Dendrobine, pharmaceutical composition and use thereof for treating diseases associated with abnormal STAT3

Abstract

The present invention discloses a dendrobium, a pharmaceutical composition and its use in treating diseases related to STAT3 abnormality, specifically the use of dendrobium in preparing a medicament for treating diseases related to STAT3 (signal transduction and activator of transcription 3) abnormality, especially the use of dendrobium in treating inflammatory diseases including sepsis, progressive inflammatory bowel disease, chronic obstructive pulmonary disease, bronchial asthma, chronic nephritis, gastritis, pancreatitis, obstructive nephropathy, diabetic glomerulopathy, retinitis and the like. The present invention is the first to discover that dendrobium has an inhibitory effect on STAT3, which can not only expand the application field of dendrobium, make traditional Chinese medicinal materials realize greater value, but also provide new drugs for treating diseases related to STAT3. The present invention finds that the combination of dendrobium and anti-inflammatory drugs, especially natural medicines such as ligustrazine, resveratrol, limonene, and matrine, has a good synergistic effect and can greatly improve the anti-inflammatory effect.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to dendrobium alkaloids, a pharmaceutical composition and use thereof in treating diseases associated with STAT3 abnormality. Background Art

[0002] STAT3 (signal transducer and activator of transcription 3) is a gene involved in regulating cellular physiology and pathology. Constitutive activation of STAT3 protein is a common pro-inflammatory and oncogenic feature found in many solid tumors, including gastric cancer. STAT3 can be phosphorylated and activated by the Janus kinase family. During IL-6 signaling, IL-6ST promotes JAK2 phosphorylation and STAT3 phosphorylation. STAT3 homodimerizes, translocates to the nucleus, binds to DNA, and activates transcription of STAT3 target genes. TFF1 (trefoil factor 1) is primarily expressed in epithelial cells lining the gastric pits. TFF1 contains a trefoil domain. TFF1 protects the gastric mucosa by forming a complete mucus layer and mucosal barrier, promoting the repair of mucosal damage. In TFF1-deficient mouse models, this mucosal protective mechanism is absent, promoting the cascade of gastric cancer development. Silencing of TFF1 in the gastric mucosa can occur through multiple molecular mechanisms, including loss of heterozygosity, promoter methylation, and transcriptional repression. TFF1 has recently been found to have anti-inflammatory and pro-apoptotic functions, protecting the gastric mucosa and maintaining its integrity. However, TFF1 deficiency activates the oncogenic NF-κB and β-catenin pathways. Research results indicate that TFF1 plays an important role in inhibiting IL6-mediated STAT3 activation.

[0003] As an inflammatory signaling pathway for stress, the Jak / Stat pathway must respond rapidly. Jak and Stat are key components of many signaling pathways that regulate cell growth, differentiation, survival, and pathogen resistance. This pathway involves the IL-6 (gp130) receptor family, which helps regulate B cell differentiation, plasma cell production, and acute phase responses.

[0004] IL-6 binding triggers receptor dimerization and simultaneous activation of receptor-bound Jak proteins. Activated Jak proteins phosphorylate the receptor and itself. These phosphorylated sites serve as binding sites for SH2-containing Stat proteins and adaptor proteins, which connect the receptor to MAPK, PI3K / Akt, and other pathways. Receptor-bound Stat proteins, phosphorylated by Jak proteins, form dimers that translocate to the cell nucleus to regulate target gene expression.

[0005] Members of the inhibitory cytokine signaling (SOCS) family attenuate receptor-transmitted signals through homologous or heterologous feedback. STATs, when unactivated, linger in the cytoplasm. Upon receiving a danger signal, they become phosphorylated and rapidly mobilize to the nucleus to perform their transcriptional functions. This mode of transcription factor mobilization should theoretically be very rapid, making it highly advantageous for responding to extracellular signals. NF-kB, the most common inflammatory cytokine, is also a transcription factor of this type, though it is regulated by the inhibitory protein IKBa.

[0006] To control inflammation, the body must precisely regulate the balance between inflammatory and anti-inflammatory genes and their products. Dr. Peter A. Ward, Professor of Pathology at the University of Michigan Medical School, is studying this complex balance. His goal is to help researchers develop new drugs to treat immune-related diseases such as COPD. A series of related experimental results were published in the June 15th issue of Immunology. In the paper, Ward and his colleagues report that an anti-inflammatory protein called Stat3 is expressed in rats with severe pneumonia.

