Traditional Chinese medicine composition for enhancing TRIM family protein as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition for enhancing TRIM family protein as well as a preparation method and application thereof, and relates to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition comprises the following components in parts by weight: 5-15 parts of ginseng, 5-15 parts of bighead atractylodes rhizome, 5-15 parts of poria cocos, 5-15 parts of liquorice, 5-15 parts of honeysuckle and 5-15 parts of astragalus membranaceus. The pharmaceutical composition disclosed by the invention is a pure traditional Chinese medicine preparation and has the effects of regulating the immune function, inducing endogenous interferon and enhancing the molecular weight of TRIM family protein, the result that the HBV-DNA is clinically converted into negative and is converted into negative is the result which cannot be displayed by clinical tests along with the increase of TRIM22 molecules, the immune system of a human body is recovered, and no antiviral drug is taken any more.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and more particularly to a traditional Chinese medicine composition for enhancing TRIM family proteins, and a preparation method and application thereof. Background Art

[0002] The tripartite motif (TRIM) family consists of many members and exists in almost all multicellular animals. It can act as an immunomodulatory protein and E3 ubiquitin ligase and participate in the body's innate immune response. The notable feature of the TRIM protein family is that the arrangement order between different domains is highly conserved.

[0003] Most TRIM proteins have a RING zinc finger structure, one or two B-box structures and a coiled coil domain (CC) from the N-terminus to the C-terminus, also known as the RBCC domain. There are usually one or two highly variable C-terminal domains after the RBCC domain, which determine the specificity of each TRIM protein. Since the first RBCC domain was discovered in the African clawed frog nuclear factor (XNF7) in 1991, there are now more than 70 members of the human TRIM protein family, and many homologous analogs have been identified in other eukaryotic organisms.

[0004] The genes encoding TRIM proteins are dispersed in the human genome, with only a few genes clustered on chromosomes 6 and 11. The proteins encoded by different TRIM genes differ in tissue distribution, with approximately 50% of TRIM genes expressed in mature tissues.

[0005] Recent studies have found that the TRIM family can participate in a variety of biological processes. It can not only maintain the normal physiological functions of the body, such as cell growth, cell differentiation, cytoskeleton reconstruction, membrane repair, signaling pathways and cell apoptosis, but also participate in the regulation of the pathogenesis of various diseases.

[0006] TRIM proteins play an important role in host resistance to viral infection. The same TRIM protein can be involved in the infection of different viruses, and the same viral infection can also be regulated by multiple TRIM proteins. TRIM proteins can directly enhance the host's antiviral function, and can also target host pattern recognition receptors to regulate the host's innate immune response, induce the production of inflammatory cytokines, chemokines and type I interferons, and indirectly enhance the antiviral function.

[0007] Take the treatment of hepatitis B virus infection as an example: Currently, these prescription drugs used to treat hepatitis B will cause the following problems after patients take them for a long time.

[0008] There are significant individual differences in the clinical efficacy of PEG IFNα treatment for patients with chronic hepatitis B. The main disadvantages of interferon used to treat hepatitis B are: relatively poor efficacy; it needs to be injected and the drug needs to be frozen; there are many adverse reactions in the early stage of medication, such as fever, leukocytopenia, etc.; it is not suitable for patients with decompensated liver disease such as cirrhosis and chronic hepatitis B patients with jaundice.

[0009] The main disadvantages of nucleoside drugs are: the course of treatment is not fixed. Patients with hepatitis B virus "big three positive" need to be treated for 6 months to 1 year after they turn into "small three positive". Some patients need 3 to 5 years or even longer. They are prone to rebound after stopping the drug. If the drug is not stopped in accordance with the standard, the virus may re-replicate. There is a possibility of viral resistance mutation during long-term use of the drug, and drug resistance is more likely to occur when the drug is not used in a standardized manner. Tenofovir used to treat HBV: Long-term use of tenofovir is prone to abnormal calcium and phosphorus metabolism in patients, and calcium loss will induce osteoporosis, and there will also be nephrotoxic reactions. Long-term use of the drug has side effects such as renal damage and low-phosphate bone disease.

