone for treating organ fibrosis.

Application of risperidone in preparing medicine for treating organ fibrosis is provided.

Preferably, risperidone has a structure as shown in formula 1;

preferably, the glycine transporter GLYT1 is inhibited by risperidone.

Preferably, risperidone or a medicament comprising risperidone is administered orally.

Preferably, the amount administered is 2-6mg/d.

Preferably for pulmonary fibrosis.

The invention has the advantages and positive effects that: the collagen synthesis can be inhibited by inhibiting glycine transporter GLYT1 by risperidone, so that the pulmonary fibrosis caused by bleomycin can be obviously improved, and the formed pulmonary fibrosis can be obviously improved; risperidone is a drug which has been used clinically for many years, and its safety and side effects have been studied for many years; risperidone acts on the glycine transporter GLYT1 with less impact on other physiological functions.

Drawings

Figure 1 change in mouse body weight in example 2;

FIG. 2 changes in weight to weight ratio of mice in example 2;

FIG. 3 results of HE staining of paraffin sections of mouse lung tissue in example 2;

FIG. 4 results of Masson staining of paraffin sections of mouse lung tissue in example 2;

FIG. 5 Sirius Red staining of mouse lung tissue paraffin sections in example 2;

FIG. 6 results of immunohistochemical treatment of lung tissue sections of mice following 1mg/kg bleomycin intervention in example 2;

FIG. 7 results of immunohistochemical treatment of lung tissue sections of mice following 2mg/kg bleomycin intervention in example 2;

FIG. 8 results of immunohistochemical treatment of lung tissue sections of mice after 5mg/kg bleomycin intervention in example 2;

FIG. 9 variation of mouse body weight in example 3;

FIG. 10 changes in mouse lung weight in example 3;

FIG. 11 results of HE staining of paraffin sections of mouse lung tissue in example 3;

FIG. 12 results of Masson staining of paraffin sections of mouse lung tissue in example 3;

FIG. 13 Sirius Red staining of mouse lung tissue paraffin sections in example 3;

FIG. 14 results of immunohistochemical treatment of lung tissue sections of mice after 1mg/kg bleomycin intervention in example 3;

FIG. 15 results of immunohistochemical treatment of lung tissue sections of mice after 5mg/kg bleomycin intervention in example 3;

FIG. 16 effect of bleomycin and risperidone on cellular activity in example 4;

FIG. 17 Western blot results of pulmonary fibrosis related indicators following risperidone intervention at different times in example 4;

FIG. 18 shows the result of Western blot gray value analysis of the pulmonary fibrosis related index in example 4;

FIG. 19 effect of GlyT1 inhibitors on cell activity in example 4;

FIG. 20 shows the results of changes in the expression levels of the indices related to pulmonary fibrosis after dry prognosis of different drugs in example 4;

FIG. 21 shows Western blot gray scale results of lung fibrosis related indicators after dry prognosis of different drugs in example 4.

Detailed Description

In chronic obstructive pulmonary disease, the elasticity of the small airways gradually decreases with the deposition of type one collagen and type III collagen in the extracellular matrix, accelerating the progression of disease progression. In addition, with the proliferation of hepatic stellate cells, type I collagen and type III collagen gradually replace type IV collagen, and the structure of hepatic blood sinus capillaries gradually changes pathologically. The change in vascular structure further aggravates the progression of fibrosis while inducing portal hypertension and related diseases. After injury to various solid organs, we can see that the level of collagen deposition in the matrix has a strong correlation with the progression of organ fibrosis. Thus, collagen deposition has a predictive function for judging the degree of organ fibrosis and for predicting the level of fibrosis.