[0007] Stat3 has become a hot topic of research because it is an important transcription factor that is involved in turning on genes required for cell division, growth, and death. Researchers have now discovered that it also regulates inflammatory responses in the lungs. The study found that Stat3 is activated after acute lung injury and appears to act as an intermediate for the production of anti-inflammatory cytokines. This is the first study to reveal a function for Stat3 in the lungs.

[0008] Researchers are interested in Stat3 because several recent studies have shown it to play a key role in inflammatory responses. For example, in an animal model of sepsis (a systemic, uncontrolled inflammatory response), Stat3 has been shown to be a key intermediate in anti-inflammatory signaling in macrophages. Furthermore, mice with a knockout of the hematopoietic-specific Stat3 gene develop progressive inflammatory bowel disease, which is often fatal. In their experiments, the researchers caused excessive accumulation of IgC immune complexes in the airways of experimental rats, triggering an inflammatory response. The rats' immune systems responded by recruiting large numbers of neutrophils and macrophages to the damaged lungs. These cells triggered a cascade of proteins and other factors that repair the damaged tissue. The researchers then analyzed the rats' lung tissue to determine the location, timing, and amount of various proteins produced during the inflammatory response.

[0009] Dendrobium is a pyrrolizidine alkaloid extracted and isolated from the stem of the orchid Dendrobium nobile Lindl. Its biological activity is similar to that of picrotoxin. Chemical and spectroscopic analysis has confirmed that dendrobium has a picrotoxane skeleton. Dendrobium is the first picrotoxane-type alkaloid to be identified.

[0010] Dendrobium, also known as Linlan, Dulan, Qiannianzhu, and Huangcao, is sweet in taste and slightly cold in nature. It enters the stomach, lung, and kidney meridians, promoting fluid production and nourishing the stomach, nourishing yin and clearing heat, and moistening the lungs and kidneys. It originates from the stems of Dendrobium annuum, Dendrobium whip, Dendrobium yellow grass, Dendrobium officinale, or Dendrobium nobile, all members of the Orchidaceae family. It is primarily produced in Sichuan, Guizhou, Yunnan, and other regions. It can be harvested year-round. After removing impurities, it is briefly scalded in boiling water or baked to soften, then rubbed and sun-dried until the leaf sheaths are clean and sun-dried. After removing some of the fibrous roots, the Dendrobium officinale is stir-fried and twisted into a spiral or spring shape, then dried. This is commonly known as "earring dendrobium." Pharmacological effects: Dendrobium contains over ten alkaloids, including dendrobine, dendrobamine, dendrobine, dendrobine, dendrobine, and 6-hydroxydendrobine, as well as mucilage and starch. Dendrobium alkaloids have analgesic and antipyretic effects, can lower heart rate and blood pressure, slow breathing, have a tonic effect and can relieve barbiturate poisoning.

[0011] Pharmacological tests have shown that oral administration of a decoction of Dendrobium can promote gastric secretion and aid digestion, increasing intestinal motility and promoting laxative effects. However, excessive dosages can lead to intestinal muscle paralysis. Dendrobium alkaloids have certain antipyretic and analgesic effects, similar to phenacetin, but with a weaker effect. Dendrobium polysaccharides can restore immune function in mice and have anti-aging and cataract-delaying effects. Two phenanthrene compounds extracted from Dendrobium nobile have anti-cancer effects on human lung cancer, ovarian adenocarcinoma, and promyelocytic leukemia. Dendrobium nobile can significantly increase SOD (superoxide dismutase) levels and reduce LPO (lipid peroxide), thereby delaying aging. An ester component contained in Dendrobium can also promote blood circulation and remove blood stasis, and has therapeutic effects on thromboangiitis obliterans, cerebral thrombosis, and arteriosclerosis occlusive.

[0012] Dendrobium alkaloids are a very important medicinal herb. They can regulate the digestive system, slow memory loss, and relieve fever and pain. In 2001, Chen Xiaomei et al. found that dendrobium alkaloids can also inhibit intestinal motility in isolated rabbits. She hypothesized that this is due to dendrobium alkaloids directly stimulating G cells, leading to increased gastrin release and elevated serum gastrin concentrations. In 2016, Liang Chuyan et al. studied the effects of Dendrobium officinale on improving memory and delaying aging. This suggests that Dendrobium officinale is an effective remedy for preventing memory loss in many middle-aged and elderly people. Dendrobium alkaloids also have certain analgesic and antipyretic effects, similar to those of phenacetin. In 1991, Li Manfei et al. discovered that the alkaloids dendrobiumine and dendrobiumine, isolated from Dendrobium officinale, inhibited rat kidney microsomal sodium-potassium ATPase, the primary enzyme responsible for heat production during basal metabolism. In 2001, Wang Chunlan et al. found that Dendrobium candidum and Dendrobium nobile inhibited the activity of the rat kidney microsomal sodium-potassium ATPase. In Traditional Chinese Medicine, yang deficiency and internal heat are manifestations of excessive sodium-potassium ATPase activity. Dendrobium alkaloids' pharmacological effect of inhibiting renal microsomal sodium-potassium ATPase activity is consistent with its yin-nourishing and heat-clearing properties.

