CN113318119A

CN113318119A - Application of Hedera aponin C in preparation of medicine for preventing and treating acute lung injury

Info

Publication number: CN113318119A
Application number: CN202110693547.4A
Authority: CN
Inventors: 高红伟; 杨世林
Original assignee: Guangxi Xinhai Pharmaceutical Technology Co Ltd
Current assignee: Guangxi Xinhai Pharmaceutical Technology Co Ltd
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2021-08-31

Abstract

The invention discloses an application of Hedera aponin C in preparing a medicament for treating and preventing acute lung injury, wherein the structural formula of the Hedera aponin C is shown as a formula (I):

the invention has certain protection effect on mouse ALI caused by LPS, and the effect of the invention not only reduces the release of TNF-alpha, IL-6 and IL-1 beta and the accumulation of neutrophils, lymphocytes and leukocytes in lung tissues, but also inhibits PIP2 signal path, NF-kappa B signal path and NL signal pathThe activation of RP3 inflammasome is involved.

Description

Application of Hedera aponin C in preparation of medicine for preventing and treating acute lung injury

Technical Field

The invention relates to the technical field of medicines. More specifically, the invention relates to an application of Hederaponin C in preparing a medicament for treating and preventing acute lung injury.

Background

Acute Lung Injury (ALI) is a common clinical critical disease with high morbidity and mortality, is caused by various direct or indirect pathogenic factors such as pathogenic microorganism infection and is a clinical syndrome mainly manifested by acute progressive respiratory distress and refractory hypoxemia. Acute inflammation of lung interstitium, such as neutrophil infiltration and proinflammatory cell aggregation in ALI lung, can block gas exchange in lung. Consequently, ALI can further progress to more severe Acute Respiratory Distress Syndrome (ARDS) if left untreated, with mortality rates up to 40%. Endotoxin LPS infection can trigger a cytokine storm leading to severe ALI, acute respiratory distress, and even multiple organ failure until death. However, there are currently no specific drugs for ALI treatment. The glucocorticoid and mechanical ventilation are used for treatment, but the glucocorticoid has good effect, but has great toxic and side effects, and can generate heavy after-effect after treatment. Hederanaponin C belongs to oleanane type triterpene saponin, and the invention mainly discusses the protective effect of Hederanaponin C on lung tissues in an ALI model.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

It is still another object of the present invention to provide a use of Hederaponin C in preparing a medicament for preventing and treating acute lung injury, which has a certain protective effect on LPS-induced mouse ALI, and the effect is not only related to the inhibition of activation of PIP2 signaling pathway, NF-. kappa.B signaling pathway and NLRP3 inflammasome, but also to the reduction of neutrophil, lymphocyte and leukocyte accumulation in lung tissue by reducing the release of TNF-. alpha.IL-6 and IL-1. beta..

To achieve these objects and other advantages in accordance with the present invention, there is provided a use of Hedera aponin C for the preparation of a medicament for the treatment of acute lung injury, the Hedera aponin C having the structural formula (I):

preferably, the acute lung injury is lung tissue injury, inflammation and infection caused by upper respiratory infection, chronic bronchitis, pulmonary edema, pneumonia, lung abscess, and by cardiac and cerebral ischemia and organ transplantation.

Preferably, the acute lung injury is caused by an influenza virus infection, a bacterial infection and/or a fungal infection.

Preferably, the medicament contains a therapeutically effective amount of Hedera aponin C and a pharmaceutically acceptable carrier.

Preferably, the medicament is formulated into a pharmaceutically acceptable dosage form.

Preferably, the agent down-regulates the level of inflammatory factors in a subject with acute lung injury, inhibiting activation of the PIP2 signaling pathway, the NF- κ B signaling pathway, and the NLRP3 inflammasome.

Preferably, Hederaponin C is administered at a dose of not less than 5mg/kg d.

