Application of eleutheroside E in the preparation of a drug for preventing or treating severe acute pancreatitis

Abstract

The present invention discloses an application of eleutheroside E or its pharmaceutical salt in the preparation of a drug for preventing or treating severe acute pancreatitis. Studies have found that eleutheroside E has a significant effect on resisting severe acute pancreatitis. The present invention has been proved by animal experiments: 3.5% sodium taurocholate was used to retrograde pancreaticobiliary injection to induce severe acute pancreatitis mouse model experiments, and it was found that eleutheroside E can significantly reduce the activity levels of serum amylase and lipase in mice, significantly reduce the iron content of pancreatic tissue in mice, reduce the oxidative stress response index MDA, ROS level, increase GSH level, increase Nrf2, HO-1, GPX4 protein expression levels, reduce the iron death marker ACSL4 protein expression level, and improve pancreatic tissue pathological damage. Therefore, eleutheroside E can prevent or treat severe acute pancreatitis.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to an application of eleutheroside E in preparing a medicine for preventing or treating severe acute pancreatitis. Background Art

[0002] Acute pancreatitis (AP) refers to a disease caused by activation of pancreatic enzymes in the pancreas due to various etiologies, with or without changes in the functions of other organs. The overall mortality rate is about 0.8%. Mild acute pancreatitis (MAP) is more common in clinical practice, and the course of the disease is self-limited. In recent years, the incidence of AP has been increasing year by year, with a global incidence of about 34 / 100,000, of which about 20% of patients will develop severe acute pancreatitis (SAP) [1] Lee PJ, Papachristou GI. New insights into acute pancreatitis [J]. Nat Rev Gastroenterol Hepatol, 2019, 16(8): 479-496. doi: 10.1038 / s41575-019-0158-2. [2] Matta B, Gougol A, Gao X, Reddy N, Talukdar R, Kochhar R, Goenka MK, Gulla A, Gonzalez JA, Singh VK, Ferreira M, Stevens T, Barbu ST, Nawaz H, Gutierrez SC, Zarnescu NO, Capurso G, Easler J, Triantafyllou K, Pelaez-Luna M,Thakkar S,Ocampo C,de-Madaria E,Cote GA,Wu BU,Paragomi P,Pothoulakis I,TangG,Papachristou GI.Worldwide Variations in Demographics,Management,andOutcomes of Acute Pancreatitis[J].Clin Gastroenterol Hepatol,2020,18(7):1567-1575.e2.doi:10.1016 / j.cgh.2019.11.017.].SAP is characterized by rapid onset and progression, and may even cause systemic inflammatory response syndrome (SIRS), which further aggravates the severity of the disease, affects prognosis, and may be complicated by multiple organ dysfunction syndrome (MODS). The mortality rate of SAP patients is as high as 30%, which brings a heavy burden to families and society. However, there is still a lack of effective treatment for the disease [3] Trikudanathan G, Wolbrink DRJ, van Santvoort HC, Mallery S, Freeman M, Besselink MG. Current Concepts in Severe Acute and Necrotizing Pancreatitis: An Evidence-Based Approach [J]. Gastroenterology, 2019, 156(7): 1994-2007.e3.doi:10.1053 / j.gastro.2019.01.269. [4] Iannuzzi JP, King JA, Leong JH, Quan J, Windsor JW, Tanyingoh D, Coward S, Forbes N, Heitman SJ, Shaheen AA, Swain M, Buie M, Underwood FE, Kaplan GG. Global Incidence of Acute Pancreatitis Is Increasing Over Time: A Systematic Review and Meta-Analysis [J]. Gastroenterology, 2022, 162 (1): 122-134. doi: 10.1053 / j.gastro.2021.09.043.]. Therefore, it is of great significance to explore the pathogenesis of SAP and find effective therapeutic drugs.

[0003] Currently, the role of ferroptosis in the pathogenesis of AP has received extensive attention. Ferroptosis is an iron-dependent, novel type of programmed cell death that is different from other types of cell death. The main mechanism of ferroptosis is that, under the action of divalent iron or esteroxygenase, it catalyzes the highly expressed unsaturated fatty acids on the cell membrane to cause lipid peroxidation, thereby inducing cell death; in addition, it is also manifested as a decrease in the expression of the antioxidant system. The main characteristics of ferroptosis are: (1) In terms of cell morphology, ferroptosis can cause the cell mitochondria to become smaller, the membrane density to increase, the cristae to decrease, while the morphological changes in the cell nucleus are not obvious. (2) In terms of cell composition, ferroptosis is manifested by increased lipid peroxidation and increased reactive oxygen species (ROS). Antioxidant molecules or enzymes such as glutathione (GSH), heme oxygenase 1 (HO-1), glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) together form an anti-ferroptosis axis to resist ferroptosis [[5]Li J, Cao F, Yin HL, Huang ZJ, Lin ZT, Mao N, Sun B, Wang G. Ferroptosis: past, present and future [J]. Cell Death Dis, 2020, 11(2): 88. doi: 10.1038 / s41419-020-2298-2.]. Studies have found that pancreatic acinar cells sensitized to ferroptosis by trypsin may be a target for the prevention and treatment of AP in mice. An unbiased drug screening experiment identified olanzapine as a novel ferroptosis inhibitor that can prevent AP in mice [[6]Liu K, Liu J, Zou B, Li C, Zeh HJ, Kang R, Kroemer G, Huang J, Tang D. Trypsin-Mediated Sensitization to Ferroptosis Increases the Severity of Pancreatitis in Mice[J]. Cell Mol Gastroenterol Hepatol, 2022; 13(2): 483-500. doi: 10.1016 / j.jcmgh.2021.09.008.].Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important stress-induced transcription factor composed of seven highly conserved epichlorohydrin-related protein domains and a basic leucine zipper, and plays an important role in regulating oxidative stress response. The Nrf2 pathway is an endogenous antioxidant mechanism in cells. It can be activated by oxidative stimulation and enter the nucleus, inducing the expression of downstream signaling molecules such as HO-1, GPX4, ferritin heavy chain 1 (FTH1) and transferrin receptor 1 (TfR1) to exert an antioxidant stress response [[7]Dodson M, Castro-Portuguez R, Zhang DD. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis [J]. Redox Biol, 2019, 23: 101107. doi: 10.1016 / j.redox.2019.101107.]. Studies have found that activating the Nrf2 pathway can inhibit ferroptosis, thereby protecting AP [[8]Lee J, Lim JW, Kim H. LycopeneInhibits IL-6Expression by Upregulating NQO1 and HO-1viaActivation of Nrf2 inEthanol / Lipopolysaccharide-Stimulated Pancreatic Acinar Cells[J]. Antioxidants(Basel),2022,11(3):519.doi:10.3390 / antiox11030519.].