The invention discloses a method for treating organ fibrosis by risperidone, which reduces organ collagen deposition by inhibiting collagen synthesis, thereby reducing organ fibrosis formation. Risperidone (risperidone) is an atypical antipsychotic for the treatment of schizophrenia. In the invention, risperidone can inhibit glycine transporter GLYT1, and reduce the transfer of glycine serving as a raw material for collagen synthesis into cells, thereby reducing collagen synthesis and effectively reducing organ fibrosis formation. Risperidone has a structure shown in formula 1;

glycine transporters are responsible for uptake of glycine into cells where it can be used to synthesize proteins, neurotransmitters and other important molecules. Recent studies have shown that glycine transporter may also play a role in the development of fibrosis, a pathological condition characterized by excessive accumulation of scar tissue in the body. One mechanism by which GlyT1 inhibitors exert an anti-fibrotic effect is to increase the availability of extracellular glycine, which has been shown to have anti-inflammatory and anti-fibrotic effects. GlyT1 inhibitors may also exert their effect by inhibiting proliferation of myofibroblasts, which are cells produced by scar tissue in fibrosis. While the results of preclinical studies are promising, more studies are needed to determine if GlyT1 inhibitors are effective and safe for treating human fibrosis. Notably, glycine transporter inhibitors are currently an active area of research for the treatment of fibrosis in humans and have not been approved for clinical use.

The collagen synthesis can be inhibited by inhibiting glycine transporter GLYT1 by risperidone, so that the pulmonary fibrosis caused by bleomycin can be obviously improved, and the formed pulmonary fibrosis can be obviously improved; risperidone has been used clinically for many years to treat schizophrenia mainly by blocking antagonism of dopamine D2 and 5-HT2 receptors, it has a high affinity for D2, 5-HT2, epinephrine-a 1 and epinephrine-a 2 and histamine H1 receptors, has a medium-low affinity for 5-HT1A, 5-HT1C and 5-HT1D receptors, has no affinity for cholinergic muscarinic receptors, β1 and β2 receptors, risperidone has been used for many years to treat schizophrenia, its safety and side effects have been studied; less impact on other physiological functions.

The following description of the present invention is made with reference to the accompanying drawings, wherein the experimental methods without specific description of the operation steps are performed according to the corresponding commodity specifications, and the instruments, reagents and consumables used in the embodiments can be purchased from commercial companies without specific description.

Example 1: risperidone relieves bleomycin-induced pulmonary fibrosis in mice

1.1 laboratory animals

C57BL/6J mice, male, 8-10W, weight 24-25g, purchased from Beijing Fukang Biotechnology Co. The mice are fed in SPF environment, the room temperature is maintained at 20-25 ℃, the humidity is controlled at 50-60%, the mice are fed normally, the illumination is regulated to 12h day and night circulation mode, and the mice are fed in the corresponding environment for 1 week before modeling. All animal experiments were compliant with the university of the Tianjin medical science animal care guidelines and guidelines, while approved by the university of the Tianjin medical science animal committee.

1.2 Experimental materials

Bleomycin (BLM, manufacturer: GLPBIO, USA), risperidone (RIS, manufacturer: zhejiang Hua Hai pharmaceutical Co., ltd., china), normal saline, avastin (tribromoethanol, manufacturer: shanghai Meilin Biochemical Co., ltd., china), 1ml syringe, 26 ¹ / ₂ G needle; the angle of the ophthalmic ointment, medical forceps, heat lamp, surgical plate is about 70 ° (from horizontal).

1.3 experimental method: bleomycin is instilled orally in mice as follows:

1. pre-diluted bleomycin was prepared and administered at 1mg/Kg, 2mg/Kg, and 5mg/Kg depending on body weight.

2. Mice were anesthetized with avermectin: weighing each mouse, and calculating the dosage of avermectin (the concentration is 1.25%, the dosage of the mouse is 0.2ml/10 g); inhibition of mouse movement by catching mouse cervical hairs, using a 1mL syringe and 26 ¹ / ₂ The G needle was intraperitoneally injected with a dose calculated from the body weight. The anesthesia time is 10-40 minutes.

3. Within 5 minutes of anesthetic injection, the mice settled down and stopped moving. The sedative effect is verified by the disappearance of the eversion and, if it is reached, the next step is continued.

4. Excessive dryness of the eyes of mice should be avoided during the experiment.

5. The required volume of bleomycin or sterile PBS is loaded into a sterile 200 μl pipette tip.

6. The mice were placed on the surgical plate and suspended on the upper incisors using a surgical threaded ring. It is ensured that there is sufficient illumination to display the vocal cords.