[0013] However, there have been no reports on the effects of dendrobium alkaloids on STAT3 (signal transducer and activator of transcription 3). Summary of the Invention

[0014] The purpose of the present invention is to study the inhibitory effect of dendrobium alkaloids on STAT3 (signal transduction and activator of transcription 3) and to provide the use of dendrobium alkaloids for treating diseases related to abnormalities in STAT3 (signal transduction and activator of transcription 3), especially inflammatory diseases including sepsis, progressive inflammatory bowel disease, chronic obstructive pulmonary disease, bronchial asthma, chronic nephritis, gastritis, pancreatitis, obstructive nephropathy, diabetic glomerulopathy, retinitis, etc.

[0015] The technical solution is as follows:

[0016] The present invention aims to provide a method for preparing a drug for treating a disease associated with abnormal STAT3 (signal transducer and activator of transcription 3). The structural formula of dendrobium is as follows:

[0017]

[0018] Preferably, the related diseases mediated by STAT3 (signal transducer and activator of transcription 3) include inflammatory diseases, autoimmune diseases, and tumors, more preferably inflammatory diseases.

[0019] More preferably, the inflammatory disease is sepsis, progressive inflammatory bowel disease, chronic obstructive pulmonary disease, bronchial asthma, chronic nephritis, gastritis, pancreatitis, obstructive nephropathy, diabetic glomerulopathy, and retinitis; the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, scleroderma, thyroid dysfunction, juvenile diabetes, psoriasis, multiple sclerosis, idiopathic thrombocytopenic purpura, ulcerative colitis, autoimmune hepatitis, and primary biliary cirrhosis; the tumor is lung cancer, multiple myeloma, head and neck squamous cell carcinoma, prostate cancer, liver cancer, esophageal cancer, gastric cancer, colon cancer, melanoma, leukemia, lymphoma, rectal cancer, brain tumor, and skin cancer.

[0020] Preferably, the inflammatory disease is sepsis, progressive inflammatory bowel disease, chronic obstructive pulmonary disease, bronchial asthma, chronic nephritis, gastritis, pancreatitis, obstructive nephropathy, diabetic glomerulopathy, or retinitis.

[0021] Preferably, the STAT3 (signal transducer and activator of transcription 3) abnormality is caused by the interleukin-6 (IL-6) receptor.

[0022] Preferably, dendrobium alkaloids are used in combination with anti-inflammatory drugs, wherein the anti-inflammatory drugs include natural medicines such as ligustrazine, resveratrol, limonene, and matrine.

[0023] More preferably, dendrobium alkaloids are used in combination with an anti-inflammatory drug, wherein the anti-inflammatory drug is ligustrazine or resveratrol.

[0024] Another object of the present invention is to provide a pharmaceutical composition for treating diseases associated with abnormal STAT3 (signal transducer and activator of transcription 3), characterized in that the pharmaceutical composition includes dendrobium alkaloids.

[0025] Preferably, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers or excipients.

[0026] Another object of the present invention is to provide a use of the pharmaceutical composition in the preparation of a drug for treating diseases associated with abnormal STAT3 (signal transducer and activator of transcription 3).

[0027] Preferably, the STAT3 (signal transducer and activator of transcription 3) abnormality is caused by the interleukin-6 (IL-6) receptor.

[0028] Beneficial effects

[0029] 1. The present invention is the first to discover that dendrobium alkaloids have an inhibitory effect on STAT3 (signal transduction and activator of transcription 3). This can not only expand the application field of dendrobium alkaloids, making traditional Chinese medicinal materials more valuable, but also provide new drugs for treating diseases related to STAT3 (signal transduction and activator of transcription 3);

[0030] 2. The present invention found that dendrobium alkaloids have a good effect on inflammatory diseases by inhibiting STAT3 (signal transduction and transcription activator 3). 50 It is 43uM, which has a good inhibitory effect.