[0004] Acanthopanax senticosus is the dried root and rhizome or stem of the plant Acanthopanax senticosus of the Araliaceae family. It tastes pungent, slightly bitter, and warm in nature. It enters the spleen, kidney, and heart meridians and has the effects of “promoting blood circulation and removing blood stasis, invigorating qi and strengthening spleen, relieving fever and pain, nourishing kidney and calming nerves, strengthening tendons and bones, and strengthening will” [[9] Liang Zihan, Wang Yiyao, Lai Yixiang, Zhang Jingyi, Song Yinhong. Research progress on the immunomodulatory effects of Acanthopanax senticosus extract and its active ingredients [J]. Chinese Herbal Medicine, 2022, 53(15): 4895-4904. doi:10.7501 / j.issn.0253-2670.2022.15.033.]. The active ingredients of Acanthopanax senticosus are flavonoids, polysaccharides, syringin, eleutheroside E (ELE), etc. The most widely used in scientific research and clinical application is Acanthopanax senticosus injection (crude extract of Acanthopanax senticosus), the main component of which is flavonoid compounds. Zhu Jianping et al. [

[10] Zhu Jianping, Li Meifang, Chen Bo, Deng Xiujian. Effects of Acanthopanax senticosus on inflammatory mediators in rats with acute pancreatitis model [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2008, 13(7): 744-746. doi:10.3969 / j.issn.1009-2501.2008.07.005.] The results showed that Acanthopanax senticosus injection can reduce the levels of serum tumor necrosis factor α (TNF-α), interleukin-1 (IL-1) and IL-6 in rat AP, and can reduce the activity levels of serum amylase, lipase and phospholipase A2 in rat AP pancreatic enzymes. Previous research results showed that Acanthopanax injection may further inhibit abnormal autophagy of pancreatic acinar cells by inhibiting the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) or nuclear factor kappa B (NF-κB) signaling pathway, thereby exerting a therapeutic effect on SAP [

[11] Wang X, Zhou G, Liu C, Wei R, Zhu S, Xu Y, Wu M, Miao Q. Acanthopanax versus 3-Methyladenineameliorates sodium taurocholate-induced severe acute pancreatitis by inhibiting the autophagic pathway in rats[J]. Mediators Inflamm, 2016, 2016:8369704.doi:10.1155 / 2016 / 8369704.].Acanthopanax injection can downregulate NF-κB p65, thereby further downregulating pro-inflammatory factors such as TNF-α and IL-6. Acanthopanax injection can upregulate the anti-inflammatory factor IL-10, which in turn downregulates NF-κB p65, further inhibiting the inflammatory response, alleviating SAP-related brain injury in rats, and reducing mortality, thereby playing a protective role against SAP-related brain injury in rats [

[12] Wang X, Zhuang X, Wei R, Wang C, Xue X, Mao L. Protective effects of Acanthopanax vs. Ulinastatin against severe acute pancreatitis-induced brain injury in rats[J]. Int Immunopharmacol, 2015, 24(2): 285-298. doi: 10.1016 / j.intimp.2014.12.020.].

[0005] Traditional Chinese medicine is profound and has a long history. It emphasizes the treatment based on syndrome differentiation and has its unique advantages. However, many Chinese medicine prescriptions and crude extracts are not recognized internationally, mainly due to problems such as unclear active substances, unknown targets, uncontrollable quality and unreliable safety. Chinese medicine monomers are different from crude extracts and compound prescriptions. They have clear molecular weights and physical and chemical properties, which are easy to quantitatively and qualitatively analyze. Therefore, technical problems and quality requirements can be strictly controlled during their preparation, thus solving the above problems [

[13] Liu Chang, Xia Shilin, Shang Dong. Research progress on Chinese medicine monomers for the treatment of acute pancreatitis [J]. Liaoning Journal of Traditional Chinese Medicine, 2018, 45(10): 2232-2236. doi:10.13192 / j.issn.1000-1719.2018.10.068.]. ELE is one of the most important active ingredients (not the main ingredient) extracted from Acanthopanax senticosus. It is a Chinese medicine monomer with the molecular formula: C 34 H 46 O 18, molecular weight: 742.72, is a phenolic glycoside compound. Studies have shown that ELE has multiple functions such as anti-oxidation, anti-inflammation, improving microcirculation, enhancing immunity, anti-radiation, anti-fatigue, anti-aging, improving body adaptability, and improving learning and memory ability [

[14] Song C, Duan F, Ju T, Qin Y, Zeng D, Shan S, ShiY, Zhang Y, Lu W. Eleutheroside Esupplementation prevents radiation-induced cognitive impairment and activates PKA signaling via gut microbiota [J]. CommunBiol, 2022, 5(1): 680. doi: 10.1038 / s42003-022-03602-7.]. However, its application in the treatment of AP has not been reported.

[0006] Wang et al. [

[15] Wang S, Yang X.Eleutheroside E decreases oxidative stressand NF-κBactivation and reprograms the metabolic response against hypoxia-reoxygenation injury in H9C2 cells[J].Int Immunopharmacol,2020,84:106513.doi:10.1016 / j.intimp.2020.106513.] showed that ELE treatment significantly inhibited hypoxia-reoxygenation-induced cardiomyocyte injury by reducing oxidative stress and NF-κB activation to reprogram metabolic responses. Moreover, the role of ELE in reprogramming metabolic responses provides a mechanistic link for the antioxidant and anti-inflammatory activities of ELE. Liu et al. [

[16] Liu Z, Gao W, Xu Y. Eleutheroside Ealleviates cerebral ischemia-reperfusion injury in a5-hydroxytryptaminereceptor 2C(Htr2c)-dependent manner in rats[J]. Bioengineered, 2022, 13(5): 11718-11731. doi: 10.1080 / 21655979.2022.2071009.] found that ELE can significantly alleviate cerebral ischemia / reperfusion injury in rats and reduce the apoptosis of hippocampal neurons. Summary of the invention

[0007] The purpose of the present invention is to provide an application of eleutheroside E in preparing a medicine for preventing or treating severe acute pancreatitis.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A first aspect of the present invention provides a use of eleutheroside E or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating severe acute pancreatitis.

[0010] The structure of eleutheroside E [

[14] Song C, Duan F, Ju T, Qin Y, Zeng D, Shan S, Shi Y, Zhang Y, Lu W. Eleutheroside E supplementation prevents radiation-induced cognitive impairment and activates PKA signaling via gut microbiota [J]. CommunBiol, 2022, 5(1): 680. doi: 10.1038 / s42003-022-03602-7.] is shown below:

[0011]

[0012] ELE molecular formula:C 34 H 46 O 18 , molecular weight: 742.72, CAS number: 39432-56-9.

[0013] The drug for preventing or treating severe acute pancreatitis refers to eleutheroside E or its pharmaceutically acceptable salt as a single active ingredient.

[0014] The weight content of the eleutheroside E as a single active ingredient is 0.1% to 99.9%.

[0015] The dosage form of the drug is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation.

[0016] The drug can be administered orally or by injection.

[0017] The clinical manifestations of severe acute pancreatitis are abdominal pain, jaundice, shock, high fever, abnormal breathing, altered consciousness, gastrointestinal bleeding, ascites, skin and mucosal bleeding, abnormal skin around the umbilicus and waist, etc. Its pathogenesis has not yet been elucidated. At present, the role of ferroptosis in the pathogenesis of acute pancreatitis has received widespread attention. Studies have shown that: (1) When acute pancreatitis occurs, abnormal iron metabolism, GPX4 deficiency or reduced activity in the patient's body are key factors inducing ferroptosis. (2) Activated trypsin can increase the severity of acute pancreatitis by increasing the sensitivity of acinar cells to ferroptosis. (3) Activation of the Nrf2 pathway can inhibit ferroptosis, thereby further protecting against acute pancreatitis. (4) In acute pancreatitis, the core protein glycan (Decorin, DCN) released by cells induced by ferroptosis can trigger the advanced glycosylation endproduct-specific receptor (AGER) / NF-κB pathway, triggering an inflammatory response and causing more cell death, thus forming a vicious cycle. (5) Ferroptosis is widely involved in the pathophysiological process of organ damage related to severe acute pancreatitis. At present, there is still a lack of effective treatment for severe acute pancreatitis. The present invention starts from the mechanism of action of ferroptosis in acute pancreatitis and selects eleutheroside E as a drug for preventing or treating severe acute pancreatitis.

