Application of tetrandrine in the treatment of secondary hemophagocytic syndrome

Abstract

The present invention relates to the field of drug application, and in particular to the application of tetrandrine in drugs for alleviating secondary hemophagocytic syndrome. The present invention proposes the application of tetrandrine in drugs for alleviating secondary hemophagocytic syndrome, solving the problem of drug selection for treating secondary hemophagocytic syndrome in the prior art. Compared with the prior art, the present invention has the following beneficial effects: tetrandrine can significantly inhibit the inflammatory expression level, lipid accumulation and oxidative stress induced by PolyI:C and LPS. Mechanistically, tetrandrine mainly plays a role in alleviating the pathological process of secondary hemophagocytic syndrome mice by inhibiting the phosphorylation of mTOR and ERK1 / 2. Tetrandrine is expected to become an effective drug for the clinical treatment of secondary hemophagocytic syndrome.

Description

Technical Field

[0001] The invention relates to the field of drug application, and in particular to the application of tetrandrine in drugs for alleviating secondary hemophagocytic syndrome. Background Art

[0002] Secondary hemophagocytic syndrome (sHLH) is a rapidly progressive acute inflammatory disease characterized by cytopenia, excessive cytokine secretion, and hyperferritinemia. Common clinical manifestations are persistent fever, lymphadenopathy, hepatosplenomegaly, and multiple organ failure, invasion of tissues by macrophages and cytotoxic T lymphocytes (CTLs), and excessive release of inflammatory cytokines. Other clinical manifestations include hepatitis, neurological symptoms (such as seizures, meningitis, decreased level of consciousness), rash, pulmonary dysfunction, and lymphadenopathy. Laboratory tests include cytopenia (affecting at least two cell lines in peripheral blood), hyperferritinemia, hypofibrinogenemia due to consumption and liver damage, hypertriglyceridemia secondary to cytokine inhibition of lipoprotein lipase, elevated transaminases and lactate dehydrogenase, hyperbilirubinemia, abnormal coagulation function, hyponatremia, and hypoproteinemia. In addition, due to excessive activation of CTLs and macrophages, soluble CD25 and CD163 may increase, and serum ferritin levels tend to be higher than the expected level of acute phase reactants and remain at 500-10000 ng / ml. Inflammation has been shown to be the main cause of sHLH. The secretion of inflammatory factors is inseparable from oxidative stress and lipid metabolism. If this stage is controlled, it is of great significance to slow down the progressive development of secondary hemophagocytic syndrome.

[0003] The HLH-2004 regimen is widely used internationally to treat secondary hemophagocytic syndrome. The 2004 regimen is based on dexamethasone, etoposide and cyclosporine, and is divided into an initial treatment period of 8 weeks and a maintenance treatment period, with an additional intrathecal injection. The use of immunoglobulin in the acute phase helps alleviate the condition. Currently, immunosuppressive drugs and hormone drugs are often used in clinical treatment of secondary hemophagocytic syndrome, but due to the diversity of sHLH manifestations and poor clinical prognosis, new treatment methods are urgently needed.

[0004] Tetrandrine (Tet) is a plant alkaloid extracted from the root of Tetrandrine fangji, a traditional Chinese medicine. It inhibits voltage-gated calcium channels and Ca 2+Activated potassium ion channels. Tetrandrine can be used as an antihypertensive drug. It is mainly used clinically to treat mild and moderate hypertension, and can also be used for hypertensive crisis. With the continuous deepening of modern research, more and more uses of tetrandrine have been discovered. Tetrandrine can reverse the resistance of CHO cells to the anti-tumor drug doxorubicin, block tumor cells in the G1 phase, increase the accumulation of chemotherapy drugs in tumor cells, and enhance the sensitivity of tumor cells to chemotherapy drugs; it can play an analgesic role by reducing the release of peroxides and the activity of phagocytes; it can loosen and degrade collagen fibers in silicosis, reduce lipids, disappear and depolymerize microtubule structures, hinder the conversion of procollagen, and appear new cells in the gaps, and then be used to treat silicosis. Tetrandrine has anti-inflammatory, analgesic, antihypertensive, anti-inflammatory, antibacterial, and anti-tumor pharmacological effects. Clinically, tetrandrine is often used to treat diseases related to the respiratory system, nervous system, circulatory system, motor system and other systems. Given that sHLH is a syndrome of excessive inflammatory response caused by abnormal immune system, the role of tetrandrine in secondary hemophagocytic syndrome is still unclear. Summary of the invention