[0031] 3. The present invention found that the combination of dendrobium alkaloids and anti-inflammatory drugs, especially natural drugs such as ligustrazine, resveratrol, limonene, and matrine, has a good synergistic effect and can greatly enhance the anti-inflammatory effect. DETAILED DESCRIPTION

[0032] The embodiments of the present invention are described in detail below. The embodiments are given to better illustrate the contents of the present invention and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0033] Experimental design

[0034] A stable RAW264.7 cell line expressing a STAT3 signaling pathway reporter gene was constructed, designated RAW-264.7-STAT3-Nano-Luc. A new expression vector containing the STAT3 enhancer sequence and the NanoLuc (NLuc) reporter gene sequence was constructed using genetic recombination techniques. Furthermore, a cell line stably expressing NLuc luciferase under STAT3 regulation was established. This cell line was used to quantitatively assay the regulatory effects of various Chinese herbal monomers on the IL-6 / STAT3 signaling pathway and to validate the inhibitory effects of Chinese herbal monomers on the IL-6 / STAT3 signaling pathway. Enzyme digestion and sequencing results confirmed the successful construction of the reporter gene expression vector pQCXIP-STAT3-NLuc.

[0035] Interleukin-6 (IL-6), a stimulator of the STAT3 transcription factor, stimulated the constructed cell line stably expressing NLuc, resulting in a specific luciferase response in a dose-dependent manner, demonstrating the successful construction of a cell line stably expressing NLuc luciferase regulated by STAT3. A reporter gene system was constructed for efficient detection of STAT3 transcriptional activity, and this system was used to successfully screen for monomeric Chinese herbal compounds that inhibit the IL-6 / STAT3 signaling pathway.

[0036] 1. Cell Culture

[0037] RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 100 U / mL penicillin and streptomycin. The incubator culture conditions were set at 5% CO2, 37°C. The medium was changed every other day, and the cell growth was observed daily.

[0038] 2. Cell Passaging

[0039] When RAW264.7 cells grow to 70-80% confluence, discard the old cell culture medium, wash the cells twice with PBS, add 0.25% trypsin, and observe cell morphology changes under an inverted microscope. When the cells show cytoplasm retraction, cell body rounding, and cell gap enlargement, discard the digestion medium and immediately add cell culture medium containing 10% serum to terminate digestion. Use a pipette to aspirate the culture medium, repeatedly and gently blow the attached cells to make them fall off and suspend them, adjust the cells to an appropriate density, and then inoculate them into a new culture dish and culture them in a 5% CO2, 37°C incubator.

[0040] 3. Cell Cryopreservation

[0041] RAW264.7 cells in the logarithmic growth phase and in good growth condition were digested with 0.25% trypsin to prepare a single-cell suspension and collected in a 15 mL centrifuge tube. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The precipitated cells were suspended in freezing solution (fetal bovine serum and DMSO mixed in a ratio of 9:1, prepared immediately before use) and the cell concentration was adjusted to (1-10) × 106 / mL. The cell suspension was divided into freezing tubes (1 mL / tube), the tube caps were tightened, the tubes were sealed, and the cell name and freezing date were marked. The cells were cooled in the order of 4°C for 30 min, -20°C for 2 h, and -80°C overnight, and finally placed in a liquid nitrogen tank for long-term storage.

[0042] 4. Cell Recovery

[0043] Remove the RAW264.7 cell cryovial that needs to be revived from the liquid nitrogen tank, place the cryovial in disposable PE gloves, and quickly place it in a water bath preheated to 37°C. Shake continuously until the cryopreservative solution is completely melted. Transfer the cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and add fresh cell culture medium containing 10% serum to make a cell suspension. Adjust the cells to an appropriate density and inoculate them into a new culture dish. Place the dish in a constant temperature and humidity incubator with 5% CO2 at 37°C for culture.

[0044] Inflammation experimental design

[0045] The RAW264.7-STAT3-Nluc cell line was cultured in complete medium until the logarithmic growth phase, and the cell count was 1*10 4 cells / 100uL / well for cell culture.

[0046] Five groups of cells were set up, including blank group, model group, high concentration of dendrobium 25uM, medium concentration 5uM, and low concentration 1uM. Each group of samples was repeated 4 times. The model group was IL-6: the concentration was 10ng / ml.

[0047] IL-6 (10 ng / ml) was added to the three drug groups (high, medium, and low dendrobium) at the same time, and the reporter gene luminescence value was measured and the average value was taken.