[0018] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0019] The present invention provides the use of eleutheroside E in the preparation of a drug for preventing or treating severe acute pancreatitis. Studies have found that eleutheroside E has a significant effect on resisting severe acute pancreatitis. The present invention has been proved by animal experiments: using 3.5% sodium taurocholate retrograde pancreaticobiliary injection to induce severe acute pancreatitis mouse model experiments, it was found that eleutheroside E can significantly reduce the activity levels of serum amylase and lipase in mice, significantly reduce the iron content of pancreatic tissue in mice, reduce the oxidative stress response index malondialdehyde (Malonaldehyde, MDA), ROS level, increase GSH level, increase Nrf2, HO-1, GPX4 protein expression levels, reduce the iron death marker ACSL4 protein expression level, and improve pancreatic tissue pathological damage. This shows that eleutheroside E may play a good role in resisting severe acute pancreatitis by regulating the iron death mediated by the Nrf2-HO-1 signaling pathway, so eleutheroside E can prevent or treat severe acute pancreatitis.

[0020] The present invention not only clarifies that eleutheroside E has the effect of resisting severe acute pancreatitis, but also provides a new means for the clinical treatment of severe acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the HE (HE×200) staining picture of the pancreatic tissue of the sham operation group (SO group) 24 hours after administration of 3.5% sodium taurocholate model in Example 1.

[0022] Figure 2 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis model group (SAP group) 24 hours after administration of 3.5% sodium taurocholate in Example 1.

[0023] Figure 3 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (ELE L+SAP group) treated with a small dose of eleutheroside E 24 hours after the administration of 3.5% sodium taurocholate in Example 1.

[0024] Figure 4 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (ELE M+SAP group) treated with medium-dose eleutheroside E 24 hours after administration of 3.5% sodium taurocholate in Example 1.

[0025] Figure 5 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (ELE H+SAP group) treated with a large dose of eleutheroside E 24 hours after the administration of 3.5% sodium taurocholate in Example 1.

[0026] Figure 6 It is a bar graph of the pancreatic tissue pathology scoring results of each group in Example 1.

[0027] Figure 7 It is a bar graph of the serum amylase activity results of each group in Example 1.

[0028] Figure 8 It is a bar graph of the serum lipase activity results of each group in Example 1.

[0029] Fig. 9 It is a bar graph of the iron content results of pancreatic tissues of each group in Example 1.

[0030] Fig.10 It is a bar graph of the MDA content results of pancreatic tissues of each group in Example 1.

[0031] Fig.11 It is a bar graph of the ROS content results of pancreatic tissues in each group in Example 1.

[0032] Fig.12 It is a bar graph showing the results of GSH content in pancreatic tissue of each group in Example 1.

[0033] Fig.13This is a typical Western blot bar graph of Nrf2, HO-1, GPX4, and ACSL4 in pancreatic tissue of each group in Example 1.

[0034] Fig.14 It is a bar graph showing the relative expression level of Nrf2 protein in pancreatic tissue of each group in Example 1.

[0035] Fig.15 It is a bar graph showing the relative expression levels of HO-1 protein in pancreatic tissue of each group in Example 1.

[0036] Fig.16 It is a bar graph showing the relative expression level of GPX4 protein in pancreatic tissue of each group in Example 1.

[0037] Fig.17 It is a bar graph showing the relative expression levels of ACSL4 protein in pancreatic tissues of each group in Example 1.

[0038] Fig.18 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis model group (SAP group) 24 hours after the administration of 3.5% sodium taurocholate in Example 2.

[0039] Fig.19 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (ELE+SAP group) treated with eleutheroside E 24 hours after administration of 3.5% sodium taurocholate in Example 2.

[0040] Fig. 20 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (Aca+SAP group) treated with Acanthopanax senticosus injection 24 hours after the modeling and administration of 3.5% sodium taurocholate in Example 2.

[0041] Fig.21 It is a bar graph of the pancreatic tissue pathology scoring results of each group in Example 2.

[0042] Fig. 22 It is a bar graph of the serum amylase activity results of each group in Example 2.

[0043] Fig.23 It is a bar graph of the serum lipase activity results of each group in Example 2.

[0044] Fig.24 It is a bar graph of the serum TNF-α level results of each group in Example 2.

[0045] Fig.25 It is a bar graph showing the results of serum IL-1β levels in each group in Example 2.

[0046] Fig.26It is a bar graph showing the results of serum IL-6 levels in each group in Example 2. DETAILED DESCRIPTION

[0047] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0048] Example 1

[0049] Therapeutic effect of eleutheroside E on severe acute pancreatitis induced by retrograde pancreaticobiliary injection of 3.5% sodium taurocholate in mice

[0050] 1 Materials and methods

[0051] 1.1 Research subjects and experimental reagents

[0052] Healthy male C57BL / 6 mice with a body weight of 22-25 g and an age of 8-10 weeks were selected and purchased from the Experimental Animal Center of Nantong University. The experimental animal use license number is SYXK (Su) 2020-0029, the experimental animal production license number is SCXK (Su) 2020-0009, and the experimental animal qualification certificate number is 202307833. The experimental animal breeding environment is SPF grade. According to the requirements of the experimental animal breeding environment, the breeding environment is well ventilated, the room temperature is maintained at 21-23 ° C, the relative humidity is maintained at 45%-55%, and the light and dark are alternated for 12 hours each. The experimental mice are free to eat and drink water, and 3-4 mice are raised per cage. 95% sodium taurocholate was purchased from Sigma, USA. Serum amylase and lipase enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Bioengineering Institute. Colorimetric assay kits for pancreatic tissue iron content, MDA, ROS, and GSH were purchased from Elabscience. Anti-Nrf2, Anti-HO-1, Anti-GPX4, and Anti-ASCL4 antibodies were purchased from abcam. Eleutherococcus senticosus (ELE) was purchased from China Food and Drug Inspection Institutes, [CAS number]: 39432-56-9, batch number: 111713-202105, specification: 20 mg, analytical standard, HPLC ≥ 98%.

[0053] 1.2 Experimental groups and treatments

[0054] This experiment set up sham operation group (SO); severe acute pancreatitis model (SAP) group; low-dose eleutheroside E (ELE)-treated SAP group (ELE L+SAP group); medium-dose ELE-treated SAP group (ELE M+SAP group); high-dose ELE-treated SAP group (ELEH+SAP group), a total of 5 groups, each with 10 mice. The dosage of ELE was obtained based on the results of the previous drug toxicity test.

[0055] A. SO group: The mice were anesthetized and opened in the same way as the SAP group. After opening the abdomen, the duodenum was turned and the pancreas was touched several times.

