Resuscitation fluid for treating ischemia reperfusion injury

Abstract

The invention discloses resuscitation fluid for treating ischemia reperfusion injury. The resuscitation fluid is prepared from HBOCs, a small-molecule reducing agent Vc, hydroxyethyl starch and a radix salviae miltiorrhizae injection. According to the formula, the volume concentration of the HBOCs is 1%-10%, the mass-volume ratio of the small molecule reducing agent Vc is 0.01%-0.1%, the volume concentration of the hydroxyethyl starch is 2%-6%, the concentration of the salvia miltiorrhiza injection is 10%-30%, and the injection dosage is equal to the rat blood loss dosage. The method comprises the following steps: firstly, infusing a hemoglobin oxygen carrier, a small molecule reducing agent Vc and hydroxyethyl starch into a patient, and infusing the salvia miltiorrhiza injection after 30 minutes; or after the hemoglobin oxygen carrier, the small molecule reducing agent Vc, the hydroxyethyl starch and the salvia miltiorrhiza injection are prepared, the patient is infused. Compared with a formula without the salvia miltiorrhiza injection, the formula can effectively relieve injury of tissues such as liver, heart, kidney and the like of ischemia reperfusion rats.

Description

Technical Field

[0001] The invention belongs to the technical field of medicaments, and in particular relates to a resuscitation fluid for treating ischemia-reperfusion injury. Background Art

[0002] Hemorrhagic shock can cause primary organ damage, including hypoperfusion of major organs such as the liver, heart, kidneys, central nervous system (CNS), and lungs, as well as strong immune response activation, manifested by excessive release of inflammasomes and activation of multiple inflammatory signaling pathways. These events lead to an immunosuppressive state and failure of host defense, ultimately leading to inflammation-mediated infection and organ dysfunction. Although subsequent resuscitation and reperfusion can improve tissue perfusion, they usually further induce a systemic inflammatory response syndrome, which further aggravates tissue / organ damage, leading to multiple organ failure (MOF) and even death. Therefore, there is an urgent need for treatments to prevent organ damage in the clinic, which will have a profound impact on the prognosis of patients undergoing resuscitation from hemorrhagic shock.

[0003] Patients who survive hemorrhagic shock are particularly susceptible to systemic inflammatory response syndrome (SIRS) in the initial stage, which subsequently triggers MOF and post-traumatic death. Among the main biochemical mediators of MOF, a large amount of reactive oxygen species (ROS) produced during hemorrhagic shock / resuscitation causes tissue damage by consuming intrinsic antioxidant activity, oxidizing plasma membranes and key proteins, and initiating systemic inflammatory cascades. After traumatic blood loss, organs undergo an ischemia-reperfusion process, which may lead to severe tissue damage. With the increasing demand for clinical blood use, blood shortages occur frequently in my country, and blood resource shortages have become an important issue in ensuring my country's blood supply. Studies have pointed out that the blood supply gap is expected to reach 21.2 million units in 2036. In addition, due to the threat of immune responses caused by traditional blood transfusions and related transfusion-transmitted diseases such as hepatitis and AIDS, the research and development of "blood substitutes" has received great attention internationally.

[0004] At present, hemoglobin-based oxygen carriers (HBOCs) are the most widely studied blood substitutes. The products are nano-scale, have no red blood cell membrane, no blood type distinction, can inactivate viruses, overcome the risk of viral infection, and are readily available on the shelf. After decades of development, HBOCs have formed various types of products. So far, 8 types of HBOCs products have been put into clinical trials. Once HBOCs products are successfully developed, they will greatly alleviate the shortage of blood supply. They will not only have immeasurable social significance and application prospects for routine medical treatment and emergency rescue, but also have considerable industrial economic benefits. However, at present, whether it is red blood cells, whole blood or HBOCs, the resuscitation of severe hemorrhagic shock will cause ischemia-reperfusion injury. We have developed a new formulation that can not only effectively resuscitate 40%-65% of hemorrhagic shock in rats, but also alleviate the damage to important organs such as the heart, liver, and kidneys of rats.