7. Gently stretching the tongue to one side and downwards towards the jawbone by using forceps with sterile belt pads so as to see vocal cords; the bleomycin loaded pipette tip is then lowered to the back of the mouth and the aspiration is followed by the delivery of fluid via the vocal cords. Waiting for the wheezing to be heard confirms that the fluid is being delivered intratracheally. Animals in the control group replaced bleomycin solution with equal amounts of sterile PBS.

8. The tongue is released and the upper incisors are carefully removed from the suspension wires. Mice were placed under a heat lamp or pad until anesthesia was restored, typically within one hour after injection of the anesthetic.

9. The forceps were cleaned with an alcohol pad before and after each use.

10. Mice were monitored daily until they were euthanized for analysis. Mice were perfused daily with either gastrorisperidone or water according to the group.

11. After the experiment reaches the proper node, the mice are euthanized, eyeballs of the mice are removed for blood collection, and then the mice are dissected for material collection.

Example 2:

different groups of mice were molded according to the method of example 1, the control group was molded with physiological saline, the experimental group was molded with bleomycin of different concentrations, and ddH was used for the next day of molding ₂ O or RIS intragastric treatment, continuous intragastric 21d; the specific experimental conditions are shown in table 1;

TABLE 1

Group of	Moulding	Stomach lavage
			Control group 1	Physiological saline	ddH ₂ O
Experiment group 1	1mg/kg bleomycin	ddH ₂ O
			Experiment group 2	1mg/kg bleomycin	RIS
Experiment group 3	2mg/kg bleomycin	ddH ₂ O
			Experiment group 4	2mg/kg bleomycin	RIS
Experiment group 5	5mg/kg bleomycin	ddH ₂ O
			Experiment group 6	5mg/kg bleomycin	RIS

The dose is converted by the ratio of the body surface area of a human body to the body surface area of a mouse, the administration dose is calculated according to the maintenance dose of 6mg/d of the human body, and the equivalent dose coefficient conversion algorithm in the pharmacological experiment methodology mainly compiled by the professor Xu Shuyun is referred to, wherein the dose of the mouse is=Xmg/kg×70k according to the body surface area conversiongx 0.0026/20g = 9.1X mg/kg, calculated as an adult 70kg body weight, the mice should be dosed at 0.79 mg/(kg X d). In this example, the RIS gastric lavage dose is 0.79mg/kg d; ddH ₂ O was lavaged in equal doses.

The body weight of the mice was measured prior to molding, monitored daily during the experiment, observed for changes in body weight, and compared to the initial experiment at the end point of the experiment. As a result, as shown in fig. 1 and fig. 2, it was found that the BLM group showed a significant dose-dependent decrease in body weight (p < 0.0001) compared to the NC group, even below its own initial body weight, and the lung weight ratio was significantly increased (p < 0.05), and the increase also showed a dose-dependent increase; and blm+ris group showed significantly increased body weight and significantly decreased change in lung weight ratio compared to BLM group (< 0.01, < 0.001).

To further carefully observe the damage of the lung tissue of the mice, the lung tissue of the mice is pathologically detected in the embodiment, and after HE staining, the mice are observed under a microscope. As a result, as shown in FIG. 3, the NC group tissue structure was found to be approximately normal, and the alveolar structure was clear. After BLM stimulation, the lung tissue structure of the mice is obviously disordered, the alveolar structure disappears, the alveolar space is thickened, the blood vessel congestion is obvious, diffuse exudation and infiltration of inflammatory cells, actual change of lung tissue and obvious fibrosis are visible. RIS intervention was initiated the next day after modeling of BLM, with a significant reduction in the extent to which BLM induced the appearance of lung tissue.

Carrying out Masson staining detection on lung tissues of mice; the Masson staining method is a test method for detecting the secretion and deposition level of collagen fibers in tissues. After treatment with different dye solutions, the whole background of the tissue slice is red, the cell nucleus is black, and the collagen fiber is blue. The content of collagen fibers can be estimated according to the shade of blue and the range of involvement. As shown in FIG. 4, compared with the NC group, a large amount of collagen deposition can be seen in the lung tissue of the BLM group mice, and the deposition is quite obvious in the 1mg/kg group, the 2mg/kg group and the 5mg/kg group, the diffuse distribution appears obvious pulmonary solid changes, and the alveolar structure disappears; collagen deposition in the blm+ris group was not apparent, and there was a large difference from that in the BLM group, and alveolar structure was well preserved without obvious changes in pulmonary transformation.