The invention at least comprises the following beneficial effects:

the research result of the invention shows that Hederaponin C protects LPS to induce the death of acute lung injury mice, inhibits the increase of RL and Re and the decrease of Cydn caused by LPS, reduces the increase of lung index, leukocyte, neutrophil and lymphocyte caused by LPS, reduces the increase of serum, BLAF and lung tissue inflammatory factors TNF-alpha, IL-6 and IL-1 beta caused by LPS, and inhibits the activation of NF-kappa B, NLRP3 inflammatory corpuscles and PIP2 signal channels in ALI lung tissues stimulated by LPS.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

Fig. 1 is a graph of the effect of hederaponin C of the present invention on survival of LPS-induced acute lung injury mice (n ═ 10);

FIG. 2 is a graph showing the effect of Hederaponin C of the present invention on the lung function-related index of LPS-induced acute lung injury mice;

FIG. 3 is a graph of the effect of Hederaponin C of the present invention on LPS-induced acute lung injury mouse lung index, leukocytes, neutrophils, and lymphocytes;

FIG. 4 is an assessment of kidney histopathology by H & E staining of LPS-induced acute lung injury mice 24 hours after Hedera aponin C of the present invention (200X);

FIG. 5 is a graph of the effect of Hederaponin C of the present invention on LPS-induced inflammatory response in mice with acute lung injury;

FIG. 6 is a graph showing the effect of Hederaponin C of the present invention on LPS-induced acute lung injury mouse tissue-associated protein.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

1 Material

1.1 Experimental animals

SPF-grade healthy BALB/C mice, with a weight of 20-22g and a week of 7-8, are male and purchased from Schlekschada laboratory animals Co., Ltd, Hunan, all the laboratory animals are raised in a controlled environment at a room temperature of 18-24 ℃ and a humidity of 40-50%, the animals eat and drink water freely during the experiment, and the circadian rhythm is normal.

1.2 major drugs and reagents

Hederapoponin C (offered by Yangshilin research platform of pharmaceutical institute of Guangxi university of traditional Chinese medicine); dexamethasone sodium phosphate injection (Henan hong pharmaceutical Co., Ltd., Chinese medicine standard H41020330); lipopolysaccharide (Shanghai Biyuntian Biotechnology Co., Ltd.); paraformaldehyde (Chinese medicine analytically pure); 0.9% sodium chloride injection (Sichuan Kelun pharmaceutical Co., Ltd.); tumor necrosis factor (TNF-alpha) kit, interleukin (IL-6, IL-1 beta) kit (Invitrogen, USA); PLC gamma 2(#3872), IP3 Receptor1(#8568), DAG Lipa alpha (#13626), PKC alpha (#2056S), p-PKC alpha (#9375S), p65(#8242T), p-p65(#3033), poly (ADP-ribose), polymerase (PARP) (#9532), NLRP3(#15101), ASC (#13833), IL-1 beta (#31202), cleared-Caspase-1 (#89332), cleared-IL-1 beta (#63124), GAPDH 5174) (Cell Signaling Technology, USA) Caspase-1(ab1872) (Abcam, USA); a quantitative nebulizer for pulmonary administration (Shanghai Yuyan scientific instruments, Inc.); an AniRes2005 animal lung function analysis system (beijing beiland bokejic, ltd.; electronic balance (mettler. toledo instruments shanghai, ltd.)), digital pipette gun (german Eppendorf), high-speed centrifuge (german Eppendorf), inverted microscope (german Leica), 4-degree refrigerator, -20-degree refrigerator, -80-degree refrigerator (macadama products), microplate reader (U.S. Bio-Tek), veterinary full-automatic blood cell analyzer (shenzhen meirui biomedical electronics ltd., ltd.), constant temperature culture shaker (shanghai-heng scientific instruments ltd.), and digital display constant temperature water bath (heshu hua electrical appliances ltd., ltd.).

2 method

2.1 animal modeling and drug delivery

168 SPF-grade BALB/C mice are male, have the weight of 20-22g, are adaptively bred for 3 days, are fed with SPF-grade feed and purified water at the room temperature of 18-24 ℃ and the relative humidity of 40-50 percent in a laboratory and are freely eaten. 12h before modeling, only water is supplied and no feed is supplied, the mice are divided into groups (the weight distribution of each group is uniform), and an acute lung injury mouse model is established according to the LPS dose of 4mg/kg or the lethal dose of 15mg/kg by tracheal instillation (i.t). Injecting 0.4% pentobarbital sodium into the abdominal cavity of a mouse according to the dose of 0.18mL/10g, after the mouse is anesthetized, lying the mouse on the back on an operating table, opening the oral cavity of the mouse by using a mouse mouth gag, injecting LPS (the same amount of sterile physiological saline is injected into a control group) into the lung from the opening of epiglottis by using a micro atomizer, completing the establishment of the model, and after the model is awakened, adding water and feed.

