Application of baicalin in preparation of medicine for inhibiting and / or treating schistosoma japonicum katsurada and medicine

Abstract

The invention discloses an application of baicalin in preparation of a medicine for inhibiting and / or treating schistosomiasis japonicas. The invention further discloses a medicine for inhibiting and / or treating schistosomiasis. Experiments prove that after praziquantel kills schistosoma japonicum adults, baicalin is used for treatment, so that liver and spleen injuries caused by schistosoma japonicum infection are effectively relieved, the liver function of a host is protected, the influence of eggs in the host and schistosoma japonicum egg protein secreted by the eggs on the host is reduced, and the survival rate of schistosoma japonicum adults is increased. The treatment effect of the praziquantel is obviously better than that of the existing praziquantel

Description

Technical Field

[0001] The present application relates to the technical field of drug combination, and in particular to the application of baicalin in the preparation of drugs for inhibiting and / or treating schistosomiasis japonicum and the drugs. Background Art

[0002] Schistosomiasis is a disease caused by schistosomes parasitizing the human body. Humans are often infected through direct skin contact with infected water. The main organs affected by Schistosoma japonicum infection include the liver and intestines. Currently, the treatment options for Schistosomiasis japonicum mainly include drug treatment options based on praziquantel and surgical treatment options based on splenectomy.

[0003] Praziquantel is currently the only first-line drug for the treatment of schistosomiasis infection. It can effectively kill adult schistosomes and plays an important role in the treatment of schistosomiasis. However, after the adult worms are killed, the large number of eggs colonized in the organs will still endanger the life and health of patients in the long term. For example, after the eggs of Schistosoma japonicum colonize in the liver, they secrete antigens such as Schistosoma japonicum egg protein (SEA), which can trigger a series of immune responses and may eventually lead to Schistosomiasis liver fibrosis or even liver failure. Praziquantel has limited therapeutic effect on such symptoms. Summary of the invention

[0004] In order to solve the above technical problems, the first purpose of the present invention is to provide the use of baicalin in the preparation of drugs for inhibiting and / or treating Japanese schistosomiasis; the second purpose of the present invention is to provide a drug; the applicant has confirmed through experiments that the use of baicalin for treatment after praziquantel kills the adult worms of Japanese schistosoma effectively alleviates the liver and spleen damage caused by Japanese schistosoma, protects the liver function of the host, and reduces the impact of the host's eggs and the schistosome egg protein (SEA) secreted by the host on the host, which is significantly better than the existing effect of praziquantel alone.

[0005] The technical solution provided by the present invention is as follows: Application of baicalin in the preparation of medicines for inhibiting and / or treating schistosomiasis japonicum.

[0006] Preferably, the drug can alleviate liver and spleen damage caused by Schistosoma japonicum.

[0007] Preferably, the drug can reduce liver fibrosis, reduce liver weight / body weight ratio, reduce liver granuloma area, reduce inflammatory factor expression in the liver, improve splenomegaly, and reduce spleen weight / body weight ratio.

[0008] Preferably, the drug can reduce transaminase, reduce the expression levels of type I collagen and α-actin, and inhibit the M2 differentiation of macrophages and the activation of hepatic stellate cells.

[0009] Preferably, baicalin is used after the administration of praziquantel is completed.

[0010] A drug for inhibiting and / or treating schistosomiasis japonicum, comprising baicalin.

[0011] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0012] Preferably, the drug is administered orally.