[0010] Hepatitis B virus (HBV) infection is a chronic viral infection with a very high infection rate. It is still an important public health problem in my country and even in the world. It can cause chronic hepatitis, cirrhosis and even liver cancer. There is currently no cure for HBV infection. HBV is a small enveloped virus with only a 3.2kb genome and a sophisticated genome structure. Important proteins in the HBV life cycle include core protein (HBcAg), E antigen (HBeAg), surface antigen (HBsAg) and X protein HBx. The expression of these proteins is regulated by HBV-related promoters.

[0011] The TRIM family has a highly conserved RBCC domain, and the sequence from N-terminus to C-terminus is a RING zinc finger structure, one or two B-box domains and a coiled-coil domain, followed by a highly variable C-terminal domain. The TRIM protein family not only maintains the normal physiological functions of the body, but also participates in the regulation of the pathogenesis of various diseases.

[0012] In recent years, the roles of various TRIM proteins in antiviral innate immunity have been discovered, such as TRIM5α, TRIM11, TRIM19, TRIM22, TRIM25, etc. Many TRIM proteins are closely related to the interferon response of cells, indicating that TRIM family proteins are a new type of widely existing antiviral innate immune molecules.

[0013] The mechanism of chronic infection of hepatitis B virus (HBV) and how to effectively resist viral replication are the focus and frontier of current immunology research. The TRIM family of innate immune molecules in innate immunity plays an important antiviral role in antiviral immunity. TRIM22 inhibits the activity of HBV core promoter, and TRIM6 interacts with HBV core promoter. By constructing a fluorescent reporter gene of HBV promoter, we found that TRIM22 can inhibit the activity of HBV core promoter and S promoter, thereby inhibiting the transcription level of HBV.

[0014] TRIM6, TRIM14, and TRIM22 have particularly significant ability to inhibit HBV. Further experiments found that TRIM6, TRIM14, and TRIM22 showed strong inhibitory effects on HBsAg, HBeAg, HBcAg in HBV, as well as the transcription level and DNA level of HBV in HepG2 and the hepatitis B cell infection model HepG2-NTCP.

[0015] It can also enhance HBV replication in cells in which TRIM6, TRIM14, and TRIM22 have been knocked out.

[0016] Therefore, how to improve the secretion of the natural immune response molecule "tripartite motif-containing protein 22, 6, 14. TRIM22, TRIM6, TRIM14" protein is a problem that technicians in this field urgently need to solve. Summary of the invention

[0017] In view of this, the present invention provides a Chinese medicine composition for enhancing TRIM family proteins, as well as a preparation method and application. The purpose of the present invention is that Chinese medicine can enhance the natural immune molecular weight of TRIM family proteins. TRIM family proteins belong to natural immune proteins that can induce endogenous interferon: TRIM family proteins are interferon-stimulated genes (ISGs); interferon-activated genes; interferon-induced genes; interferon-responsive genes. They play an important role in viral infections, tumors, and neurodegenerative diseases.

[0018] In order to achieve the above object, the present invention adopts the following technical solution:

[0019] A traditional Chinese medicine composition for enhancing TRIM family proteins, comprising the following components in parts by weight:

[0020] 5-15 parts of ginseng, 5-15 parts of atractylodes, 5-15 parts of poria, 5-15 parts of licorice, 5-15 parts of honeysuckle, and 5-15 parts of astragalus.

[0021] A method for preparing a traditional Chinese medicine composition for enhancing TRIM family proteins comprises the following steps:

[0022] The raw Chinese medicinal materials are weighed according to the amount; the raw Chinese medicinal materials are dried and processed into micron Chinese medicinal powder by a deep-cooled vibration ultrafine grinding unit at a low temperature of minus 80°C.

[0023] Furthermore, the micron Chinese medicine powder has a particle diameter of 0.1 to 75 mm, an average particle diameter generally less than 10 mm, and is mainly distributed in the range of 1 to 20 mm. Under a microscope, 98% of the micron Chinese medicine particles are cell fragments, and the organizational structure of the medicine cannot be seen, so it is also called cell-level or subcellular-level micronized Chinese medicine.

[0024] Furthermore, the micron Chinese medicine powder is added with lactose excipients, granulated by spray granulation and other methods, and made into micron Chinese medicine granules through steps of granulation and total mixing. The micron Chinese medicine granules can improve bioavailability, maintain the material basis of pharmacodynamics, and improve the efficacy.