The following 3 experiments were performed according to the above molding and administration method:

(1) modeling with LPS (15mg/kg) with lethal dose, randomly dividing into 6 groups according to 10 mice as one group, respectively setting blank control group, LPS model group (15mg/kg), Hedera aponin C (5, 10, 20mg/kg) group, and positive drug group (dexamethasone, DEX, 5mg/kg), injecting Hedera aponin C (5, 10, 20mg/kg) into tail vein at 0h, 12h, 24h, 48h, 72h after modeling, injecting equal amount of sterile physiological saline into mice in the blank control group by tail vein injection, modeling DEX group according to the dose, and dexamethasone injection (5mg/kg) is injected into the body of the mouse for 1 time in the mode of intraperitoneal injection 0h after the model is made, the same amount of physiological saline was injected into mice in the form of intraperitoneal injection for 12h, 24h, 48h and 72 h. Observing the survival condition of the mouse within 156 h;

(2) modeling is carried out by using 4mg/kg LPS, 6 mice are taken as one group and are randomly divided into 6 groups, the 6 groups are respectively set as a blank control group, an LPS model group (4mg/kg), a Hedera aponin C (5, 10 and 20mg/kg) group and a positive drug group (dexamethasone, DEX and 5mg/kg), Hedera aponin C (5, 10 and 20mg/kg) is given by tail vein injection 0h and 12h after modeling, the blank control group is injected with equal amount of sterile physiological saline into the bodies of the mice by tail vein injection, the DEX group is modeled according to the dose, and equal amount of physiological saline is injected into the bodies of the mice by intraperitoneal injection 12h after dexamethasone injection (5mg/kg) is given 1 time by intraperitoneal injection 0h after modeling. After 24h of molding, detecting the lung respiratory function of the mouse by using a respirator;

(3) modeling is carried out by using 4mg/kg LPS, 12 mice are taken as one group and are randomly divided into 6 groups, the 6 groups are respectively set as a blank control group, an LPS model group (4mg/kg), a Hedera aponin C (5, 10 and 20mg/kg) group and a positive drug group (dexamethasone, DEX and 5mg/kg), Hedera aponin C (5, 10 and 20mg/kg) is given by tail vein injection 0h and 12h after modeling, the blank control group is injected with equal amount of sterile physiological saline into the bodies of the mice by tail vein injection, the DEX group is modeled according to the dose, and equal amount of physiological saline is injected into the bodies of the mice by intraperitoneal injection 12h after dexamethasone injection (5mg/kg) is given 1 time by intraperitoneal injection 0h after modeling. After modeling for 24 hours, after blood is taken from modeled eyeballs, the change trend of leukocytes and neutrophils is detected by the blood routine, the expression levels of cytokines TNF-alpha, IL-6 and IL-1 beta in serum, alveolar lavage fluid and lung tissues are detected by an ELISA method, the pathological changes of the lung are detected by an HE staining method, and the expression of lung tissue-related proteins is detected by a Western blotting method.

2.2 detection of indicators

2.2.1 mouse survival Rate

After modeling by tracheal instillation (i.t) of LPS (15mg/kg), survival of mice was observed and recorded every 12 h.

2.2.2 detection of Lung respiratory function in mice

After LPS (4mg/kg) tracheal instillation (i.t) modeling, a mouse lung respiratory function analyzer was used 24h later. Injecting 0.4% sodium pentobarbital into a mouse body in an intraperitoneal injection mode according to the dose of 0.18mL/10g, performing trachea intubation after anesthesia, connecting a lung function analyzer of the mouse, and detecting the lung Resistance (RL), the respiratory resistance (Re) and the dynamic lung compliance (Cydn) of the mouse.

2.2.3 routine blood analysis

After 24 hours of the model fabrication by tracheal instillation (i.t) of LPS (4mg/kg), the mouse eyeballs were bled, 40. mu.L of blood was taken, and the numbers of lymphocytes (Lym), neutrophils (Neu) and leukocytes (WBC) in the blood were measured by a Meyer's blood analyzer.

2.2.4 detection of serum TNF-alpha, IL-6 and IL-1 beta

LPS (4mg/kg) is instilled by trachea (i.t) for 24h for molding, mouse eyeball whole blood is taken, when supernatant is separated out, the mouse eyeball whole blood is placed in a centrifuge (4 ℃, 3000rpm/min) for centrifugation for 15min, the supernatant is taken, and the level of IL-6, TNF-alpha and IL-1 beta inflammatory factors in serum is detected strictly according to ELISA operation instructions.