[0013] The applicant has experimentally confirmed that the use of baicalin for treatment after praziquantel kills adult Schistosoma japonicum effectively alleviates liver and spleen damage caused by Schistosoma japonicum, protects the host's liver function, and reduces the impact of worm eggs in the host and the schistosome egg protein (SEA) secreted by the host on the host, which is significantly better than the effect of existing praziquantel alone. At the same time, the above ingredients are administered orally, which is convenient and cheap. The application also confirms that the use of baicalin at a therapeutic dose has no obvious liver and kidney toxicity, and has great practical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 A flow chart of the animal model preparation protocol for this application; Figure 2 The following are the gross photos of the mouse liver and spleen, the count of worm eggs in liver tissue, and the statistical graph of the corresponding isolated organ mass / body weight ratio in the embodiments of the present invention; Figure 3 It is a hematoxylin-eosin staining diagram of mouse liver tissue, a statistical diagram of granuloma area, a statistical diagram of serological test results, and a statistical diagram of qPCR test results of inflammation-related factors in mouse liver tissue in the embodiment of the present invention; Figure 4 The Masson staining diagram of mouse liver tissue, the collagen volume fraction (CVF, the ratio of the blue collagen area to the total tissue area in the calculated section, used to reflect the degree of fibrosis) statistical diagram and the statistical diagram of the qPCR detection results of mouse liver tissue fibrosis-related factors in the embodiments of the present invention; Figure 5 The immunohistochemical staining diagram and result statistics of mouse liver tissue in the embodiment of the present invention are used to detect fibrosis-related proteins; Figure 6The immunofluorescence staining diagram of mouse liver tissue and the statistical diagram of the results in the embodiment of the present invention are used to detect the relevant markers of macrophages and their typing; Figure 7 The statistical graph of the qPCR detection results of mouse macrophages cultured in vitro and the gating scheme of flow cytometry detection in the embodiment of the present invention; Figure 8 The flow cytometry test results and statistical graphs of mouse macrophages cultured in vitro in the embodiment of the present invention are shown; Fig. 9 It is a schematic diagram of the indirect co-culture of mouse liver stellate cells and mouse macrophages in vitro in an embodiment of the present invention, and a statistical diagram of the qPCR detection and Western Blot detection results of mouse liver stellate cell specimens after indirect co-culture. DETAILED DESCRIPTION

[0016] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0017] Experimental protocol After one week of adaptive feeding, 6-8 week-old C57 black mice were infected with Schistosoma japonicum cercariae. On the 35th and 36th days after cercariae infection, praziquantel was given by gavage for two consecutive days (500 mg / kg / d), and then randomly divided into three groups, with 5 mice in each group: quality control group (SJ-Control), low-dose baicalin group (SJ-lowBA) and high-dose baicalin group (SJ-highBA). The low-dose group and the high-dose group were treated with the traditional Chinese medicine ingredient baicalin (Baicalin) by gavage every day from the 37th day, with 50 mg / kg / d of baicalin in the low-dose group and 100 mg / kg / d in the high-dose group; the quality control group was treated with carboxymethyl cellulose solution without drug ingredients (i.e., baicalin solvent, as a placebo) by gavage from the 37th day. The model construction scheme is as follows. Figure 1 shown.

[0018] Eight weeks after infection, all mice were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital solution and weighed. The abdomen was then opened and peripheral blood samples from mice in different groups were obtained using the inferior vena cava blood collection method. After collecting blood in sterile EP tubes containing sodium heparin, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (Cre) in the mouse blood were detected by enzyme-linked immunosorbent assay (ELISA) to reflect the differences in their liver and kidney functions.

[0019] After blood sampling, mice in each group were euthanized, and the whole liver and spleen were separated and weighed respectively. The left lobe of the liver was uniformly taken and fixed with tissue fixative for subsequent sectioning and pathological examination. The size of the granuloma in the liver was measured by hematoxylin-eosin staining; the collagen area was detected by Masson staining and type I collagen (Collagen I, represented by Col1a1 gene), α-actin (α-SMA, encoded by Acta2 The expression level of gene encoding) was used to measure the degree of fibrosis; the expression and distribution of F4 / 80, CD163 and iNOS were detected by immunofluorescence to measure the typing of intrahepatic macrophages in mice. The right lobe of the liver was uniformly taken, weighed and soaked in 5ml 4% potassium hydroxide (KOH) at 37℃ for 24h. After mixing, 20ul of homogenate was taken from each specimen three times for counting eggs under a microscope. A small amount of liver tissue was frozen at -80℃ for subsequent real-time fluorescence quantitative PCR (RT-qPCR) experiments. The relative expression of inflammatory-related factors and fibrosis-related factors at the transcriptional level was detected to analyze the degree of inflammatory response and fibrosis in the liver of each group of mice.