[0005] In view of this, the present invention provides the use of tetrandrine in drugs for alleviating secondary hemophagocytic syndrome, thereby solving the problem of drug selection for treating secondary hemophagocytic syndrome in the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of tetrandrine in preparing medicine for alleviating and / or treating hemophagocytic syndrome.

[0008] In some specific embodiments of the present invention, the hemophagocytic syndrome comprises secondary hemophagocytic syndrome.

[0009] In some specific embodiments of the present invention, the tetrandrine inhibits and / or improves inflammatory response.

[0010] The most typical manifestation of secondary hemophagocytic syndrome is that the body secretes a large amount of pro-inflammatory factors. In some specific embodiments of the present invention, the tetrandrine inhibits any of the following:

[0011] (I), Poly I:C and / or LPS-induced expression of IL-6 and / or IL-1β in macrophages; and / or

[0012] (II) Inhibiting the expression of IL-6 and / or IL-1β in animals.

[0013] In some specific embodiments of the present invention, the tetrandrine prolongs the survival time of an animal.

[0014] In some specific embodiments of the present invention, the tetrandrine prolongs the improvement of toxic reactions in animals.

[0015] When inflammation occurs in the body, lactate dehydrogenase is over-secreted and two or three lines of whole blood cells are reduced. In some specific embodiments of the present invention, the tetrandrine

[0016] (I), reducing the content of lactate dehydrogenase; and / or

[0017] (II) Suppressing the number of leukocytes and / or platelets.

[0018] The hallmark of secondary hemophagocytic syndrome is the increase of triglyceride and serum ferritin content. In some specific embodiments of the present invention, the tetrandrine

[0019] (I), inhibiting lipid accumulation; and / or

[0020] (II), reducing serum ferritin levels; and / or

[0021] (III) Inhibit oxidative stress.

[0022] in:

[0023] (I), the inhibition of lipid accumulation comprises reducing the content of triglycerides;

[0024] (II) The inhibiting of oxidative stress comprises reducing the expression of oxidative stress-related proteins SOD1 and / or SOD2.

[0025] The mTOR and ERK1 / 2 signaling pathways may be involved in inflammation, oxidative stress, and lipid metabolism processes associated with secondary hemophagocytic syndrome. In some specific embodiments of the present invention, the tetrandrine inhibits the phosphorylation level of mTOR and / or ERK1 / 2, thereby inhibiting the overactivation of the mTOR and / or ERK1 / 2 signaling pathways and improving the state of the animal.

[0026] Compared with the prior art, the present invention has the following beneficial effects: Tetrandrine A can significantly inhibit the inflammatory expression level, lipid accumulation and oxidative stress induced by Poly I:C and LPS. Mechanistically, Tetrandrine A mainly alleviates the pathological process of secondary hemophagocytic syndrome mice by inhibiting the phosphorylation of mTOR and ERK1 / 2. Tetrandrine A is expected to become an effective drug for the clinical treatment of secondary hemophagocytic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0028] Figure 1 It was shown that tetrandrine inhibited the expression of IL-6 and IL-1β in macrophages induced by Poly I:C and LPS;

[0029] Figure 2 It was shown that tetrandrine prolongs the survival time of mice with secondary hemophagocytic syndrome;

[0030] Figure 3 It was shown that tetrandrine inhibited the expression of IL-6 and IL-1β in mice with secondary hemophagocytic syndrome;