[0025] The technical scheme of the present invention is to add or subtract the Sijunzi Decoction, which is known as the "first prescription for replenishing qi and blood in heaven and earth" in traditional Chinese medicine. The prescription is derived from the "Hejijufang" and is a classic prescription for nourishing blood and softening liver, strengthening spleen and stomach, clearing brain and calming, preventing cancer and lowering blood sugar, improving immunity, relieving fatigue, and restoring vitality. The prescription with ginseng, white atractylodes, poria and licorice as main ingredients is a tonic, which has the effects of replenishing qi and strengthening spleen.

[0026] Ginseng: It can promote the activity of the natural killer cell (NKC)-interferon-interleukin-2 (IL-2) regulatory network. Ginseng flower saponins can promote the activity of mouse spleen natural killer cells in vitro, and can synergize with mitogens to induce interferon and interleukin-2, with a significant dose-effect relationship.

[0027] Astragalus: Adding Astragalus decoction to cultured mouse spleen cells can induce interferon-γ. However, some studies have shown that Astragalus itself does not directly stimulate mice to produce interferon, but promotes the ability of viruses to induce interferon. Similar reports have also been reported in clinical studies. In addition, Astragalus can also increase the sensitivity of cells to interferon.

[0028] Licorice: It was observed in vitro that a certain concentration of licorice polysaccharides can promote the production of gamma-interferon by spleen cells, and its titer is 3.8 times higher than that of concanavalin A alone; licorice polysaccharides can also promote the production of gamma-interferon induced by phytohemagglutinin in human whole blood, and its titer is 6.5 to 13 times higher than that induced by phytohemagglutinin alone; it can induce gamma-interferon in human mononuclear cells, and its titer is 4.9 times higher than that induced by phytohemagglutinin, and can promote the induction of alpha-interferon in human whole blood by Newcastle disease virus (NDV). Its titer is 12.4 times higher than that produced by NDV alone.

[0029] Honeysuckle: Enhances the activity of natural killer cells. Both the aerosol and injection of Shuanghuanglian (honeysuckle, scutellaria, and forsythia) can enhance the activity of NK cells in human peripheral blood and the ability of lymphocytes to produce α-interferon. The latest biological research confirms that the effective antiviral component of honeysuckle is 2911 plant element (MIR2911). Honeysuckle contains a broad-spectrum antiviral plant element component: MIR2911, including anti-influenza A, Ebola virus, and new coronavirus.

[0030] The present invention can effectively enhance the content of TRIM22, TRIM6 and TRIM14 family protein molecules to achieve the clinical cure standard of HBV.

[0031] TRIM22, TRIM6, and TRIM14 proteins are involved in all stages of HBV virus replication and have antiviral functions. The discovery of the antiretroviral function of TRIM22, TRIM6, TRIM14 molecules and other TRIM proteins provides new ideas for the prevention of hepatitis B (HBV) virus infection and the development of clinical therapeutic drugs.

[0032] TRIM22 is a recently discovered member of the TRIM family. Preliminary experiments have found that TRIM22 expression is downregulated in the liver tissue of patients with chronic hepatitis B, but upregulated during the acute infection period. The TRIM22 molecule can inhibit HBV replication.

[0033] TRIM22 was highly expressed and negatively correlated with HBV DNA and HBeAg; TRIM22 levels were positively correlated with the enrichment of NOD2 / NF-κB signaling pathway genes, and high expression of TRIM22 increased the levels of inflammatory cytokines IL-1β and IL-8. This suggests that TRIM22, as one of the interferon-stimulated genes, is an important factor affecting the therapeutic efficacy of exogenous PEG IFNα, and is related to immune activation and enhancing the antiviral response induced by PEG IFNα.

[0034] TRIM22 of the three-motif protein (TRIM) family and its negative regulator miR-548c-3p may be important factors affecting the efficacy of PEG IFNα. Studies have shown that TRIM22 is highly expressed in patients who respond to PEG IFNα treatment and is negatively correlated with HBV DNA and HBeAg levels. TRIM22 overexpression significantly increases the levels of inflammatory cytokines IL-1β and IL-8 in the NOD2 / NF-κB pathway and enhances the antiviral activity of PEG IFNα, while miR-548c-3p is a key negative regulator of TRIM22, which reduces the efficacy of PEG IFNα in treating HBeAg-positive chronic hepatitis B patients by inhibiting TRIM22 expression.