2.2.5 detection of inflammatory factors in alveolar lavage fluid (BALF)

Selecting 6 pieces of the above raw materials in sequence, killing after blood collection, rapidly opening thoracic cavity and exposing neck trachea, injecting 0.3mL of physiological saline (ice) into trachea with 1mL injector, repeatedly withdrawing and injecting for 3-5 times, repeating for 3 times, mixing 3 times of alveolar lavage fluid, and placing on ice. Centrifugation was carried out at 4 ℃ and 1600rpm/min for 15min, the supernatant was removed, and the levels of IL-6, TNF- α, and IL-1 β inflammatory factors in alveolar lavage fluid were determined strictly according to ELISA protocol.

2.2.6 Lung tissue index determination

(1) Lung index: the whole lung was removed, washed in physiological saline, then blotted dry with filter paper and weighed. (lung index ═ lung mass/mouse mass 100%)

(2) Taking the lower lobe of the right lung, fixing (soaking the right lung in 4% paraformaldehyde), after 24 hours, removing old paraformaldehyde, changing into fresh 4% paraformaldehyde, then sequentially dehydrating, transparentizing, embedding in paraffin, slicing, staining with eosin-hematoxylin (H & E), and observing by a microscope to obtain the morphological change of the right lung tissue of the mouse.

(3) Washing the rest lung with cold normal saline, drying surface water with filter paper, precisely weighing, homogenizing with tissue grinder, storing in-80 deg.C refrigerator, and detecting IL-6, IL-1 beta, and TNF-alpha contents; western blotting is used for detecting the expression of the proteins related to the signal paths such as NF-kappa B, PIP2, NLRP3 and the like.

2.2.7 statistical analysis

Statistical analysis was performed using GraphPad Prism 6.0 software, and comparisons between groups were performed by one-way anova. P <0.05 is a significant event.

3 results of the experiment

3.1 Effect of Hedera aponin C on the protection of LPS-induced death in mice with acute Lung injury

As shown in FIG. 1, the blank Control group was indicated by Control, the LPS model group (15mg/kg) was indicated by LPS, the Hedera aponin C (5, 10, 20mg/kg) group was indicated by LPS + Hedera aponin C (5mg/kg), LPS + Hedera aponin C (10mg/kg), LPS + Hedera aponin C (20mg/kg), and the positive drug group (dexamethasone, DEX, 5mg/kg) was indicated by LPS + DEX (5mg/kg), respectively. At 60h after LPS modeling treatment, the mortality rate of an LPS group is 20%, the mortality rate of a Hedera aponin C (5mg/kg) group is 10%, the mortality rate of the Hedera aponin C (10mg/kg) group is 10%, and the mortality rate of a DEX group is 10%; at 72h, the mortality rate of LPS group is 40%, and the mortality rate of Hedera aponin C (5mg/kg) group is 20%; the mortality rate of LPS group is 50% at 84h, and the mortality rate of Hederaperonin C (10mg/kg) group is 20% at 96 h; at 120h, the mortality rate of LPS group is 70%, the mortality rate of Hedera aponin C (20mg/kg) group is 20%, and the mortality rate of DEX group is 20%; at 132h, the mortality rate of the Hedera aponin C (5mg/kg) group is 30%, the mortality rate of the Hedera aponin C (10mg/kg) group is 30%, and the mortality rate of the Hedera aponin C (20mg/kg) group is 30%; at 144h, the mortality rate of LPS group was 90%, the mortality rate of Hedera aponin C (5mg/kg) group was 50%, and the mortality rate of Hedera aponin C (10mg/kg) group was 40%; at 156h, the mortality rate was 100% for LPS group, 60% for Hedera aponin C (5mg/kg) group and 30% for DEX group. The results indicate that Hederaponin C protects LPS from inducing death in mice with acute lung injury.