[0031] Figure 4 It was shown that tetrandrine inhibited the number of leukocytes and platelets in mice with secondary hemophagocytic syndrome;

[0032] Figure 5 It was shown that tetrandrine inhibited the level of lactate dehydrogenase in mice with secondary hemophagocytic syndrome;

[0033] Figure 6 It was shown that tetrandrine inhibited the serum triglyceride level in mice with secondary hemophagocytic syndrome;

[0034] Figure 7 It was shown that tetrandrine inhibited the serum ferritin level in mice with secondary hemophagocytic syndrome;

[0035] Figure 8 It was shown that tetrandrine inhibited the oxidative stress level in the liver of mice with secondary hemophagocytic syndrome;

[0036] Fig. 9 The results showed that tetrandrine inhibited the phosphorylation levels of mTOR and ERK1 / 2 in mice with secondary hemophagocytic syndrome.

[0037] Among them: Ctrl is the control group, Tet is the tetrandrine group, PL is the Poly I:C and LPS combined modeling group, and PLTet group is the modeling plus tetrandrine group; * It means P < 0.05 compared with Ctrl group in PL group; # P<0.05 compared with the PLTet group; ** It means that P < 0.01 was compared between PL group and Ctrl group; ## P<0.01 compared with the PLTet group; *** It means P < 0.001 compared with Ctrl group in PL group; ### The difference between PL group and PLTet group was P<0.001. DETAILED DESCRIPTION

[0038] The present invention discloses the use of tetrandrine in drugs for alleviating secondary hemophagocytic syndrome. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0039] In the application of tetrandrine provided by the present invention in the drug for alleviating secondary hemophagocytic syndrome, the raw materials and reagents used can be purchased from the market.

[0040] The present invention will be further described below in conjunction with embodiments:

[0041] Example 1 Effect of tetrandrine on Poly I:C and LPS-induced macrophage inflammatory factors

[0042] (1) Experimental cells

[0043] Mouse monocytic macrophage leukemia cell line RAW264.7.

[0044] (2) Experimental cell treatment

[0045] The above cells were inoculated into 6-well plates (2×10 6 / well), cells were treated as follows:

[0046] Ctrl group;

[0047] Tet group: treated with Tet (4 μmol / L);

[0048] PL group: first treated with Poly I:C (10 ng / mL) for 24 h, then treated with LPS (5 ng / mL) for 24 h;

[0049] PLTet group: pretreated with Tet (4 μmol / L) for 12 h, then co-treated with Tet (4 μmol / L) and Poly I:C (10 ng / mL) for 24 h, and then co-treated with Tet and LPS (5 ng / mL) for 24 h.

[0050] (3) Experimental results

[0051] (3.1) Tetrandrine inhibits the expression of IL-6 and IL-1β in macrophages induced by Poly I:C and LPS.

[0052] The most typical manifestation of secondary hemophagocytic syndrome is the secretion of a large number of proinflammatory factors, which in turn cause immune disorders. After macrophages were induced by Poly I:C and LPS (abbreviated as PL in the figure), we explored the improvement effect of tetrandrine on the inflammatory response of RAW264.7 cells. The content of secretory proteins in the cell supernatant was detected by ELISA (the results are shown in Table 1). The expression levels of IL-6 and IL-1β secretory proteins in the PL group were significantly upregulated, and Tet significantly inhibited their expression levels. Figure 1 The results suggest that Tet can improve PL-induced inflammation.

[0053] Table 1 Expression of IL-6 and IL-1β in the cell supernatant of each group

[0054]

[0055]

[0056] Note: ** It means that P < 0.01 was compared between PL group and Ctrl group; ## P<0.01 compared with the PLTet group; *** It means P < 0.001 compared with Ctrl group in PL group; ### The difference between PL group and PLTet group was P<0.001.

[0057] Example 2 Effect of tetrandrine in the treatment of secondary hemophagocytic syndrome

[0058] (1) Experimental animals

[0059] Male C57BL / 6J mice aged 7-8 weeks were kept in an environmental temperature controlled at 22-24°C, and the experimental animals were allowed to eat and drink freely.