[0056] B. SAP group: The SAP model in mice was established by retrograde pancreaticobiliary injection of 3.5% sodium taurocholate. The model was established according to the method of Cheng Zhenxing et al. [

[17] Cheng Zhenxing, Tang Zhongming, Yu Weiping, Zhang Nan, Zheng Shuyun, Ou Xilong. Improvement of a method for establishing a mouse model of severe acute pancreatitis caused by gallstones [J]. Chinese Journal of Critical Care Medicine, 2016, 28(4): 308-313. doi:10.3760 / cma.j.issn.2095-4352.2016.04.004.] The specific operation was as follows: the experimental mice were fasted for 8 h before surgery and had free access to water. After the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (10 mL / kg), the abdomen was opened 0.5 cm below the xiphoid process, and then the descending part of the duodenum was lifted out of the incision with blunted ophthalmic forceps to see the pink pancreatic tissue surrounded by the duodenum. The direction of the common bile duct and its opening position in the descending part of the duodenum were observed with the naked eye. The 8-0 needle suture was passed through the common bile duct in a "U" shape at the pancreatic head close to the intestinal wall and tied with a loose single knot; the left index finger was placed under the descending part of the duodenum, and a small hole was lightly poked with the needle of an insulin syringe (31G) in the avascular area of ​​the intestinal wall opposite to the opening of the common bile duct, and then the metal needle with a catheter was inserted into the intestinal cavity through this hole. The round needle could slide into the common bile duct without damage at the approximate position of the common bile duct opening, and the feeling of emptying was obvious. The needle entered about 3 mm, tightened the single knot and tied 3 overlapping knots to fix it. After the catheter is fixed, a micro artery clamp is used to clamp the pancreatic and bile duct at the porta hepatis to prevent the pancreatic and bile duct injection fluid from flowing back to the liver. A micro pump is used to control the injection of freshly prepared 3.5% sodium taurocholate 1mL / kg, and the infusion time is maintained for 5min. After the infusion is completed, the device is retained for 5min. Bright red flake bleeding can be seen in the pancreatic tissue. Loosen the micro artery clamp, remove the fixed suture and withdraw the needle, close the intestinal wall puncture hole with 8-0 suture, and close the abdomen in two layers. After surgery, the mouse is placed in a warming blanket, and 20mL / kg of normal saline is injected subcutaneously on the back for resuscitation. This model is a classic mouse SAP model. Studies have confirmed that 3h after the injection of 3.5% sodium taurocholate into the pancreatic and bile duct, the pancreas bleeds and necroses, that is, SAP modeling is successful.

[0057] C. ELE L+SAP group: The modeling method was the same as that of the SAP group. After modeling (after injection of 3.5% sodium taurocholate into the pancreaticobiliary duct), the mice were intraperitoneally injected with ELE 15 mg / kg.

[0058] D. ELE M+SAP group: The modeling method was the same as that of the SAP group. After modeling (after injection of 3.5% sodium taurocholate into the pancreaticobiliary duct), 30 mg / kg of ELE was intraperitoneally injected.

[0059] E. ELE H+SAP group: The modeling method was the same as that of the SAP group. After modeling (after injection of 3.5% sodium taurocholate into the pancreaticobiliary duct), 60 mg / kg of ELE was intraperitoneally injected.

[0060] 24 hours after SAP modeling (3.5% sodium taurocholate injection into the pancreatic bile duct), the surviving mice in each group were killed, and blood samples and pancreatic tissue specimens of the surviving mice in each group were collected for subsequent testing.

[0061] 1.3 Comparison of survival rates among groups

[0062] 24 hours after SAP modeling (after 3.5% sodium taurocholate injection into the pancreaticobiliary duct), the number of surviving mice in each group was counted, the survival rate of each group was calculated, and the survival rates of mice in each group were compared pairwise.

[0063] Table 1 Scoring criteria for local pancreatic lesions in SAP under light microscopy

[0064]

[0065]

[0066] 1.4 Hematoxylin-eosin (HE) staining and pathological scoring of pancreatic tissue in each group

[0067] The pancreatic tissue of each group of mice was taken and fixed in 4% paraformaldehyde solution. After the tissue blocks were taken out and rinsed with running water, they were placed in 70% alcohol for 2 hours, 80% alcohol overnight, 90% alcohol for 2 hours, and 100% alcohol for 1 hour for dehydration. Then the tissue blocks were immersed in xylene, a mixture of xylene and paraffin, and paraffin for paraffin embedding. The sample wax blocks were cut into 5μm slices with a slicer and spread on a glass slide. After drying, the slices were taken out and soaked in xylene for 15 minutes for dewaxing. Then the slices were soaked in 95%, 85%, and 75% alcohol for 2 minutes. After washing with distilled water, the slices were stained in hematoxylin solution for 5 minutes, and after washing with distilled water, they were placed in 1% hydrochloric acid alcohol for 3 seconds. The slices were taken out and washed with distilled water, and then stained in eosin staining solution for 3 minutes. The slices were taken out, the liquid was wiped off, and the slices were covered on coverslips after sealing and dried at room temperature. Observed and photographed under a microscope. The pancreatic tissue was scored pathologically (edema, inflammation, and glandular cell necrosis were scored 0 to 4 points each, and hemorrhage was scored 0 to 1 point. The total score of the four items was 13 points) according to the microscopic pathological scoring method of Schmidt et al. [

[18] Schmidt J, Rattner DW, Lewandrowski K, Compton CC, Mandavilli U, Knoefel WT, Warshaw AL. A better model of acute pancreatitis for evaluating therapy [J]. Ann Surg, 1992, 215(1): 44-56. doi: 10.1097 / 00000658-199201000-00007.] (edema, inflammation, and glandular cell necrosis were scored 0 to 4 points each, and hemorrhage was scored 0 to 1 point. The total score of the four items was 13 points). See Table 1 for details.

[0068] 1.5 Determination of serum amylase and lipase activities in each group

[0069] The ELISA method was used to detect the activities of serum amylase and lipase in each group of mice, and the specific method was carried out strictly according to the instructions.

[0070] 1.6 Determination of iron content in pancreatic tissue of each group

[0071] The pancreatic tissue of each group of mice was homogenized and centrifuged to obtain the supernatant, and the procedure was carried out strictly according to the instructions of the kit.

[0072] 1.7 Determination of oxidative stress response index levels in pancreatic tissue of each group.

[0073] The pancreatic tissue of each group of mice was homogenized and centrifuged to obtain the supernatant. The levels of MDA, ROS and GSH were detected by colorimetry. The specific method was strictly followed the operating steps in the kit instructions.