[0005] The current technical formula is aimed at clinical severe hemorrhagic shock resuscitation, and has achieved very good results in animal experiments, which is comparable to the whole blood of rats in the treatment of severe hemorrhagic shock in rats. However, due to ischemia-reperfusion injury and the difference in oxygen supply pathways between nano-oxygen-carrying drugs and red blood cells, it will cause damage to organs including the liver, heart, and kidneys, affecting the prognosis of rats. Based on the above analysis, the present invention provides a resuscitation solution for treating ischemia-reperfusion injury, which includes HBOCs, a small molecule reducing agent Vc, hydroxyethyl starch and Danshen injection, and can achieve a good resuscitation effect by re-infusing an equal amount of blood loss to ischemic rats. Summary of the invention

[0006] In view of the above-mentioned deficiencies, the purpose of the present invention is to reduce the adverse reactions caused by the resuscitation process, including liver damage, heart damage and kidney damage, while ensuring the resuscitation effect of rats with severe hemorrhagic shock.

[0007] The present invention is achieved by the following technical means:

[0008] The present invention first discloses a resuscitation fluid for treating ischemia-reperfusion injury, comprising:

[0009] Hemoglobin oxygen carriers (HBOCs), small molecule reducing agent Vc, hydroxyethyl starch and Danshen injection; among which:

[0010] The mass concentration of HBOCs is 1%-10%, the mass fraction of small molecule reducing agent Vc is 0.01%-0.1%, the concentration of hydroxyethyl starch is 2%-6%, and the volume fraction of Danshen injection liquid is 10%-30%. Compared with the formula without Danshen injection liquid, the formula can effectively alleviate the damage of liver, heart, kidney and other tissues in rats with ischemia-reperfusion.

[0011] Furthermore, the mass concentration of HBOCs is 1%-6%, the mass content of small molecule reducing agent Vc is 0.01%-0.05%, and the volume content of Danshen injection is 15%-20%.

[0012] Furthermore, the hemoglobin-based oxygen carriers include, but are not limited to, oxygen carriers made from human placental blood, human peripheral blood, bovine blood, and pig blood.

[0013] The present invention also discloses a method for preparing a resuscitation solution for treating ischemia-reperfusion injury, comprising:

[0014] The mass concentration of hemoglobin oxygen carrier is adjusted to 1%-6%, Vc is added to make its mass concentration 0.01%-0.05%, and then Danshen injection is added to make the final volume concentration of Danshen injection be 10%-30%, and the optimal concentration is 15%-20%.

[0015] The invention also discloses a resuscitation fluid for treating ischemia-reperfusion injury, which is used for treating animals or humans with a blood loss of 40%-65%.

[0016] The beneficial effects of the present invention are:

[0017] This patent mainly analyzes that the main causes of reperfusion injury caused by HBOCs are inflammation and oxidative stress. Among them, Danshen injection (SMI) has antioxidant and anti-free radical effects. Its active ingredient, tanshinone, can remove superoxide anion free radicals through xanthine and xanthine oxidase reaction system, and tanshinone can effectively remove lipid peroxides on damaged cell mitochondria, thereby completing its anti-free radical effect in the body.

[0018] The current technical formula is for clinical severe hemorrhagic shock resuscitation. It has achieved very good results in animal experiments and is comparable to the whole blood of rats in the treatment of severe hemorrhagic shock in rats. However, the resuscitation process goes through the process of ischemia and reperfusion, and there will be ischemia and reperfusion damage. In order to reduce the damage to important organs caused by the resuscitation fluid in technology one, we have studied another technology, HBOCs, small molecule reducing agent Vc, and hydroxyethyl starch, which can achieve a good resuscitation effect by re-infusing equal amounts of blood loss to ischemic rats. This technology significantly reduces problems such as organ damage, including heart damage, liver damage, and kidney damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Comparison of the survival rates of the two groups of rats at 72 hours;

[0020] Figure 2 : Comparison of ALB between the two groups of rats at baseline and 24 hours after resuscitation;

[0021] Figure 3: Comparison of LDL-C between the two groups of rats at baseline and 24 hours after resuscitation;

[0022] Figure 4 : Comparison of HDL-C between the two groups of rats at baseline and after 24 hours of resuscitation;

[0023] Figure 5 : Comparison of TC between the two groups of rats at baseline and 24 hours after resuscitation;

[0024] Figure 6 : Comparison of AST between the two groups of rats at baseline and 24 hours after resuscitation;

[0025] Figure 7 : Comparison of LDH between the two groups of rats at baseline and 24 hours after resuscitation;

[0026] Figure 8 : Comparison of the hearts of the two groups of rats after 24 hours of resuscitation, A: HE staining of heart sections of rats in the HBOC+HES+SMI group; B: HE staining of heart sections of rats in the HBOC+HES group;

[0027] Fig. 9 : Comparison of the livers of the two groups of rats after 24 hours of resuscitation. A: HE staining of liver sections of rats in the HBOC+HES+SMI group; B: HE staining of liver sections of rats in the HBOC+HES group. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

[0029] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application for which protection is sought, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0030] The chemical reagents used in the following examples are all conventional reagents and can be obtained commercially. Experimental methods not specifically described are all conventional methods known in the art.