Staining and detecting the lung tissue Sirius Red of the mice; sirius Red staining is also a test method for detecting the level of collagen fiber secretion and deposition in tissues. The alkaline group in the collagen fiber is combined with the strong acid sirius red dye solution, the nucleus is blue under a common optical microscope, the collagen is red, and the birefringence phenomenon occurs under a polarizer, so that the type I collagen fiber and the type III collagen fiber can be distinguished. As shown in fig. 5, no obvious collagen deposition was found in the lung tissue sections of NC mice, the alveolar structure was relatively clear, and slight collagen deposition was visible around bronchi; obvious collagen deposition and diffuse distribution are visible in lung tissue sections of mice after BLM stimulation; after administration of RIS, collagen deposition is significantly reduced and alveolar structure is also relatively normal and clear, but there may still be slight collagen deposition around the bronchi.

To further analyze the effect of RIS on collagen, immunohistochemical analysis was performed on mouse lung tissue sections, and the expression levels of type I collagen and type III collagen were analyzed, and the detection results are shown in FIGS. 6 to 8; the results show that compared with NC group, after BLM intervention, the deposition of type I collagen and type III collagen in the lung tissue of the mice is obvious, and the expression level is higher; after RIS treatment, the deposition of type I collagen and type III collagen is obviously reduced compared with that of BLM group, and the expression level is obviously reduced.

Example 3:

different groups of mice were molded according to the method of example 1, the control group was molded with physiological saline, the experimental group was molded with bleomycin of different concentrations, and after 14d molding, ddH was used, respectively ₂ O or RIS intragastric treatment, continuous intragastric administration 14d; the specific experimental conditions are shown in table 2;

TABLE 2

Group of	Moulding	Stomach lavage
			Control group 2	Physiological saline	ddH ₂ O
Experiment group 7	1mg/kg bleomycin	ddH ₂ O
			Experiment group 8	1mg/kg bleomycin	RIS
Experiment group 9	5mg/kg bleomycin	ddH ₂ O
			Experiment group 10	5mg/kg bleomycin	RIS

The dose is converted by the ratio of human body surface area to mouse body surface area, the administration dose is calculated according to the maintenance dose of 6mg/d of human, and the equivalent dose coefficient conversion algorithm in pharmacological experiment methodology compiled by the main teaching of Xu Shuyun is referred to, wherein the dose of the mouse is calculated according to the body surface area conversion, the dose of the mouse is calculated according to 70 kg/X70 kg X0.0026/20 g=9.1X mg/kg, and the administration dose of the mouse is calculated according to 70kg body weight of an adult, and the administration dose of the mouse is 0.79 mg/(kg X d). In this example, the RIS gastric lavage dose is 0.79mg/kg d; ddH ₂ O was lavaged in equal doses.

The body weight of the mice was measured prior to molding, monitored daily during the experiment, observed for changes in body weight, and compared to the initial experiment at the end point of the experiment. As a result, as shown in fig. 9 and 10, it was found that the BLM group showed a significant decrease in the body weight of the mice in the NC group (< 0.0001) in the dose-dependence, even below its own initial body weight, and the lung weight ratio was significantly increased (< 0.05) in the dose-dependence; and blm+ris group showed significantly increased body weight and significantly decreased change in lung weight ratio compared to BLM group (< 0.01, < 0.001). Such a change is effective whether RIS is applied throughout.

To further carefully observe the damage of the lung tissue of the mice, the lung tissue of the mice is pathologically detected in the embodiment, and after HE staining, the mice are observed under a microscope. As a result, as shown in FIG. 11, it was found that the NC group tissue structure was approximately normal and the alveolar structure was clear. After BLM stimulation, the lung tissue structure of the mice is obviously disordered, the alveolar structure disappears, the alveolar space is thickened, the blood vessel congestion is obvious, diffuse exudation and infiltration of inflammatory cells, actual change of lung tissue and obvious fibrosis are visible. RIS intervention was initiated 14 days after molding of BLM, and could also attenuate BLM-induced actual changes in lung tissue.