3.2 Effect of Hedera aponin C on LPS-induced acute Lung injury mouse respiratory function

As shown in fig. 2, the blank Control group was indicated by Control, the LPS model group (15mg/kg) was indicated by LPS, the hederaponin C (5, 10, 20mg/kg) group was indicated by LPS + hederaponin C (5mg/kg), LPS + hederaponin C (10mg/kg), LPS + hederaponin C (20mg/kg), respectively, the positive drug group (dexamethasone, DEX, 5mg/kg) was indicated by LPS + DEX (5mg/kg), (a) mouse lung Resistance (RL); (B) mouse respiratory resistance (Re); (C) dynamic lung compliance (Cydn) in mice; compared with the blank control group, the # p is less than 0.001; p <0.05, p <0.01, p <0.001(n 6) compared to LPS group. Compared with a blank control group, the lung Resistance (RL) and the respiratory resistance (Re) of the LPS group are obviously increased, the dynamic lung compliance (Cydn) is obviously reduced, and compared with an LPS model group, Hederaponin C (5, 10 and 20mg/kg) and DEX groups RL and Re are obviously reduced, and Cdyn is obviously increased (p is less than 0.05, p is less than 0.01, and p is less than 0.001).

The results show that LPS can cause the increase of RL and Re and the decrease of Cydn of the respiratory function of mice, while Hederaponin C can inhibit the decrease of RL, Re and Cydn caused by LPS, and the effect of Hederaponin C (20mg/kg) on the inhibition of Re and the increase of Cydn is better than that of Dex group.

3.3 Hedera aponin C improves LPS-induced mouse ALI

During the process of LPS induction of mouse ALI, leukocytes, neutrophils and lymphocytes rapidly enter the alveolar space after being activated by inflammatory factors and pathogens. As shown in fig. 3-4, the blank Control group was represented by Control, the LPS model group (15mg/kg) was represented by LPS, the hederaponin C (5, 10, 20mg/kg) group was represented by LPS + hederaponin C (5mg/kg), LPS + hederaponin C (10mg/kg), LPS + hederaponin C (20mg/kg), respectively, and the positive drug group (dexamethasone, DEX, 5mg/kg) was represented by LPS + DEX (5mg/kg), wherein in fig. 3, (a) lung index (═ lung mass/mouse mass × 100%) (B) White Blood Cell (WBC) number in blood of mice; (C) the number of neutrophils (neutrophiles) (D) lymphocytes (lymphocytes) in the blood of the mice, in FIG. 4, # p <0.001 compared to the blank control group; p <0.05, p <0.01, p <0.001 (n-12) compared to LPS group. As shown in fig. 3A-D, LPS increased pulmonary index, white blood cells, neutrophils and lymphocytes in the blood of mice compared to the blank control group, whereas DEX, heierasporin C decreased pulmonary index, white blood cells, neutrophils and lymphocytes levels ([ p ] <0.05, [ p ] <0.01, [ p ] < 0.001). Furthermore, as shown in fig. 4, H & E staining results showed that LPS caused destruction of the alveolar normal structure of lung tissue, alveolar interstitial exudation with thickened alveolar septa, partial alveolar structural disturbance, and unclear borders, compared to the blank control group, whereas DEX, hederaponin C treatment reversed LPS-induced lung tissue damage.

The results show that LPS can cause the increase of the pulmonary index, the level of white blood cells, the level of neutrophils and the level of lymphocytes of BALB/C mice, and Hederaponin C can reduce the pulmonary index, the level of white blood cells, the level of neutrophils and the level of lymphocytes.

3.4 Effect of Hedera aponin C on LPS-induced ALI mouse inflammatory factor

As shown in FIG. 5, the blank Control group was indicated by Control, the LPS model group (15mg/kg) was indicated by LPS, the Hedera aponin C (5, 10, 20mg/kg) group was indicated by LPS + Hedera aponin C (5mg/kg), LPS + Hedera aponin C (10mg/kg), LPS + Hedera aponin C (20mg/kg), and the positive drug group (dexamethasone, DEX, 5mg/kg) was indicated by LPS + DEX (5mg/kg), respectively, wherein (A) the TNF-. alpha.release level in the serum of mice; (B) levels of IL-6 release in mouse serum; (C) levels of IL-1 β release in mouse serum; (D) levels of TNF- α release in mouse alveolar lavage (BLAF); (E) levels of IL-6 release in mouse alveolar lavage (BLAF); (F) levels of IL-1 β release in mouse alveolar lavage fluid; (G) levels of TNF- α release in mouse lung tissue; (H) levels of IL-6 release in mouse lung tissue; (I) levels of IL-1 β release in mouse lung tissue; compared with the blank control group, the # p is less than 0.001; p <0.01, p <0.001 (n-12) compared to LPS group. LPS-induced lung inflammation is a typical form of ALI, and causes the release of lung inflammatory cytokines such as TNF- α, IL-1 β, and IL-6. To detect the effect of Hedera aponin C on the ALI inflammatory response, serum, BALF and lung tissue were collected and the level of inflammatory response was detected using an ELISA kit. The results showed that the expression of IL-6, IL-1 β, TNF- α was significantly higher in the LPS group BLAF, serum, and lung tissue than in the control group 24h after LPS treatment, indicating that the ALI model induced by LPS (4mg/kg, 24h) was successful, whereas each dose of DEX, Hederaponin C inhibited the levels of TNF- α, IL-6, and IL-1 β in serum, TNF- α, IL-6, and IL-1 β in BALF, and TNF- α, IL-1 β, and IL-6 in lung tissue (P <0.05, P <0.01, P < 0.001).