[0035] TRIM22, TRIM6, and TRIM14 protein family members interfere with various stages of HBV virus replication, and TRIM22 protein expression is induced by interferon, all of which indicate that this family of proteins may be important regulators of antiviral innate immune responses and are involved in a series of biological processes of innate immunity. For retroviruses, multiple TRIM proteins including TRIM22, TRIM6, and TRIM14 act at various stages of viral replication when expressed in vitro and have antiviral activity. TRIM22 inhibits HBV core promoter activity, TRIM6 interacts with HBV core promoter, and TRIM14 can inhibit the activity of HBV core promoter and S promoter.

[0036] Before the virus is assembled into mature virus particles, TRIM22, TRIM6, and TRIM14 may also interact with the HIV-1 Gag protein through their N-terminal RING domain to reduce the production of mature virus particles, thereby reducing the number of viruses and the ability to infect when HBV is reinfected.

[0037] The expression level of TRIM22 gene in peripheral blood monocytes of HBV-positive and HBV-negative subjects was studied by quantitative PCR. The results showed that compared with HBV-positive subjects, the expression of TRIM family protein genes in HBV-negative subjects was quite high.

[0038] TRIM22 (Staf50) inhibits HBV viral replication by inhibiting the transcription of the long terminal repeat of viral HBV-DNA. It interferes with the assembly and release of HBV virions, thereby inhibiting the aggregation of HBV virions on the surface of HOS, UCOVID-192OS143B and HeLa cell lines. After type I IFN induction, TRIM22 can also inhibit the budding of HBV virus by interfering with the transport of HIV-1 virus Gag protein to the cell membrane. The results also show that TRIM22 is a key regulator in the HBV restriction response induced by type I IFN.

[0039] TRIM22 also exhibits inhibitory activity against HBV virus, inhibiting the replication of the virus by interfering with the core promoter of hepatitis B virus (HBV).

[0040] The correlation between TRIM22 and patients' serum routine virological or serological indicators such as HBV-DNA, HBsAg, HBeAg, ALT, and AST levels showed that TRIM22 was negatively correlated with serum HBV DNA and HBeAg levels, suggesting that increased TRIM22 expression may be related to the better efficacy of PEG IFNα.

[0041] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0042] The present invention belongs to a method of natural immune molecule treatment. The selected Chinese medicine complex has the function of bidirectional immune regulation and has the function of protein biological production inhibitor. When the TRIM22 molecule increases, the order of hepatitis B virus test turning negative is: HBV-DNA, HBeAg, anti-HBc, HBsAg, and cccDNA turning negative.

[0043] In the peripheral blood of HBeAg-positive CHB patients, extremely low miR-548c-3p may indicate high expression of TRIM22 and may result in better response to PEG IFNα.

[0044] High expression of TRIM22 can inhibit HBV replication and gene expression, and enhance the antiviral effect of IFNα by upregulating the expression of antiviral proteins. Higher TRIM22 levels during treatment are an effective biomarker for predicting PEG IFNα treatment response.

[0045] The pharmaceutical composition of the present invention is a pure Chinese medicine preparation, which has the function of regulating immunity, inducing endogenous interferon, and enhancing the molecular weight of TRIM family proteins. As the TRIM22 molecule increases, the hepatitis B virus HBV-DNA clinically converts to negative, and the conversion to negative is a result that cannot be shown in clinical tests. The human immune system is restored and no antiviral drugs are needed.

[0046] Recent studies have shown that in addition to the key role played by T cell responses in fighting HBV infection, innate immune molecules in innate immunity, such as the TRIM family, play an important antiviral role in antiviral immunity. TRIM22 is a recently discovered member of the TRIM family. Preliminary studies have found that TRIM22 expression is downregulated in the liver tissue of chronic hepatitis B patients, while it is upregulated during acute infection. TRIM22 molecules can inhibit HBV replication.