[0074] 1.8 Western blot detection of the expression levels of Nrf2, HO-1, GPX4, and ACSL4 proteins in pancreatic tissue of each group of mice

[0075] The pancreatic tissue of each group of mice was added with an appropriate amount of PBS and prepared into a tissue homogenate, and then an appropriate amount of RIPA lysis buffer containing PMSF was added thereto and lysed on ice for 5 minutes. After centrifugation at 4°C and 12,000 rpm for 5 minutes, the supernatant was collected, which was the total protein extract. The protein concentration of the extract was determined using the BCA protein quantification kit according to the method in the instructions. The protein concentration was adjusted to the same level with PBS according to the test results. Polyacrylamide gel preparation, sample electrophoresis, transfer, membrane blocking and antibody incubation (including blocking, primary antibody and secondary antibody incubation) were performed respectively. In a dark room, ECL luminescent liquid A and B were mixed at a ratio of 1:1 for standby use, added dropwise to the PVDF membrane and covered with plastic wrap. The PVDF membrane was transferred into a dark box and covered with a photosensitive film for development. The developed film was scanned, and the optical density value of the target band was analyzed using grayscale analysis software, and the relative expression of Nrf2, HO-1, GPX4 and ACSL4 proteins was calculated. Relative protein expression = (target protein band grayscale - background grayscale) / (β-actin protein band grayscale - background grayscale).

[0076] 1.9 Statistical methods

[0077] SAS 8.1 statistical software was used. The differences in the means of each group were tested by one-way analysis of variance, and the least significant difference (LSD) method was used for multiple comparisons between groups. The Fisher exact probability method was used for comparison of mortality, and P < 0.05 was considered statistically significant.

[0078] 2 Results

[0079] 2.1 Comparison of 24-hour survival rate of mice in each group: There were 10 mice in each group. All mice in the SO group survived, with a survival rate of 100%; 6 mice in the SAP group survived, with a survival rate of 60%; 6 mice in the ELE L+SAP group survived, with a survival rate of 60%; 7 mice in the ELE M+SAP group survived, with a survival rate of 70%; 8 mice in the ELE H+SAP group survived, with a survival rate of 80%.

[0080] The results of hematoxylin-eosin (HE) staining of pancreatic tissue are as follows Figure 1 to Figure 5 As shown, Figure 1 This is the HE (HE×200) staining picture of the pancreatic tissue of the sham operation group (SO group) 24 hours after administration of 3.5% sodium taurocholate model in Example 1. Figure 2This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis model group (SAP group) 24 hours after administration of 3.5% sodium taurocholate in Example 1. Figure 3 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (ELE L+SAP group) treated with a small dose of eleutheroside E 24 hours after the administration of 3.5% sodium taurocholate in Example 1. Figure 4 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (ELE M+SAP group) treated with medium-dose eleutheroside E 24 hours after administration of 3.5% sodium taurocholate in Example 1. Figure 5 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (ELE H+SAP group) treated with a large dose of eleutheroside E 24 hours after the administration of 3.5% sodium taurocholate in Example 1. Figure 1 No obvious pathological changes were found in the pancreatic tissue of the SO group. Figure 2 Chinese SAP Group, Figure 3 In the ELE L+SAP group, pathological changes of varying degrees were observed, including pancreatic acinar edema, fat liquefaction, interstitial widening, enlarged interlobular spaces, neutrophil infiltration, focal or patchy pancreatic hemorrhage and necrosis, cell lysis, hemorrhage, necrosis, and lobular contour damage. Figure 4 ELE M+SAP group, Figure 5 The pathological damage degree of ELE H+SAP group was higher than that of Figure 2 The SAP group was relieved.

[0081] 2.2 Comparison of pancreatic histopathological scores of mice in each group

[0082] Pancreatic histopathological scoring Figure 6 As shown, Figure 6 is a bar graph of the pancreatic tissue pathology scoring results of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). As can be seen from the figure, the pancreatic histopathological score of the SAP group was significantly higher than that of the SO group (P<0.01), and the pancreatic histopathological scores of the ELE M+SAP group and the ELE H+SAP group were significantly lower than those of the SAP group (P<0.01). The data are shown in Table 2.

[0083] 2.3 Comparison of serum amylase activity in each group of mice

[0084] Serum amylase activity Figure 7 As shown, Figure 7 is a bar graph of the serum amylase activity results of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, BP<0.01). As can be seen from the figure, the serum amylase activity in the SAP group was significantly higher than that in the SO group (P<0.01), and the serum amylase activity in the ELE M+SAP group and the ELE H+SAP group was significantly lower than that in the SAP group (P<0.01). The data are shown in Table 2.

[0085] 2.4 Comparison of serum lipase activity in each group of mice

[0086] Serum lipase activity Figure 8 As shown, Figure 8 is a bar graph of the results of serum lipase activity in each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). As can be seen from the figure, the serum lipase activity in the SAP group was significantly higher than that in the SO group (P<0.01), and the serum lipase activity in the ELE H+SAP group was significantly lower than that in the SAP group (P<0.01). The data are shown in Table 2.

[0087] Table 2 Pancreatic histopathological scores, serum amylase activity and serum lipase activity of mice in each group and comparison

[0088]

[0089] Note: Compared with SO group, A P<0.01; compared with the SAP group, B P<0.01. SO group: sham operation group; SAP group: severe acute pancreatitis model group; ELE L+SAP group: severe acute pancreatitis group treated with low dose of eleutheroside E (15 mg / kg of eleutheroside E was intraperitoneally injected); ELE M+SAP group: severe acute pancreatitis group treated with medium dose of eleutheroside E (30 mg / kg of eleutheroside E was intraperitoneally injected); ELE H+SAP group: severe acute pancreatitis group treated with high dose of eleutheroside E (60 mg / kg of eleutheroside E was intraperitoneally injected).

[0090] As shown in Table 2, the pancreatic histopathological scores, serum amylase and lipase activity levels in the SAP model group were significantly higher than those in the sham operation group, proving that the modeling was successful. With the increase in the dose of eleutheroside E, the pancreatic histopathological scores, serum amylase and lipase activity levels in the severe acute pancreatitis group treated with high-dose eleutheroside E (60 mg / kg) were significantly lower than those in the model group. Figures 1 to 8 This indicates that Eleutherococcus senticosus glycoside E has protective and therapeutic effects on severe acute pancreatitis and can be used as a drug for the protection and treatment of severe acute pancreatitis.

[0091] 2.5 Comparison of relative expression levels of iron content in pancreatic tissue of mice in each group

[0092] Pancreatic tissue iron content Fig. 9 As shown, Fig. 9 is a bar graph of the pancreatic tissue iron content results of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). As can be seen from the figure, the iron content of pancreatic tissue in the SAP group was significantly higher than that in the SO group (P<0.01), and the iron content of pancreatic tissue in the ELE M+SAP group and the ELE H+SAP group was significantly lower than that in the SAP group (P<0.01). The data are shown in Table 3.

[0093] 2.6 Comparison of relative expression levels of oxidative stress response indicators MDA, ROS and GSH in pancreatic tissue of mice in each group

[0094] The results are as follows Figures 10-12 As shown, Fig.10 is a bar graph of the MDA content in pancreatic tissue of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). Fig.11 is a bar graph of the ROS content in pancreatic tissue of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). Fig.12 is a bar graph of the pancreatic tissue GSH content results of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). As can be seen from the figure, the relative expression levels of MDA and ROS in the pancreatic tissue of the SAP group were significantly higher than those in the SO group, while the relative expression level of GSH was significantly lower than that in the SO group (P<0.01). The relative expression levels of MDA and ROS in the pancreatic tissue of the ELE M+SAP group and the ELE H+SAP group were significantly lower than those in the SAP group, while the relative expression level of GSH in the pancreatic tissue of the ELE H+SAP group was significantly higher than that in the SAP group (P<0.01). The data are shown in Table 3.