Carrying out Masson staining detection on lung tissues of mice; as shown in fig. 12, compared with NC group, a large amount of collagen deposition was seen in the lung tissue of BLM group mice, and significant lung transition occurred, and alveolar structure disappeared; collagen deposition in the blm+ris group was not apparent, and there was a large difference from that in the BLM group, and alveolar structure was well preserved without obvious changes in pulmonary transformation. In combination with the results of example 2, in a bleomycin-induced model of pulmonary fibrosis in mice, collagen in the lung tissue of mice is secreted in large amounts, and administration of RIS intervention significantly reduces bleomycin-induced collagen deposition in the lung tissue of mice.

Staining and detecting the lung tissue Sirius Red of the mice; as shown in fig. 13, no obvious collagen deposition was found in the lung tissue sections of NC mice, the alveolar structure was relatively clear, and slight collagen deposition was visible around bronchi; obvious collagen deposition and diffuse distribution are visible in lung tissue sections of mice after BLM stimulation; after administration of RIS, the collagen deposition is significantly reduced, the alveolar structure is also relatively normal and clear, but there is still a slight collagen deposition around the bronchi, this intervention being effected whether it is carried out the next day after moulding or after 14 days.

To further analyze the effect of RIS on collagen, immunohistochemical analysis was performed on mouse lung tissue sections, and the expression levels of type I collagen and type III collagen were analyzed, and the detection results are shown in FIGS. 14 to 15; the results show that compared with NC group, after BLM intervention, the deposition of type I collagen and type III collagen in the lung tissue of the mice is obvious, and the expression level is higher; in combination with the results of example 2, both the following day of RIS treatment and the 14 days of RIS treatment, the type I and type III collagen deposition were significantly reduced compared to the BLM group, and the expression level was significantly reduced.

Example 4: risperidone relieves bleomycin-induced A549 cell destruction

Experimental materials: a549 cells; bleomycin (BLM, manufacturer: medChemExpress Co., USA); risperidone (RIS, manufacturer: medChemExpress, usa); glyT1Inhibitor (GLYT 1IN, manufacturer: medChemExpress Co., USA); COL1A1 primary antibody (ebotec biotechnology limited, china); COL3A1 primary antibody (ebotec biotechnology limited, china); COL4A1 primary antibody (ebotec biotechnology limited, china); beta-actin primary antibody (abbotac biotechnology limited, china); e-cadherin primary antibody (Aibotai Biotechnology Co., ltd., china); alpha-SMA primary antibody (ebotec biotechnology limited, china).

4.1 cytotoxicity assay (CCK 8)

Cell proliferation and cytotoxicity were detected using the CCK-8 kit. The working principle is as follows: in the presence of an electron coupling reagent, WST-8 (chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt) can be reduced by intramitochondrial dehydrogenase to produce a highly water-soluble orange-yellow formazan product, the shade of the color of which is proportional to cell proliferation and inversely proportional to cytotoxicity, and the shade of the color and the number of cells are in linear relation to the same cells. The OD value measured at a wavelength of 450nm using an enzyme-labeled instrument can indirectly reflect the number of living cells.

The CCK8 experiment is utilized to detect the activity of the A549 cells after being treated by BLM and RIS with different concentrations for 72H, the BLM with the activity of about 80% of the A549 cells is selected for stimulation, and the A549 cells are stimulated and molded by the BLM with the activity of 25 mu M; RIS with about 88% A549 cell viability was selected for stimulation, and 20. Mu.M RIS was used as the drug treatment concentration in this example. The results are shown in figure 16, A, B, which shows that the cell viability of a549 cells decreases with increasing BLM, RIS concentration, and that a 10 μm bleomycin stimulation resulted in statistical differences, and a 15 RIS μm stimulation resulted in statistical differences. 4.2 in vitro construction of bleomycin-induced pulmonary fibrosis model