[0048] The non-toxic dose of dendrobium alkaloids to RAW264.7 was determined to be 50uM by CCK8 assay, and its IC 50 :43uM. (Note: This data is half inhibition rate, but the experimental data later showed that the inhibition rate was more than half at 25uM. I feel that there is a contradiction between the two. IC 50 Should be less than 25uM)

[0049] The pharmaceutical composition is prepared by a method similar to the above.

[0050] Experimental data:

[0051] In the first experiment, the IL-6 recombinant protein in the model group promoted inflammation by approximately 13.1-fold. Dendrobium at a high concentration of 25 μM inhibited inflammation by 73%, at a medium concentration of 5 μM by 51%, and at a low concentration of 1 μM by 47%. Dendrobium inhibited the expression of the signaling pathway reporter gene at 10 ng / ml of IL-6 by 63%, 56%, and 37% respectively, in a dose-dependent manner.

[0052] In the second experiment, the IL-6 recombinant protein in the model group promoted inflammation by approximately 12.3-fold. Dendrobium at a high concentration of 25 μM inhibited inflammation by 73%, at a medium concentration of 5 μM by 66%, and at a low concentration of 1 μM by 57%. Dendrobium inhibited the expression of the signaling pathway reporter gene protein, induced by IL-6 at 10 ng / ml, by 70%, 65%, and 51% in a dose-dependent manner.

[0053] In the third experiment, the iL-6 recombinant protein stimulated inflammation in the model group by approximately 13.3-fold. Dendrobium at a high concentration of 25 μM inhibited inflammation by 83%, at a medium concentration of 5 μM by 66%, and at a low concentration of 1 μM by 47%. Dendrobium inhibited the expression of the signaling pathway reporter gene protein, induced by iL-6 at 10 ng / ml, by 80%, 61%, and 42%, respectively, in a dose-dependent manner.

[0054] The above experiments show that dendrobium alkaloids have a strong inhibitory effect on the STAT3-signaling pathway, especially on the signaling pathway caused by I L6.

[0055] In the fourth experiment, the IL-6 recombinant protein in the model group promoted inflammation by approximately 12.7-fold. Ligustrazine at a high concentration of 25 μM inhibited inflammation by 62%, at a medium concentration of 5 μM by 46%, and at a low concentration of 1 μM by 27%. Ligustrazine inhibited the expression of the signaling pathway reporter protein, induced by IL-6 at 10 ng / ml, by 58%, 41%, and 22%, respectively, from 100%.

[0056] In the fifth experiment, the iL-6 recombinant protein promoted inflammation by approximately 13.1-fold in the model group. The high-concentration resveratrol (25 μM) group inhibited inflammation by 42%, the medium-concentration resveratrol (5 μM) group inhibited inflammation by 36%, and the low-concentration resveratrol (1 μM) group inhibited inflammation by 15%. Resveratrol inhibited the expression of the signaling pathway reporter protein, induced by iL-6 at 10 ng / ml, by 39%, 25%, and 8% respectively, from 100%.

[0057] In the sixth experiment, the iL-6 recombinant protein stimulated inflammation in the model group by approximately 12.6-fold. Dendrobium and ligustrazine were administered at a 1:1 ratio of 25uM, 5uM, and 1uM. The high 25uM concentration inhibited inflammation by 96%, the medium 5uM concentration by 88%, and the low 1uM concentration by 62%. The expression of the signaling pathway reporter protein, induced by 10ng / ml of iL-6, decreased from 100% to 95%, 88%, and 59%, respectively.

[0058] In the seventh experiment, the iL-6 recombinant protein in the model group increased inflammation by approximately 13.0-fold. Dendrobium and resveratrol were administered at a 1:1 ratio of 25uM, 5uM, and 1uM. The high 25uM concentration inhibited inflammation by 98%, the medium 5uM concentration inhibited inflammation by 92%, and the low 1uM concentration inhibited inflammation by 71%. The expression of the signaling pathway reporter protein, induced by iL-6 at 10ng / ml, decreased from 100% to 96%, 90%, and 65%, respectively.

[0059] It can be seen from the above experiments that the combination of dendrobium and ligustrazine, and dendrobium and resveratrol can greatly improve the inhibition rate of inflammation.

[0060] Obviously, the above embodiments are merely examples for the purpose of illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, to the extent such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to encompass such modifications and variations.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for treating inflammatory diseases, characterized in that: The pharmaceutical composition consists of dendrobium alkaloids and resveratrol, and in the pharmaceutical composition, dendrobium alkaloids and resveratrol are configured in a ratio of 1:1 to a concentration of 5 μM or 1 μM.

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