[0060] (2) Experimental groups

[0061] Forty healthy SPF C57BL / 6J male mice aged 8 weeks were selected and randomly divided into 4 groups (10 mice in each group): control group (Ctrl), tetrandrine group (Tet), model group (PL), and model drug group (PLTet). Within three days, the Tet group and PLTet group were gavaged with 200 mg / kg body weight of Tet every day, and the Ctrl group and PL group were gavaged with 200 mg / kg body weight of 0.5% CMC-Na every day. On the second day, the PL group and PLTet group were intraperitoneally injected with 10 mg / kg body weight of Poly I:C, and the Ctrl group and Tet group were intraperitoneally injected with 10 mg / kg body weight of normal saline. On the third day, the PL group and PLTet group were intraperitoneally injected with 3 mg / kg body weight of LPS, and the Ctrl group and Tet group were intraperitoneally injected with 3 mg / kg body weight of normal saline. The state and survival of the mice were observed and recorded until the mice died naturally.

[0062] According to the survival experiment described below (3.1), it was determined that tetrandrine A could prolong the survival of mice. Then the next batch of animal experiments were carried out. The grouping and modeling methods were the same as above, with 8 mice in each group. The mice were killed 12 hours after intraperitoneal injection of LPS and tissues and serum were collected.

[0063] (3) Experimental results

[0064] (3.1) Tet prolongs the survival of sHLH mice

[0065] In order to explore whether Tet can improve the survival time of PL-induced sHLH mice, we first conducted a mouse survival experiment and observed the status of the mice. We found that the control group and the tetrandrine group were normal; the model group was more depressed than the control group; the PLTet group was improved compared with the PL group; Figure 2 As shown in the results, the survival time of mice in the model-treated group was higher than that in the model group, and the results were statistically significant. This preliminarily indicates that tetrandrine has a protective effect on PL-induced secondary hemophagocytic syndrome model mice.

[0066] (3.2) Tet improves PL-induced inflammation in mice

[0067] To further explore whether Tet inhibits PL-induced sHLH inflammation, the levels of IL-6 and IL-1β secreted proteins in mouse serum were detected (the results are shown in Table 2). Compared with the Ctrl group, the expression levels of the above indicators in the PL model group were upregulated. After Tet treatment, the expression levels of inflammatory proteins in the PL group mice were reduced ( Figure 3 ).

[0068] Table 2 Comparison of IL-6 and IL-1β expression levels in the serum of mice in each group

[0069]

[0070] Note: *** It means P < 0.001 compared with Ctrl group in PL group; ### The difference between PL group and PLTet group was P<0.001.

[0071] (3.3) Tet improves PL-induced toxicity in mice

[0072] The results of complete blood routine examination are shown in Table 3. Compared with the Ctrl group, the number of white blood cells and platelets in the PL group mice was significantly reduced ( Figure 4 ); the number of platelets in the PLTet group was higher than that in the PL group. The results of serum biochemical analysis (as shown in Table 4) showed that the lactate dehydrogenase (LDH) in the PL group was significantly higher than that in the Ctrl group. After Tet treatment, the LDH content decreased ( Figure 5 ).

[0073] Table 3 WBC and PLT contents of mice in each group

[0074]

[0075] Note: ## P<0.01 compared with the PLTet group; *** It means P < 0.001 compared with Ctrl group in PL group; ### The difference between PL group and PLTet group was P<0.001.

[0076] Table 4 LDH content of mice in each group

[0077]

[0078] Note: # P<0.05 compared with the PLTet group; *** The difference between PL group and Ctrl group was P<0.001.