To explore the effect of RIS on alveolar epithelial cells under the destructive action of bleomycin, the present example constructed a bleomycin-induced pulmonary fibrosis model in vitro, divided into four groups: NC group (control group: treatment with physiological saline), BLM group (bleomycin group: treatment with BLM of 25. Mu.M for 72 hours), BLM+RIS 20. Mu.M for 48 hours after group (bleomycin and risperidone group: treatment with BLM of 25. Mu.M for 72 hours, RIS of 20. Mu.M for 24 hours) and BLM+RIS 20. Mu.M together group (bleomycin and risperidone group: treatment with BLM of 25. Mu.M for 72 hours, RIS of 20. Mu.M for 72 hours). And detecting the expression level of the pulmonary fibrosis related indexes col1α1, col3α1 and alpha-SMA protein by adopting a Western blot method. As shown in fig. 17-18, the expression levels of col1a1, col3a1, a-SMA proteins in a549 cells were significantly increased after bleomycin stimulation compared to the control, whereas the increases were significantly down-regulated by RIS treatment for 24h and 72 h.

From the above results of the cell and animal model studies, risperidone (RIS) can significantly improve organ fibrosis. From the in vivo pulmonary fibrosis model, the risperidone treatment can obviously improve collagen deposition after pulmonary fibrosis, the collagen content is obviously reduced, the pulmonary tissue structure is slightly changed, the actual changes are less, the pulmonary alveolus structure is more complete, in addition, the weight of a mouse severely reduced due to fibrosis is increased, and the weight ratio of the increased pulmonary weight is reduced; from the in vitro fibrosis model, risperidone treatment can obviously change the expression of the pulmonary fibrosis related index at different times, and can obviously weaken the expression increase caused by bleomycin stimulation.

Example 5: risperidone inhibits GLYT1 mechanism research of glycine transporter

To further verify whether this down-regulation was mediated by the risperidone inhibitory glycine transporter GLYT1, this example examined the activity of A549 cells after 72h treatment with different concentrations of GlyT1Inhibitor (GLYT 1 IN) using the CCK8 kit, and the results showed that the activity of A549 cells gradually decreased with increasing GLYT1IN concentration, as shown IN FIG. 19. Under 380nM conditions, the activity of A549 cells was greater than 50%, and 190nM GLYT1IN was selected as the treatment concentration IN the study for easier stable experimental results.

To further verify whether this down-regulation is mediated by risperidone inhibiting glycine transporter GLYT1, A549 cells were divided into four treatment groups: NC group (control group: treatment with physiological saline), BLM group (bleomycin group: treatment with BLM of 25. Mu.M for 72 hours), BLM+RIS group (bleomycin and risperidone group: treatment with BLM of 25. Mu.M for 72 hours, RIS of 20. Mu.M for 72 hours) and BLM+GLYT1IN group (bleomycin and risperidone group: treatment with BLM of 25. Mu.M for 72 hours, GLYT1IN of 190nM for 72 hours).

And detecting the expression levels of pulmonary fibrosis related indexes col1α1, col3α1, col4α1, E-cadherein (E-Cad) and alpha-SMA protein by using a Western blot method. As shown IN fig. 20-21, after BLM stimulation, the expression levels of col1a1, col3a1, col4a1, a-SMA protein were significantly increased, the expression level of E-cadherein protein was significantly decreased, and the expression level of E-cadherein protein was significantly increased IN the case of the combination of RIS and GLYT1IN intervention as compared to the BLM group. It is speculated that RIS might improve the change in fibrin caused by BLM by inhibiting GLYT1.

In summary, RIS may improve lung fibrosis caused by BLM by inhibiting GLYT1, and in vivo model experiments, lung fibrosis is improved regardless of duration of risperidone action; in vitro experiments, whether the application of RIS for a short period or the prior application of RIS intervention, can significantly affect the expression level of a pulmonary fibrosis related indicator.

The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.

Claims

1. Application of risperidone in preparing medicine for treating pulmonary fibrosis is provided.

2. The use according to claim 1, characterized in that: risperidone has a structure shown in formula 1;

formula 1.

3. The use according to claim 1, characterized in that: glycine transporter GLYT1 is inhibited by risperidone.

4. The use according to claim 1, characterized in that: risperidone or a medicament comprising risperidone is administered orally.

5. The use according to claim 4, characterized in that: the dosage is 2-6mg/d.