[0047] In the acute HBV infection mouse model and the HBV transgenic chronic infection mouse model, the inhibitory function of TRIM22 molecules on HBV-DNA was first detected in HBV-DNA. TRIM22 can significantly inhibit the activity of the hepatitis B virus core promoter, and HBV-DNA turns negative, thereby achieving the purpose of inhibiting viral replication.

[0048] Tripartite motif (TRIM) proteins are a family of E3 ubiquitin ligases that play key roles in a variety of cellular processes, such as innate immunity, cell differentiation, and apoptosis. TRIM proteins have a ternary domain structure, including a RING domain, a B-box domain, and a coil-coil domain, as well as a variable C-terminal domain. The RING domain confers E3 ligase activity, allowing TRIM proteins to ubiquitinate target proteins, leading to their degradation by the proteasome or lysosome, or altering their subcellular localization or function. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Example 1

[0051] Weigh the following weights of raw materials: 9 grams of Poria cocos, 9 grams of ginseng, 9 grams of Atractylodes macrocephala, 6 grams of roasted licorice, 9 grams of Astragalus, and 9 grams of honeysuckle.

[0052] The above raw Chinese medicinal materials are dried, deep-cold and ultra-finely crushed at low temperature, and processed into micron Chinese medicinal powder. The particle diameter of micron Chinese medicinal preparation is 0.1-75mm, the average particle size is generally less than 10mm, and is mainly distributed in 1-20mm.

[0053] 9 grams of lactose was added to the micron Chinese medicine powder, spray-dried at 80°C, and processed into a micron particle preparation.

[0054] Spray drying technology is a heat treatment method widely used in pharmaceutical, food and other industries. It can be used to dry liquid, semi-solid or paste materials into powder or granules. In the field of traditional Chinese medicine, spray drying technology is also widely used in the drying of medicinal extracts, liquid medicines, traditional Chinese medicine compounds and other materials.

[0055] Direct spray drying method:

[0056] Direct spray drying is a method of directly spray drying a Chinese herbal medicine extract or liquid medicine into powder or granules. The main steps of this method include atomizing the Chinese herbal medicine extract or liquid medicine into fine droplets through a nozzle, and then contacting it with hot air to rapidly evaporate the water to obtain dry powder or granules.

[0057] The advantages of direct spray drying include fast drying speed, high production efficiency, high product purity, and avoiding the destruction of the active ingredients of medicinal materials by heating.

[0058] 1. Materials

[0059] 1.1 Drug Bifendate (provided by Jiangsu Wujiang County Pharmaceutical Factory)

[0060] 1.2 Animals Kunming mice and SD rats were provided by the Institute of Zoology, Chinese Academy of Sciences

[0061] 2. Methods and Results

[0062] 2.1 Effects of carbon tetrachloride (CCl4)-induced acute liver injury in mice and rats

[0063] Take 50 mice, half of them were ♀ and half of them were ♂, and their body weight was 20-22g, and they were divided into 5 groups: normal (ig physiological saline 20ml / kg) group; CCl4 (0.1ml / kg) model group; CCl4 plus bifendate (100mg / kg) group; CCl4 plus three dose groups of micron Chinese medicine powder of the present invention (10g / kg, 20g / kg, 40g / kg). The mice were given oral administration once a day for 10 consecutive days, and CCl4 was injected intraperitoneally 1h after the last administration, and then fasted for 12h, and the animals were decapitated, and serum was taken to determine AST and ALT values.

[0064] Fifty rats, half ♀ and half ♂, weighing 140-160 g, were divided into 5 groups. The dosing and detection methods were the same as the CCl4 acute liver injury test in mice, with doses of 7.5 g / kg, 15 g / kg, and 30 g / kg. The results are shown in Tables 1 and 2.

[0065] Table 1 Effects of carbon tetrachloride (CCl4) on acute liver injury in mice (x±s)

[0066]

[0067] Compared with the normal control group, ##P<0.01; compared with the model group, **P<0.01

[0068] As can be seen from the above table, compared with the normal control group, the ALT and AST values ​​of the model group were significantly increased (P < 0.01); compared with the model group, the ALT and AST values ​​of the bifendate group were significantly decreased (P < 0.01); compared with the model group, the ALT and AST values ​​of the three dosage groups of the present invention were significantly decreased (P < 0.01).