[0095] Table 3 Comparison of iron content in pancreatic tissue and relative expression levels of oxidative stress response indicators MDA, ROS and GSH in each group of mice

[0096]

[0097] Group Number of cases Iron content MDA ROS GSH SO group 10 <![CDATA[1.03±0.07 B ]]> <![CDATA[1.07±0.09 B ]]> <![CDATA[1.02±0.09 B ]]> <![CDATA[1.08±0.10 B ]]> SAP Group 6 <![CDATA[1.91±0.20 A ]]> <![CDATA[1.72±0.14 A ]]> <![CDATA[1.84±0.13 A ]]> <![CDATA[0.66±0.09 A <!-- 10 -->]]> ELEL+SAP group 6 <![CDATA[1.81±0.19 A ]]> <![CDATA[1.65±0.14 A ]]> <![CDATA[1.79±0.24 A ]]> <![CDATA[0.71±0.10 A ]]> ELEM+SAP Group 7 <![CDATA[1.64±0.13 A,B ]]> <![CDATA[1.54±0.13 A,B ]]> <![CDATA[1.58±0.11 A,B ]]> <![CDATA[0.72±0.06 A ]]> ELEH+SAP group 8 <![CDATA[1.36±0.12 A,B ]]> <![CDATA[1.31±0.07 A,B ]]> <![CDATA[1.34±0.07 A,B ]]> <![CDATA[0.85±0.07 A,B ]]>

[0098] Note: Compared with SO group, A P<0.01; compared with the SAP group,B P<0.01. SO group: sham operation group; SAP group: severe acute pancreatitis model group; ELE L+SAP group: severe acute pancreatitis group treated with low dose of eleutheroside E (15 mg / kg of eleutheroside E was intraperitoneally injected); ELE M+SAP group: severe acute pancreatitis group treated with medium dose of eleutheroside E (30 mg / kg of eleutheroside E was intraperitoneally injected); ELE H+SAP group: severe acute pancreatitis group treated with high dose of eleutheroside E (60 mg / kg of eleutheroside E was intraperitoneally injected).

[0099] 2.7 Comparison of relative expression levels of Nrf2, HO-1, GPX4 and ACSL4 proteins in pancreatic tissue of mice in each group

[0100] The results are as follows Figures 13 to 17 As shown, Fig.13 This is a typical Western blot bar graph of Nrf2, HO-1, GPX4, and ACSL4 in pancreatic tissue of each group in Example 1. Fig.14 It is a bar graph showing the relative expression level of Nrf2 protein in pancreatic tissue of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). Fig.15 It is a bar graph showing the relative expression level of HO-1 protein in pancreatic tissue of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). Fig.16 It is a bar graph showing the relative expression level of GPX4 protein in pancreatic tissue of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01). Fig.17 is a bar graph showing the relative expression levels of ACSL4 protein in pancreatic tissue of each group in Example 1 (Note: Compared with the SO group, A P<0.01; compared with the SAP group, B P<0.01).

[0101] As can be seen from the figure, the relative expression levels of Nrf2, HO-1 and GPX4 proteins in the pancreatic tissue of the SAP group were significantly lower than those in the SO group, while the relative expression level of ACSL4 protein was significantly higher than that in the SO group (P<0.01). The relative expression levels of Nrf2, HO-1 and GPX4 proteins in the pancreatic tissue of the ELE H+SAP group were significantly higher than those in the SAP group, while the relative expression level of ACSL4 protein in the pancreatic tissue of the ELE H+SAP group was significantly lower than that in the SAP group (P<0.01), as shown in Table 4.

[0102] Table 4 Comparison of relative expression levels of ferroptosis indicators Nrf2, HO-1, GPX4 and ACSL4 proteins in pancreatic tissue of mice in each group

[0103]

[0104] Group Number of cases Nrf2 / β-actin HO-1 / β-actin GPX4 / β-actin ACSL4 / β-actin SO group 10 <![CDATA[1.00±0.06 B ]]> <![CDATA[1.08±0.08 B ]]> <![CDATA[1.05±0.07 B ]]> <![CDATA[1.12±0.10 B ]]> SAP Group 6 <![CDATA[0.65±0.10 A ]]> <![CDATA[0.46±0.09 A ]]> <![CDATA[0.56±0.08 A ]]> <![CDATA[4.48±0.37 A ]]> ELEL+SAP group 6 <![CDATA[0.71±0.12 A ]]> <![CDATA[0.50±0.06 A ]]> <![CDATA[0.61±0.07 A ]]> <![CDATA[4.24±0.40 A ]]> ELEM+SAP Group 7 <![CDATA[0.73±0.08 A ]]> <![CDATA[0.51±0.09 A ]]> <![CDATA[0.63±0.08 A ]]> <![CDATA[4.13±0.41 A ]]> ELEH+SAP group 8 <![CDATA[0.82±0.07 A,B ]]> <![CDATA[0.74±0.07 A,B ]]> <![CDATA[0.79±0.07 A,B ]]> <![CDATA[3.17±0.35 A,B ]]>

[0105] Note: Compared with SO group, A P<0.01; compared with the SAP group, B P<0.01. SO group: sham operation group; SAP group: severe acute pancreatitis model group; ELE L+SAP group: severe acute pancreatitis group treated with low dose of eleutheroside E (15 mg / kg of eleutheroside E was intraperitoneally injected); ELE M+SAP group: severe acute pancreatitis group treated with medium dose of eleutheroside E (30 mg / kg of eleutheroside E was intraperitoneally injected); ELE H+SAP group: severe acute pancreatitis group treated with high dose of eleutheroside E (60 mg / kg of eleutheroside E was intraperitoneally injected).

[0106] As shown in Tables 3 and 4, Acanthopanax senticosus glycoside E can significantly reduce the iron content in pancreatic tissue of mice, reduce the levels of oxidative stress response indicators MDA and ROS, increase the level of GSH, increase the expression levels of Nrf2, HO-1, and GPX4 proteins, and reduce the expression level of ACSL4 protein, a marker of ferroptosis. Figures 1 to 8 The results in Table 2 indicate that eleutheroside E may play a protective and therapeutic role in SAP in mice by regulating ferroptosis mediated by the Nrf2-HO-1 signaling pathway.

[0107] Example 2

[0108] Comparative study on the therapeutic effects of Acanthopanax senticosus glycoside E and Acanthopanax senticosus injection on mice with severe acute pancreatitis induced by retrograde pancreaticobiliary injection of 3.5% sodium taurocholate

[0109] 1 Materials and methods

[0110] 1.1 Research subjects and experimental reagents

[0111] Healthy male C57BL / 6 mice were selected, with a body weight of 22-25 g and an age of 8-10 weeks. They were purchased from the Experimental Animal Center of Nantong University. The experimental animal use license number is SYXK (Su) 2020-0029, the experimental animal production license number is SCXK (Su) 2020-0009, and the experimental animal qualification certificate number is 202307833. The experimental animal breeding environment is SPF grade. According to the requirements of the experimental animal breeding environment, the breeding environment is well ventilated, the room temperature is kept at 21-23 ° C, the relative humidity is 45%-55%, and the light and dark are alternated for 12 hours each. The experimental mice are free to eat and drink, and 3-4 mice / cage are raised. 95% sodium taurocholate was purchased from Sigma, USA, serum amylase and lipase enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Bioengineering Institute, and serum TNF-α, IL-1β, and IL-6 ELISA kits were purchased from Shanghai Jianglai Biotechnology Co., Ltd. Acanthopanax glycoside E (ELE) was purchased from China Food and Drug Inspection Institute, [CAS No.]: 39432-56-9, batch number: 111713-202105, specification: 20 mg, analytical standard, HPLC ≥ 98%. 0.2% Acanthopanax injection was purchased from Heilongjiang Wusulijiang Pharmaceutical Co., Ltd., national medicine standard Z23023216, specification: 250 mL / bottle, total flavonoid content 2 mg / mL.