[0031] Example 1

[0032] A resuscitation solution for treating ischemia-reperfusion injury

[0033] The raw materials and formulation are as follows:

[0034] 90 ml of blood substitute with human placental hemoglobin as raw material (mass concentration is 6.7%), 180 ml of hydroxyethyl starch (mass concentration is 6.7%), 1 ml of vitamin C (mass concentration is 3.0%), and 29 ml of salvia miltiorrhiza injection.

[0035] The specific configuration method is:

[0036] Under aseptic conditions, first add 180 ml of hydroxyethyl starch to a disposable sterile oxygen-barrier bag, then add 90 ml of the above-mentioned blood substitute, 1 ml of vitamin C, and 29 ml of Danshen injection respectively. After the injection is completed, seal the disposable sterile oxygen-barrier bag; shake the oxygen-barrier bag to allow the drugs to stagnate and mix fully in the oxygen-barrier bag.

[0037] Example 2

[0038] A resuscitation solution for treating ischemia-reperfusion injury

[0039] The raw materials and formulation are as follows:

[0040] 90ml of blood substitute with human placental hemoglobin as raw material (mass concentration is 6.7%), 180ml of hydroxyethyl starch (mass concentration is 6.7%), 2ml of vitamin C (mass concentration is 3.0%), 3ml of glycine (mass concentration is 50%), 25ml of salvia miltiorrhiza injection, and a disposable sterile oxygen-isolating bag.

[0041] The specific configuration method is: under sterile conditions, first add 180 ml of hydroxyethyl starch to a disposable sterile oxygen-barrier bag, and then add 90 ml of the above-mentioned blood substitute, 2 ml of vitamin C, and 25 ml of Danshen injection, 3 ml of glycine, and seal the disposable sterile oxygen-barrier bag after the injection; shake the oxygen-barrier bag to allow the drugs to be stored and mixed fully in the oxygen-barrier bag.

[0042] Example 3

[0043] A resuscitation solution for treating ischemia-reperfusion injury

[0044] The raw materials and formulation are as follows:

[0045] 80ml of blood substitute with human placental hemoglobin as raw material (mass concentration is 6.7%), 160ml of hydroxyethyl starch (mass concentration is 6.7%), 3ml of vitamin C (mass concentration is 3.0%), 3ml of glycine (mass concentration is 50%), 24ml of salvia miltiorrhiza injection, and a disposable sterile oxygen-isolating bag.

[0046] The specific configuration method is:

[0047] Under aseptic conditions, first add 180 ml of hydroxyethyl starch to a disposable sterile oxygen-barrier bag, and then add 90 ml of the above-mentioned blood substitute, 2 ml of vitamin C, 25 ml of salvia miltiorrhiza injection, and 3 ml of glycine. After the injection, seal the disposable sterile oxygen-barrier bag; shake the oxygen-barrier bag to allow the drugs to stagnate and mix fully in the oxygen-barrier bag.

[0048] Test Example 1

[0049] Rat animal experiment

[0050] The rats were divided into two groups. After anesthesia, the femoral vein and femoral artery of the rats were cannulated, and blood was let out at a uniform rate to create a 50% hemorrhagic shock model in the rats. The control group was resuscitated with 2% HBOCs + 4% hydroxyethyl starch resuscitation solution in a dose equal to the amount of blood loss, and the experimental group was resuscitated with 2% HBOCs + 4% hydroxyethyl starch + 15% Danshen injection resuscitation solution in a dose equal to the amount of blood loss.

[0051] 1. Compare the survival rates of the two groups of rats at 72 hours. For details, see Figure 1 :

[0052] according to Figure 1 The results showed that the experimental group did not show a difference in survival rate compared with the control group, indicating that adding Danshen injection to the formula had no effect on the overall survival rate of rats.

[0053] 2. Detect the liver-related indicators of the two groups of rats, including the basic values ​​of ALB, LDL, HDL, TC and the comparison after 24 hours of resuscitation. Figure 2-Figure 5 :

[0054] according to Figure 2 The results showed that after 24 hours of resuscitation, the ALB content of the rats in the experimental group was significantly lower than that in the control group, indicating that adding Danshen injection to the formula can effectively reduce the ALB content of rats after 24 hours of resuscitation and alleviate liver damage.