[0069] Table 2 Effects of carbon tetrachloride (CCl4)-induced acute liver injury in rats (x±s)

[0070]

[0071] Compared with the normal control group, ##P<0.01; compared with the model group, **P<0.01

[0072] As can be seen from the above table, compared with the normal control group, the ALT and AST values ​​of the model group were significantly increased (P < 0.01); compared with the model group, the ALT and AST values ​​of the bifendate group were significantly decreased (P < 0.01); compared with the model group, the ALT and AST values ​​of the three dosage groups of the present invention were significantly decreased (P < 0.01).

[0073] 3. Conclusion

[0074] The invention has obvious therapeutic effect on chemical liver damage in rats and mice.

[0075] Continuous intragastric administration of 10g / kg for 20 days can increase the phagocytic rate, phagocytic index and helper T cell count of mouse phagocytes, and promote the production of IL-2. Continuous intragastric administration of 1.0g / ml herbal medicine decoction for 3 weeks can increase the percentage of nonspecific esterase staining (ANAE) positive cells in peripheral blood lymphocytes (LC) of normal and hydrocortisone-induced immunosuppressed mice, increase the percentage of CD4+T lymphocytes, reduce the percentage of CD8+T lymphocytes and adjust the CD4+ / CD8+ ratio to normal, thereby enhancing the body's cellular immune function. Its prescription polysaccharide 40 and 120μg / ml promotes the proliferation of normal human peripheral blood T lymphocytes induced by phytohemagglutinin (PHA), mainly acting on the S phase of the cell cycle.

[0076] Poria, ginseng, Atractylodes macrocephala, licorice, astragalus, honeysuckle powder:

[0077] Animal grouping and treatment methods 72 mice were randomly divided into 6 groups: Group 1 was the normal control group, which was given water and feed in a fixed amount; Group 2 was the Cy control group, which was injected with Cy on the 4th day of the experiment, at 50 mg / kg, for 7 consecutive days; Groups 3 and 4 were the Chinese medicine immunomodulator groups (referred to as the Chinese medicine groups), and the mice were given the drug through drinking water, once a day, at twice the equivalent dose of the animal calculated by body surface area, 12 mL / (d・;mouse), for 15 consecutive days; Groups 5 and 6 were the Yupingfeng Powder groups, at 12 mL / (d・;mouse). Groups 3 and 5 were only given the drug but not injected with Cy, while Groups 4 and 6 were given the drug and injected with Cy 1 hour after drinking the drug on the 4th day (the dosage and course of treatment were the same as those of Group 2)

[0078] Preparation of drug-containing serum: Blood was collected by eyeball picking 1 hour after the last administration (fasting for 12 hours before blood collection). The blood was placed in a 1 mL centrifuge tube, left to stand for 2 hours, centrifuged at 3000 min for 20 minutes, and the serum was separated aseptically. After inactivation in a 56'C water bath for 30 minutes, it was stored in a 4'C refrigerator and used within 24 hours.

[0079] Preparation of mouse spleen lymphocytes The spleens of mice in each group were sterilely removed.

[0080] The spleen tissue was prepared into a single spleen cell suspension in cold Hank's solution, centrifuged at 1500 min for 10 min, the supernatant was discarded, 6 mL / L TrisNH4C1 was added, the red blood cells were lysed in a 37'C water bath for 5 min, and then centrifuged at 1500 min for 10 min, the supernatant was discarded, and an appropriate amount of Hank's buffer was added for washing 3 times, each time centrifuged at 1500 min for 10 min, the supernatant was discarded, and the cell density was adjusted to 5X109 / L with serum-free RPMI1640 culture medium, and the cell viability was detected by trypan blue staining to be >95%.