[0112] 1.2 Experimental groups and treatments

[0113] This experiment set up a severe acute pancreatitis (SAP) group, an Acanthopanax E-treated SAP group (ELE+SAP) group, and an Acanthopanax injection (Aca)-treated SAP group (Aca+SAP), with 10 mice in each group. The dosage of ELE was the same as that of the high-dose group in Example 1, i.e., 60 mg / kg intraperitoneal injection. The dosage of Acanthopanax injection was the same as that of the previous study [

[11] Wang X, Zhou G, Liu C, Wei R, Zhu S, Xu Y, Wu M, Miao Q. Acanthopanax versus 3-Methyladenine ameliorates sodium taurocholate-induced severe acute pancreatitis by inhibiting the autophagic pathway in The dose in the study was 35 mg / kg in rats[J].MediatorsInflamm,2016,2016:8369704.doi:10.1155 / 2016 / 8369704.]. Since the subjects of this study were rats, the reference literature [

[19] Xu Shuyun, Bian Rulian, Chen Xiu (eds.). Pharmacological Experimental Methodology[M]. 2nd edition. Beijing: People's Medical Publishing House, 1992:19.], according to the body surface area method, the dose for mice was 1.44 times that for rats, so the dose of Aca for mice was 50.4 mg / kg.

[0114] A. SAP group: The SAP model of mice was established by retrograde pancreaticobiliary injection of 3.5% sodium taurocholate. The method of Example 1 was used to establish the SAP model of mice.

[0115] B. ELE+SAP group: After modeling (3.5% sodium taurocholate sodium injection into the pancreaticobiliary duct), the mice were given an intraperitoneal injection of 60 mg / kg ELE.

[0116] C. Aca+SAP group: After modeling (3.5% sodium taurocholate sodium injection into the pancreaticobiliary duct), Aca 50.4 mg / kg was injected into the tail vein.

[0117] 24 hours after SAP modeling (3.5% sodium taurocholate injection into the pancreatic bile duct), the surviving mice in each group were killed, and blood samples and pancreatic tissue specimens of the surviving mice in each group were collected for subsequent testing.

[0118] 1.3 Comparison of survival rates among groups

[0119] 24 hours after SAP modeling (3.5% sodium taurocholate injection into the pancreaticobiliary duct), the number of surviving mice in each group was counted, the survival rate of each group was calculated, and the survival rates of mice in each group were compared pairwise.

[0120] 1.4 Hematoxylin-eosin (HE) staining and pathological scoring of pancreatic tissue in each group

[0121] HE staining and pathological scoring of pancreatic tissue were performed according to the method of Example 1.

[0122] 1.5 Determination of serum amylase and lipase activities in each group

[0123] The ELISA method was used to detect the activities of serum amylase and lipase in each group of mice, and the specific method was carried out strictly according to the instructions.

[0124] 1.6 Determination of serum TNF-α, IL-1β and IL-6 levels in each group

[0125] The ELISA method was used to detect the levels of serum TNF-α, IL-1β and IL-6 in each group of mice. The specific method was carried out strictly according to the instructions.

[0126] 1.7 Statistical methods

[0127] SAS 8.1 statistical software was used. The differences in the means of each group were tested by one-way analysis of variance, and the least significant difference (LSD) method was used for multiple comparisons between groups. The comparison of mortality rates was performed using the Fisher exact probability method. P<0.05 was considered statistically significant.

[0128] 2 Results

[0129] 2.1 Comparison of 24h survival rate of mice in each group: There were 10 mice in each group. In the SAP group, 5 mice survived, with a survival rate of 50%; in the ELE+SAP group, 8 mice survived, with a survival rate of 80%; and in the Aca+SAP group, 7 mice survived, with a survival rate of 70%.

[0130] The results of hematoxylin-eosin (HE) staining of pancreatic tissue are as follows Figures 18 to 20 As shown, Fig.18 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis model group (SAP group) 24 hours after the administration of 3.5% sodium taurocholate in Example 2. Fig.19 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (ELE+SAP group) treated with eleutheroside E 24 hours after administration of 3.5% sodium taurocholate in Example 2. Fig. 20 This is a HE (HE×200) staining image of the pancreatic tissue of the severe acute pancreatitis group (Aca+SAP group) treated with Acanthopanax senticosus injection 24 hours after the modeling and administration of 3.5% sodium taurocholate in Example 2. Fig.18In the medium SAP group, pathological changes of varying degrees were observed, including pancreatic acinar edema, fat liquefaction, interstitial widening, enlarged interlobular spaces, neutrophil infiltration, focal or patchy pancreatic hemorrhage and necrosis, cell lysis, hemorrhage, necrosis, and lobular contour damage. Fig.19 ELE+SAP group, Fig. 20 The pathological damage degree of Aca+SAP group was higher than that of Fig.18 The SAP group was relieved.

[0131] 2.2 Comparison of pancreatic histopathological scores of mice in each group

[0132] Pancreatic histopathological scoring Fig.21 As shown, Fig.21 is a bar graph of the pancreatic tissue pathology scoring results of each group in Example 2 (Note: Compared with the SAP group, A P<0.01.). As can be seen from the figure, the pancreatic histopathological scores of the ELE+SAP group and the Aca+SAP group were significantly lower than those of the SAP group (P<0.01), and there was no significant difference between the ELE+SAP group and the Aca+SAP group (P>0.05). The data are shown in Table 5:

[0133] Table 5 Pancreatic histopathological scores, serum amylase activity and serum lipase activity of mice in each group and comparison

[0134]

[0135] Note: Compared with the SAP group, A P<0.01. SAP group: severe acute pancreatitis model group; ELE+SAP group: severe acute pancreatitis group treated with Acanthopanax senticosus glycoside E (administered with 60 mg / kg Acanthopanax senticosus glycoside E intraperitoneal injection); Aca+SAP group: severe acute pancreatitis group treated with Acanthopanax senticosus injection (administered with 50.4 mg / kg Acanthopanax senticosus injection via tail vein injection).

[0136] 2.3 Comparison of serum amylase activity in each group of mice

[0137] Serum amylase activity Fig. 22 As shown, Fig. 22 is a bar graph of the serum amylase activity results of each group in Example 2 (Note: Compared with the SAP group, A P<0.01.). As can be seen from the figure, the serum amylase activities of the ELE+SAP group and the Aca+SAP group were significantly lower than those of the SAP group (P<0.01), while there was no significant difference between the ELE+SAP group and the Aca+SAP group (P>0.05); the data are shown in Table 5.