[0020] Mouse macrophages (RAW264.7 cell line was used in this project) were used for in vitro experiments. After macrophages were stimulated with SEA, two doses of baicalin were used for intervention. The SEA concentration was 20 ng / ml, which was used to simulate the colonization of Schistosoma japonicum eggs in vitro and to continuously release SEA to stimulate macrophages; the concentration of baicalin in the low-dose group was 25 μM, and the concentration of baicalin in the high-dose group was 50 μM. After 24 hours of intervention, the transcription levels of M2 macrophage markers such as CD206 and Arg1, as well as the results of flow cytometry, were detected to reflect the M2 polarization of macrophages. The supernatant in the macrophage culture system was collected for subsequent indirect co-culture experiments.

[0021] In vitro experiments were conducted using mouse hepatic stellate cells (JS-1 cell line was used in this project). TGF-β was used to stimulate mouse hepatic stellate cells to simulate the activated state of hepatic stellate cells (a state that can lead to liver tissue fibrosis), and then two different concentrations of baicalin were used to continue to treat mouse hepatic stellate cells (the concentrations of baicalin were 25uM and 50uM, respectively) to determine whether baicalin could directly affect hepatic stellate cells. The supernatant of the above macrophage culture system was used to indirectly co-culture mouse hepatic stellate cells, and the expression of baicalin in hepatic stellate cell specimens was detected. Acta2 , Col1a1 The transcriptional expression level and protein expression level of hepatic stellate cells reflect that hepatic stellate cells are affected by macrophage secretions.

[0022] Experimental Results First, the gross appearance of mouse liver and spleen After treatment with baicalin, the fibrosis area on the liver surface of mice infected with Schistosoma japonicum was significantly reduced (gray-white particles on the liver surface in the figure, Figure 2 A) The ratio of liver mass to body weight in vitro decreased significantly (indicating that the degree of fibrosis was reduced, Figure 2 E) and there was no significant change in the weight of mice among the groups ( Figure 2 D), indicating that baicalin at therapeutic doses did not affect the digestion or energy metabolism-related functions of mice, but as the dose of baicalin increased, the degree of liver fibrosis was reduced. At the same time, the size of the spleen of mice was significantly reduced after baicalin treatment ( Figure 2 B), and the ratio of spleen mass to body weight decreased significantly ( Figure 2 F), indicating that after baicalin treatment, the complications of hypersplenism caused by liver fibrosis were significantly improved. There was no significant difference in the number of worm eggs in the liver tissue of mice among the groups ( Figure 2 C), which indicates that baicalin treats liver fibrosis in mice induced by Schistosoma japonicum infection not by directly reducing the number of worm eggs.

[0023] Second, evaluation of the degree of inflammatory response in mouse liver tissue and detection of liver and kidney function Hematoxylin-eosin staining of mouse liver sections showed that the granuloma area was significantly reduced after baicalin treatment ( Figure 3 A, 3B); Serological tests found that transaminase in blood samples decreased significantly after baicalin treatment, while there was no significant difference in creatinine values ​​among the groups ( Figure 3 CE); qPCR detection found inflammatory markers in liver tissue specimens after baicalin treatment Il1b , Il10 , Tgb , Tnf The expression at the transcriptional level was significantly decreased ( Figure 3 FI). This suggests that baicalin treatment can effectively reduce the formation of liver tissue granulomas caused by Schistosoma japonicum infection, alleviate the intrahepatic inflammatory response, and protect liver function. At the same time, therapeutic doses of baicalin will not cause additional renal damage.

[0024] Third, assessment of liver fibrosis in mice Masson staining of mouse liver sections showed that the blue fibrosis area was significantly reduced after baicalin treatment ( Figure 4 A), collagen volume fraction (CVF) decreased significantly ( Figure 4 B); qPCR detection of mouse liver tissue specimens revealed that the gene encoding the fibrosis-related protein α-actin (α-SMA) was expressed at the transcriptional level Acta2 and type I collagen (Col1a1) encoding genes Col1a1 The expression was significantly decreased ( Figure 4C, 4D); Immunohistochemical detection of mouse liver sections showed that the expression levels of α-SMA and Col1a1 proteins also decreased significantly ( Figure 5 ). This suggests that baicalin treatment can effectively alleviate liver fibrosis in mice induced by Schistosoma japonicum infection.