The results show that LPS can cause the increase of the levels of BALB/C mouse serum, BLAF and lung tissue TNF-alpha, IL-6 and IL-1 beta, and DEX and Hedera aponin C reduce the increase of the inflammatory factors and have the function of regulating the ALI inflammation progression.

3.5 Effect of Hedera aponin C on LPS Induction of ALI mouse Lung tissue-associated proteins

ALI is a highly fatal lung disease that can lead to edema, hypoxia and respiratory failure. Recent studies have shown that NF-. kappa.B has an irreplaceable effect on the development of ALI. LPS activates the NF-. kappa.B signaling pathway in lung tissue, and subsequently NF-. kappa.B is activated, resulting in the release of inflammatory cytokines and chemokines such as IL-1. beta., IL-6 and TNF-. alpha.in lung tissue, the production of which plays a critical role in the development of ALI. As shown in FIG. 6, the blank Control group was indicated by Control, the LPS model group (15mg/kg) was indicated by LPS, the Hedera aponin C (5, 10, 20mg/kg) group was indicated by LPS + Hedera aponin C (5mg/kg), LPS + Hedera aponin C (10mg/kg), LPS + Hedera aponin C (20mg/kg), and the positive drug group (dexamethasone, DEX, 5mg/kg) was indicated by LPS + DEX (5mg/kg), respectively, wherein (A) TAK1/P-TAK1, PKC α/P-PKC α and P65/P-65 protein expression in lung tissue was detected by Western blotting; (B) detecting the expression of NLRP3/ASC/pro-Caspa/Caspa1 p10/IL-1 beta p17 protein in lung tissues by using a Western blotting method; (C) detecting PIP2/PLC gamma 2/DAG/IP3 protein expression in lung tissue by using a Western blotting method, (D) carrying out statistical analysis on PIP2/PLC gamma 2/DAG/IP3 protein, wherein histograms from left to right of any experimental group are PIP2, PLC gamma 2, DAG and IP3 respectively; compared with a blank control group, the # p is less than 0.05; p <0.05, p <0.01, p <0.001 (n-12) compared to LPS group. As shown in fig. 6A, lung tissues in LPS group showed significantly increased phosphorylated protein expression of TAK1, PKC α and P65, with unchanged total protein expression, while DEX group and heierasporin C group significantly reduced phosphorylated protein expression of TAK1, PKC α and P65, without changing total protein expression.

Following activation of NLRP3 inflammasome, overexpression of clear-caspase-1 and mature IL-1 β results in neutrophil recruitment, which in turn promotes the development of ALI. Therefore, inhibition of activation of NLRP3 inflammasome may provide an alternative strategy for the treatment of ALI. As shown in FIG. 6B, LPS stimulated activation of the inflammatory bodies of NLRP3 in lung tissue compared to the blank control group, with NLRP3, clear-caspase-1 and mature IL-1 β overexpression, and NLRP3, clear-caspase-1 and clear-IL-1 β expression in DEX and Hederasponin C treated lung tissue significantly decreased compared to the LPS group. To further explore the mechanism of activation of inflammatory bodies of NF-. kappa.B and NLRP3 in ALI, we examined the expression of PIP2 signal pathway-related proteins in lung tissues by Western blotting method, as shown in FIGS. 6C-D, the expression of PIP2, DAG and IP3 proteins was up-regulated and PLC. gamma.2 protein expression was down-regulated under LPS stimulation, while the DEX group and Hederaponin C group significantly reversed the expression of these proteins, reducing the activation of PIP2 signal pathway.

Taken together, it was shown that NF- κ B, NLRP3 inflammasome and activation of PIP2 signaling pathways in LPS stimulated ALI lung tissue, whereas DEX and heierasporin C had inhibitory activity of these signaling pathways.