[0081] Determination of splenic lymphocyte proliferation response

[0082] Effect of drug-containing serum on transformation of normal mouse spleen lymphocytes When the serum concentration was 50mL / L, 50p L 5X109 / L mouse spleen lymphocytes, 45uL serum-free RPMI1640 and 5uL drug-containing serum were added to each well of a 96-well cell culture plate; when the serum concentration was 100mL / L, 50u L 5X 109 / L mouse spleen lymphocytes, 40uL serum-free RPMI1640 and 10uL drug-containing serum were added to each well of a 96-well cell culture plate. Each group of serum was set up with 4 replicate wells and compared with the same concentration of calf serum RPIM1640 solution, and a culture medium blank control was set up at the same time. Lymphocyte transformation test of mouse spleen cells in each test group: In a 96-well cell culture plate, 95u L of 5X109 / L mouse spleen lymphocytes diluted with RPMI1640 containing 100mL / L calf serum was added to each well, 5u L of RPMI1640 solution without serum was added to each well of 4 horizontal rows A, B, C, and D, and 5uL of 200mg / L PHA was added to each well of 4 horizontal rows E, F, G, and H. Four replicate wells were set for each group of cells, and RPMI1640 containing 100mL / L calf serum was used as a blank control. The cells were cultured at 37°C, 50 mL / L CO2 and saturated humidity for 44 h, 10 μL of 5 g / L MTT was added to each well, and cultured for another 4 h. Then, 50 μL of SDSDMF solution was added. A570 was measured with an ELISA instrument 30 min later. The results were expressed as stimulation index (SI): SI = mean value of A in test wells / mean value of A in control wells × 100%. Data processing was analyzed using statistical software.

[0083] All data are expressed as mean ± standard deviation (x ± s).

[0084] The effect on the cellular immune function of mice was studied. 72 Kunming mice were randomly divided into 6 groups, namely the normal saline control group, low, medium and high doses of ultrafine powder groups and low, medium and high doses of coarse powder groups. After each group was continuously gavaged for 6 days, the changes in peripheral blood T cell subsets (CD4+ / CD8+) were detected. The results showed that the prescription can increase the proportion of CD4+ / CD8+T lymphocyte subsets in the peripheral blood of mice, and the low dose of micropowder can achieve the same effect as the decoction dose.

[0085] Tripartite motif-containing protein 25 (TRIM25) plays a key role in fish nonspecific immunity. In this experiment, different proportions of Chinese herbal medicine compound (Poria, ginseng, Atractylodes macrocephala, Licorice, Astragalus, Honeysuckle) were added to the feed of large yellow croaker. After feeding for 30 days, spleen tissue was used as experimental material. Real-time fluorescence quantitative PCR technology was used to analyze the relative expression changes of TRIM25 gene after feeding Chinese herbal medicine. The results showed that compared with the control group, the expression of this gene in each experimental group decreased to varying degrees (except for the E5 group). The expression level of the E3 group decreased the most (64%), followed by the E1 group (47%). The expression level of the E5 group increased by 19% compared with the control group, but there was no significant difference between each group (P>0.05). The results of this study provide basic data for the application of Chinese herbal medicine as a feed additive in large yellow croaker feed, and also provide a preliminary research basis for Chinese herbal medicine as a fish immune enhancer. Summary of Chinese herbal medicine From the results of this experiment, it was concluded that after yellow croaker was fed with Chinese herbal medicine, the expression of TRIM25 gene in its spleen was increased except for the E5 group.

[0086] Tripartite motif-containing protein 25 (TRIM25) plays an important role in the innate immune response. In this experiment, different proportions of Chinese herbal medicine compound (Pseudostellaria heterophylla, Morinda officinalis, Angelica sinensis, Lycium barbarum, Poria cocos, Atractylodes macrocephala, etc.) were added to the feed of yellow croaker. After feeding for 30 days, the kidney tissue of yellow croaker was used as the experimental material, and the relative expression changes of TRIM25 gene after feeding with Chinese herbal medicine were analyzed by real-time fluorescence quantitative PCR technology. The results showed that compared with the control group, the expression of this gene in each experimental group decreased to varying degrees (except for the E2 group), and the expression level of the E3 group decreased the most (81%), followed by the E1 group (68%), and the expression level of the E5 group increased by 654% compared with the control group, but there was no significant difference among the groups (P greater than 0.05). The results of this study provide basic data for the application of Chinese herbal medicine as a feed additive in yellow croaker feed, and also provide a preliminary research basis for Chinese herbal medicine as a fish immune enhancer.