[0025] Fourth, identification of macrophage types in mouse liver tissue It is well known in the art that macrophages have two differentiation directions, among which M2 differentiation results in the promotion of fibrosis. Immunofluorescence staining of mouse liver sections (F4 / 80 is a universal marker for mouse macrophages, CD163 is a characteristic marker for M2 macrophages, and iNOS is a characteristic marker for M1 macrophages) showed that the proportion of M2 macrophage infiltration was significantly reduced after baicalin treatment ( Figure 6 A, 6C), while the proportion of M1 macrophage infiltration did not change significantly ( Figure 6 B, 6D). This suggests that baicalin may play a key role in the inhibition of macrophage M2 polarization in the treatment of liver fibrosis caused by Schistosoma japonicum infection.

[0026] Fifth, in vitro experimental results of mouse macrophages SEA was used to stimulate mouse macrophages to simulate the environment of mouse liver macrophages in vitro, and baicalin was used for continued treatment. qPCR detection showed that after SEA stimulation, M2 macrophage-related markers Il10 , Tgb , Mrc1 (CD206 encoding gene), Arg1 The expression of these indicators was significantly increased at the transcriptional level, while baicalin treatment could reduce the expression of these indicators ( Figure 7 AD); at the same time, a macrophage flow cytometry gate scheme was constructed (the gate positions of CD206 and Arg1 were determined by the minus-one control, Figure 7 E), flow cytometry showed that CD206 + Macrophages (i.e., M2 macrophages) were significantly reduced, and Arg1 + The proportion of cells also decreased significantly (an important functional marker of M2 macrophages, Figure 8 ), which indicates that baicalin can directly inhibit the polarization and function of M2 macrophages.

[0027] 6. Results of in vitro experiments on mouse liver stellate cells It is well known in the art that hepatic stellate cells are usually the final target cells of various factors that cause liver fibrosis. Fig. 9A is a diagram of the indirect co-culture scheme of mouse macrophages and mouse liver stellate cells. qPCR detection shows that after SEA treatment of macrophages, the conditioned medium prepared by mixing the macrophage culture supernatant with fresh complete culture medium at a volume ratio of 1:1 can lead to the indirect co-culture of mouse macrophages and liver stellate cells. Acta2 , Col1a1 Compared with macrophages treated with SEA alone, the conditioned medium prepared by co-treatment with SEA and baicalin significantly reduced the expression of β-catenin in hepatic stellate cells. Acta2 , Col1a1 Expression at the transcriptional level ( Fig. 9 D, 9E). Western Blot analysis confirmed that compared with macrophages treated with SEA alone, the conditioned medium prepared by co-treatment with baicalin and SEA could lead to a decrease in the expression levels of α-SMA and Col1a1 proteins in hepatic stellate cells ( Fig. 9 FH). In addition, mouse TGF-β recombinant protein was used to directly induce mouse hepatic stellate cell activation, and then different concentrations of baicalin were used for treatment. Acta2 , Col1a1 There was no significant change in transcription level ( Fig. 9 B, 9C). This indicates that baicalin cannot directly affect mouse liver stellate cells, but it can effectively inhibit the activation of mouse liver stellate cells by affecting macrophage polarization and paracrine secretion, thereby achieving the purpose of alleviating liver fibrosis.

[0028] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of baicalin in the preparation of drugs for inhibiting and / or treating schistosomiasis japonicum.

2. The use according to claim 1, characterized in that: The drug can alleviate liver and spleen damage caused by Schistosoma japonicum.

3. The use according to claim 2, characterized in that: The drug can reduce liver fibrosis, reduce the liver weight / body weight ratio, reduce the liver granuloma area, reduce the expression of inflammatory factors in the liver, improve splenomegaly, and reduce the spleen weight / body weight ratio.

4. The use according to claim 2, characterized in that: The drug can reduce transaminase, reduce the expression levels of type I collagen and alpha-actin, and inhibit the M2 differentiation of macrophages and the activation of liver stellate cells.

5. The use according to any one of claims 1 to 4, characterized in that: Baicalin should be used after the administration of praziquantel is completed.

6. A drug for inhibiting and / or treating schistosomiasis japonicum, characterized in that: Including baicalin.

7. The drug according to claim 6, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The drug according to claim 6, characterized in that The drug is used orally.

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