[0055] according to Figure 3 The results showed that after 24 hours of resuscitation, the LDL content of rats in the experimental group was significantly lower than that in the control group, indicating that adding Danshen injection to the formula can effectively reduce the LDL content of rats 24 hours after resuscitation and alleviate liver damage.

[0056] according to Figure 4 The results showed that after 24 hours of resuscitation, the HDL content of rats in the experimental group was significantly increased compared with that in the control group, indicating that adding Danshen injection to the formula can effectively increase the HDL content of rats after 24 hours of resuscitation and alleviate liver damage.

[0057] according to Figure 5 The results showed that 24 hours after resuscitation, the TC content of the rats in the experimental group was significantly lower than that in the control group, indicating that adding Danshen injection to the formula can effectively reduce the TC content of rats 24 hours after resuscitation and alleviate liver damage.

[0058] 3. Detect the heart-related indicators of the two groups of rats, including the basic values ​​of AST and LDH and the comparison after 24 hours of resuscitation. Figure 6-Figure 7 :

[0059] according to Figure 6 The results showed that AST content in the experimental group was significantly lower than that in the control group 24 hours after resuscitation, indicating that adding Danshen injection to the formula can effectively reduce AST content in rats 24 hours after resuscitation and alleviate cardiac damage.

[0060] according to Figure 7 The results showed that after 24 hours of resuscitation, the LDH content of the rats in the experimental group was significantly lower than that in the control group, indicating that adding Danshen injection to the formula can effectively reduce the LDH content of rats after 24 hours of resuscitation and have a mitigating effect on heart damage.

[0061] 4. Compare the heart sections of the two groups of rats after 24 hours of resuscitation. Figure 8 :

[0062] according to Figure 8 The results of a and 8b show that after 24 hours of resuscitation, the inflammatory response of the heart slices of the rats in the experimental group was reduced compared with that in the control group, indicating that adding Danshen injection to the formula can effectively reduce cardiac inflammation in rats after 24 hours of resuscitation and have a relieving effect on cardiac damage.

[0063] 5. Compare the liver sections of the two groups of rats after 24 hours of resuscitation. Fig. 9 :

[0064] according to Fig. 9 The results of a and 9b show that after 24 hours of resuscitation, the liver inflammatory response of the rats in the experimental group was reduced compared with that in the control group, indicating that adding Danshen injection to the formula can effectively reduce liver inflammation in rats 24 hours after resuscitation and have a relieving effect on liver damage.

[0065] The present invention is not limited to the above optional implementations, and anyone can derive other various forms of products under the enlightenment of the present invention. The above specific implementations should not be understood as limiting the scope of protection of the present invention. The scope of protection of the present invention should be based on the definition in the claims, and the description can be used to interpret the claims.

Claims

1. A resuscitation fluid for treating ischemia-reperfusion injury, comprising: Hemoglobin oxygen carrier, small molecule reducing agent Vc, hydroxyethyl starch and Danshen injection; among them: The mass concentration of the hemoglobin oxygen carrier is 1%-10%, the mass concentration of the small molecule reducing agent Vc is 0.01%-0.1%, the concentration of hydroxyethyl starch is 2%-6%, and the volume fraction of the salvia miltiorrhiza injection liquid is 10%-30%.

2. The resuscitation solution for treating ischemia-reperfusion injury according to claim 1, wherein: The mass concentration of the hemoglobin oxygen carrier is 1%-6%, the mass concentration of the small molecule reducing agent Vc is 0.01%-0.05%, and the volume content of the salvia miltiorrhiza injection is 15%-20%.

3. The resuscitation solution for treating ischemia-reperfusion injury according to claim 1 or 2, wherein: The hemoglobin oxygen carriers include oxygen carriers made from human placental blood, human peripheral blood, bovine blood and pig blood.

4. A use of a resuscitation solution for treating ischemia-reperfusion injury according to any one of claims 1 to 3, comprising: Used to treat animals or humans with blood loss of 40%-65%.

5. The use according to claim 4, wherein: The method of using the resuscitation fluid is selected from any of the following: First, infuse the patient with hemoglobin oxygen carrier, small molecule reducing agent Vc, and hydroxyethyl starch, and then infuse Danshen injection 30 minutes later; or Hemoglobin oxygen carrier, small molecule reducing agent Vc, hydroxyethyl starch and Danshen injection are prepared and infused into the patient.

Images