[0138] 2.4 Comparison of serum lipase activity in each group of mice

[0139] Serum lipase activity Fig.23 As shown, Fig.23 is a bar graph of the serum lipase activity results of each group in Example 2 (Note: Compared with the SAP group, A P<0.01.). As can be seen from the figure, the serum lipase activity of the ELE+SAP group and the Aca+SAP group was significantly lower than that of the SAP group (P<0.01), while there was no significant difference between the ELE+SAP group and the Aca+SAP group (P>0.05). The data are shown in Table 5.

[0140] As shown in Table 5, the pancreatic histopathological scores, serum amylase and lipase activity levels in the ELE+SAP group and the Aca+SAP group were significantly lower than those in the SAP group. Figures 18 to 20 The results showed that both eleutheroside E and Acanthopanax injection had protective and therapeutic effects on severe acute pancreatitis and could be used as drugs for the protection and treatment of severe acute pancreatitis. There were no significant differences in the pancreatic tissue pathology scores and serum amylase and lipase activity levels between the ELE+SAP group and the Aca+SAP group, indicating that the anti-severe acute pancreatitis effect of eleutheroside E was equivalent to that of Acanthopanax injection. Moreover, eleutheroside E as a Chinese medicine monomer had obvious advantages over Acanthopanax injection (Acanthopanax crude extract, the main component of which was flavonoid compounds).

[0141] As a crude extract of traditional Chinese medicine, Acanthopanax injection has been reported to have many adverse reactions in clinical application, such as anaphylactic shock, systemic reaction, skin and appendage damage, respiratory system reaction, digestive system reaction, cardiovascular system reaction, visual impairment, etc. References [

[20] Li Yi, Gui Wandi. Literature analysis of 78 cases of allergic reactions caused by Acanthopanax injection [J]. Central South Pharmacy, 2016, 14(12): 1398-1400. doi:10.7539 / j.issn.1672-2981.2016.12.028.

[21] Zhong Qiaoni, Chen Xuanying. Clinical rational application of traditional Chinese medicine from the perspective of adverse events of traditional Chinese medicine [J]. Drug Evaluation, 2012, 9(20): 26-30. doi:10.3969 / j.issn.1672-2981.2016.12.028. 72-2809.2012.20.006.

[22] Zheng Huojun. Epidemiological analysis of adverse reactions of Acanthopanax senticosus injection[J]. China Pharmaceutical Industry, 2013, 22(1):34-35.doi:10.3969 / j.issn.1006-4931.2013.01.017.

[23] Wang Huifang, Wang Weiping, Luan Jiajie, Jiang Jia, Xu Zhenyu, Zuo Jian, Zhou Dexi. Analysis of influencing factors of 43 cases of adverse events of Acanthopanax senticosus injection[J]. Chinese Licensed Pharmacists, 2016, 13(4):43-47.doi:10.3969 / j.issn.1672-5433.2016.04.009.], and the further application of Acanthopanax senticosus glycoside E as a monomer of traditional Chinese medicine in clinical practice can avoid the occurrence of these adverse reactions.

[0142] 2.5 Comparison of serum TNF-α, IL-1β and IL-6 levels in each group of mice

[0143] The results are as follows Figure 24 to Figure 26 As shown, Fig.24 is a bar graph of the serum TNF-α levels of each group in Example 2 (Note: Compared with the SAP group, A P<0.01.). Fig.25 is a bar graph of the serum IL-1β levels of each group in Example 2 (Note: Compared with the SAP group, A P<0.01.). Fig.26 is a bar graph of the serum IL-6 levels of each group in Example 2 (Note: Compared with the SAP group, A P<0.01.). As can be seen from the figure, the levels of serum TNF-α, IL-1β and IL-6 in the ELE+SAP group and the Aca+SAP group were significantly lower than those in the SAP group (P<0.01), while there was no significant difference between the ELE+SAP group and the Aca+SAP group (P>0.05). The data are shown in Table 6:

[0144] Table 6 Comparison of serum TNF-α, IL-1β and IL-6 levels in mice of each group

[0145] Group Number of cases TNF-α(ng / L) IL-1β (ng / L) IL-6 (ng / L) SAP Group 5 363.08±42.26 157.65±15.79 562.8±39.08 ELE+SAP group 8 <![CDATA[257.08±37.85 A ]]> <![CDATA[108.20±12.86 A ]]> <![CDATA[375.31±41.95 A ]]> Aca+SAP group 7 <![CDATA[264.24±42.07 A ]]> <![CDATA[116.56±20.73 A ]]> <![CDATA[394.20±55.36 A ]]>

[0146] Note: Compared with the SAP group, A P<0.01. SAP group: severe acute pancreatitis model group; ELE+SAP group: severe acute pancreatitis group treated with Acanthopanax senticosus glycoside E (given 60 mg / kg of Acanthopanax senticosus glycoside E by intraperitoneal injection); Aca+SAP group: severe acute pancreatitis group treated with Acanthopanax senticosus injection (given 50.4 mg / kg of Acanthopanax senticosus injection by tail vein injection).

[0147] As shown in Table 6, the serum TNF-α, IL-1β and IL-6 levels in the ELE+SAP group and the Aca+SAP group were significantly lower than those in the SAP group. Figure 24 to Figure 26 This indicates that both eleutheroside E and eleuthero injection have the effect of inhibiting inflammation in severe acute pancreatitis. There was no significant difference in the levels of TNF-α, IL-1β and IL-6 between the ELE+SAP group and the Aca+SAP group, indicating that eleutheroside E and eleuthero injection have the same effect of inhibiting inflammation.

[0148] Combined with Example 1 and Example 2, it is shown that eleutheroside E has the effect of protecting and treating severe acute pancreatitis, and can be used as a drug for protecting and treating severe acute pancreatitis.

[0149] Acanthopanax injection (Acanthopanax crude extract) is currently approved by the National Drug Administration for clinical use based on the total flavonoid content. Other ingredients are not specified, and the active ingredient is the total flavonoids. Reference: Announcement of the National Drug Administration on the revision of the instructions for Acanthopanax injection (No. 62, 2018). In the literature [

[10] Zhu Jianping, Li Meifang, Chen Bo, Deng Xiujian. Effects of Acanthopanax senticosus on inflammatory mediators in rats with acute pancreatitis model [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2008, 13(7): 744-746. doi:10.3969 / j.issn.1009-2501.2008.07.005.], Acanthopanax senticosus injection is described as follows: 20g contains 100mg total flavonoids, produced by Heilongjiang Wandashan Pharmaceutical Factory, batch number: Z23030810, the active ingredient is total flavonoids, other ingredients are not described, which is different from the Acanthopanax glycoside E component of this application.

[0150] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Use of eleutheroside E or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating severe acute pancreatitis.

2. Use of eleutheroside E or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing or treating severe acute pancreatitis, characterized in that: The drug for preventing or treating severe acute pancreatitis refers to eleutheroside E or its pharmaceutically acceptable salt as a single active ingredient.

3. Use of eleutheroside E or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing or treating severe acute pancreatitis, characterized in that: The weight content of the eleutheroside E as a single active ingredient is 0.1% to 99.9%.

4. Use of eleutheroside E or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing or treating severe acute pancreatitis, characterized in that: The dosage form of the drug is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation.

5. Use of eleutheroside E or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing or treating severe acute pancreatitis, characterized in that: The drug can be administered orally or by injection.

Images