[0087] Each bag is 10 grams, twice a day, 90 days as a course of treatment. 13 patients receiving treatment for hepatitis B virus infection were selected. Among them, 8 were male and 5 were female, aged 23 to 48 years old, with an average age of 28.4±2.4 years old. The treatment results are as follows.

[0088] The test report form of the Microbiology Laboratory of the Liver Research Institute of Tianjin Third Central Hospital shall be based on the test results. The relevant symptoms will not be repeated. The test results of Beijing, Tianjin and Hebei are mutually recognized.

[0089] The application of pure natural immune granules to hepatitis B patients can play an effective clinical role and promote the improvement of patients' immunity. The following cases were observed after the TRIM22 molecule was elevated:

[0090] Experimental Principle

[0091] This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotin-labeled detection antibodies, and HRP enzyme conjugates are added to the microwells pre-coated with human Trim22 molecule (TRIM22) capture antibodies in sequence. After incubation and washing, the substrate TMB is used for color development. TMB is converted into blue under the catalysis of peroxidase (HRP) and into the final yellow under the action of acid. The depth of color is positively correlated with the human Trim22 molecule (TRIM22) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme reader to calculate the sample concentration.

[0092] Sensitivity: 0.151 ng / mL

[0093] Specificity: It can detect human TRIM22 in samples without obvious cross-reaction with its analogs.

[0094] Provided by Shanghai Jianglai Biotechnology Co., Ltd.

[0095] The following case records were used for clinical efficacy observation after TRIM22 molecule elevation (approved by the Ethics Committee of Handan Traditional Chinese Medicine Hospital):

[0096] Shanghai Jianglai Biotechnology Co., Ltd. is responsible for providing the test kits.

[0097] Table 3: Clinical case 1: Observation of the efficacy of HBV-DNA conversion to negative

[0098]

[0099]

[0100] Table 4: Clinical case 2: Observation of the efficacy of HBV-DNA conversion to negative

[0101] Date and time HBV-DNA (cs / ml) HBV-IC TRIM22 (OD value) July 26, 2018 6.104E1 29.14 10 October 18, 2018 ＜2.000E1 25.94 20↑

[0102] Table 5: Clinical case 3: Observation of the efficacy of HBVeAg conversion to negative:

[0103] Date and time HBVeAg TRIM22 February 19, 2021 64.00mcu / mI 5 April 20, 2021 ≤0.50mcu / mI 10↑

[0104] Table 6: Clinical case 4: Observation of the efficacy of HBVeAg conversion to negative:

[0105] Date and time HBVeAg TRIM22 January 5, 2021 5.49mcu / mI 5 February 10, 2021 ≤0.50mcu / mI 10↑

[0106] Table 7: Clinical case 5: Observation of the efficacy of HBVsAg conversion to negative:

[0107] Date and time HBVsAg TRIM22 January 5, 2021 44.01mcu / mI 10 February 10, 2021 3.38mcu / mI 20↑ April 22, 2021 0mcu / mI 20↑

[0108] Table 8: Clinical case 6: Observation of the efficacy of HBVsAg conversion to negative:

[0109]

[0110]

[0111] 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. A Chinese medicine composition for enhancing TRIM family proteins, characterized in that: In parts by weight, it includes the following components: 5-15 parts of ginseng, 5-15 parts of atractylodes, 5-15 parts of poria, 5-15 parts of licorice, 5-15 parts of honeysuckle, and 5-15 parts of astragalus.

2. A method for preparing a traditional Chinese medicine composition for enhancing TRIM family proteins, characterized in that: The following steps are involved: Weigh the raw Chinese medicinal materials according to the amount described in claim 1; dry the raw Chinese medicinal materials, and process them into micron Chinese medicinal powders by deep-cooling vibration ultrafine grinding unit at a low temperature of minus 80°C.

3. The method for preparing a traditional Chinese medicine composition for enhancing TRIM family proteins according to claim 2, characterized in that: The micron Chinese medicine powder has a particle diameter of 0.1 to 75 mm and an average particle diameter of less than 10 mm.

4. The method for preparing a traditional Chinese medicine composition for enhancing TRIM family proteins according to claim 2, characterized in that: The micron Chinese medicine powder is added with lactose auxiliary material, granulated by spray granulation or other methods, and then granulated and mixed to prepare micron Chinese medicine granules.

Images