[0079] (3.4) Tet improves PL-induced lipid accumulation, serum ferritin elevation, and oxidative stress levels in mice

[0080] The hallmark indicators of HLH are the increase in triglyceride (TG) and serum ferritin content, so this study detected the expression levels of serum TG and ferritin in mice (the results are shown in Tables 5 and 6). Compared with the Ctrl group, the TG and ferritin levels in the PL modeling group were significantly increased, but after Tet treatment, the levels of both in the PL group decreased ( Figure 6 and Figure 7). To explore the role of Tet in oxidative stress in sHLH, Western blot was used to detect the expression of oxidative stress-related proteins SOD1 and SOD2 (as shown in Table 7). The results showed that PL could induce oxidative stress in mice. Compared with the PL group, the oxidative stress level in the PLTet group was reduced ( Figure 8 ). In summary, tetrandrine can improve the over-secretion of triglycerides and ferritin expression levels in model mice and inhibit oxidative stress.

[0081] Table 5 Triglyceride levels in liver serum of mice in each group

[0082]

[0083] Note: ### Means P < 0.001 compared with PLTet group; *** The difference between PL group and Ctrl group was P<0.001.

[0084] Table 6 Comparison of serum ferritin levels in liver of mice in each group

[0085]

[0086]

[0087] Note: * It means P < 0.05 compared with Ctrl group in PL group; # P<0.05 compared with the PL group and the PLTet group.

[0088] Table 7 Relative expression of SOD1 and SOD2 proteins in each group of mice

[0089]

[0090] Note: * It means P < 0.05 compared with Ctrl group in PL group; # P<0.05 compared with the PL group and the PLTet group.

[0091] (3.5) Tet improves PL-induced overactivation of mTOR and ERK1 / 2 signaling pathways

[0092] Studies have shown that mTOR and ERK1 / 2 signaling pathways may be involved in inflammation, oxidative stress, and lipid metabolism associated with secondary hemophagocytic syndrome. Therefore, we detected the protein expression of p-ERK / ERK and p-mTOR / mTOR by Western blot (the results are shown in Table 8). Fig. 9As shown in the figure, the phosphorylation levels of ERK and mTOR in the PL group mice were increased, and the phosphorylation levels were reduced after treatment with tetrandrine. We speculate that tetrandrine may inhibit the inflammatory response and improve the state of the model mice by affecting the excessive activation of the mTOR and ERK1 / 2 signaling pathways induced by PL.

[0093] Table 8 p-ERK / ERK and p-mTOR / mTOR protein expression in the liver of mice in each group

[0094]

[0095] Note: * It means P < 0.05 compared with Ctrl group in PL group; # P<0.05 compared with the PL group and the PLTet group.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of tetrandrine as the sole active ingredient in the preparation of a drug for relieving and / or treating hemophagocytic syndrome; The hemophagocytic syndrome is secondary hemophagocytic syndrome.

2. The use according to claim 1, characterized in that The tetrandrine inhibits and / or improves inflammatory response.

3. The use according to claim 1, characterized in that The tetrandrine inhibits any of the following: (I), Poly I:C and / or LPS-induced expression of IL-6 and / or IL-1β in macrophages; and / or (II) Inhibit the expression of IL-6 and / or IL-1β in animals.

4. The use according to claim 1, characterized in that The tetrandrine prolongs the survival time of animals.

5. The use according to claim 1, characterized in that The tetrandrine improves the toxic reaction of animals.

6. The use according to claim 5, characterized in that Tetrandrine (I), reducing the content of lactate dehydrogenase; and / or (II) Suppressing the number of leukocytes and / or platelets.

7. The use according to claim 1, characterized in that Tetrandrine (I), inhibiting lipid accumulation; and / or (II), reducing serum ferritin levels; and / or (III) Inhibit oxidative stress.

8. The use according to claim 7, characterized in that (I) The inhibition of lipid accumulation comprises reducing the content of triglycerides; (II) The inhibiting of oxidative stress comprises reducing the expression of oxidative stress-related proteins SOD1 and / or SOD2.

9. The use according to claim 1, characterized in that The tetrandrine inhibits the phosphorylation of mTOR and / or ERK1 / 2.

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