Blank control group, LPS model group (15mg/kg), Hedera aponin C (5, 10, 20mg/kg), and positive drug group (dexamethasone, DEX, 5mg/kg)

Discussion 4

ALI is a disease with loss of lung function and high mortality, caused by an uncontrolled inflammatory response, leading to hypoxic respiratory insufficiency, with severe cases with Acute Respiratory Distress Syndrome (ARDS). Studies have shown that the pathogenesis of ALI is summarized as early alveolar inflammation with lung injury. The study simulates an ALI model by adopting a method of tracheal instillation of LPS, and researches on the protective effect and mechanism of B5 on ALI. The death protection experiment result shows that Hedera aponin C can improve the survival rate of ALI mice. The lung function detection result shows that Hederaponin C has a relieving effect on the respiratory function of the ALI mice. In addition, the lung tissue boundary of the LPS group is not clear, the alveolar structure is damaged, a large amount of inflammatory infiltration is generated, and the lung injury caused by LPS is obviously relieved by the Hederaperonin C group.

Under the action of pathogenic microorganisms, target cells in the lung are activated, and the cells are promoted to secrete inflammatory cytokines such as TNF-alpha, IL-6, IL-1 beta and the like, so that endothelial cells are activated, and pulmonary edema is caused. TNF-alpha levels are elevated in serum, BALF and lung tissue of ALI patients, and TNF-alpha content is directly related to lung injury degree. In addition, ALI patients have higher IL-6 levels in serum, BALF and lung tissue, poor prognosis and high mortality. IL-6 can activate pro-inflammatory responses, with higher levels of IL-6 indicating a greater risk of ALI death. Macrophages release the active cytokine IL-1 β, and IL-1 β release is increased in the serum, BALF and lung tissues of ALI patients. It has been found that a continuous increase in IL-1 β levels is associated with a poor prognosis. In addition, neutrophils, lymphocytes and leukocytes play irreplaceable roles in mediating inflammatory and immune responses, not only killing invading pathogenic microorganisms, but also exacerbating tissue and organ damage. Therefore, agents that reduce inflammatory cytokines, neutrophils, lymphocytes, and leukocytes are highly desirable in ALI treatment. Research results show that Hederaponin C can inhibit the release of TNF-alpha, IL-6 and IL-1 beta and the massive recruitment of neutrophils, lymphocytes and leukocytes so as to play an anti-inflammatory role.

NF- κ B is a pluripotent regulator of various cellular signaling pathways, involved in cellular responses to various inflammatory stimuli. Recently, NF-. kappa.B has been shown to induce the release of inflammatory factors (TNF-. alpha., IL-6) in ALI by inhibiting LPS. Studies have shown that intracellular Ca2+ balance is disrupted and excessive release causes activation of NLRP3 inflammasome. The activated NLRP3 inflammatory corpuscle mediates caspase-1 activation, secretes proinflammatory cytokine IL-1 beta/IL-18, and promotes accumulation of neutrophils, lymphocytes and leukocytes to lung tissues, thereby promoting ALI. Therefore, inhibition of activation of NLRP3 inflammasome may provide an alternative strategy for the treatment of ALI. The results of the study show that Hederaponin C inhibits activation of NLRP3 inflammasome by reducing calcium production through PIP2 signaling pathway, and also inhibits activation of NF- κ B by preventing PKC α phosphorylation activation by reducing DAG production.

The invention utilizes Lipopolysaccharide (LPS) to establish a mouse Acute Lung Injury (ALI) model, discusses the protection effect and possible mechanism of Hederanaponin C on acute lung injury, and provides basis for preventing and treating ALI. The invention randomly divides 168 mice into 6 groups: control group, model group (LPS group), heierasporin C (5, 10, 20mg/kg) group (heierasporin C group), positive drug group (dexamethasone, DEX group, 5mg/kg), 28 per group. (1) Mice were modeled with a lethal dose of LPS (15mg/kg) and observed for survival over 156 h. Compared with the control group, 156h mice in the LPS group all died, and the mortality rates of the mice in the Hedera aponin C (5, 10 and 20mg/kg) group are respectively 60 percent, 50 percent and 40 percent lower than those of the LPS group; (2) and (3) modeling by using 4mg/kg LPS, and detecting the lung respiratory function of the mouse by using a breathing machine. Compared with a control group, the lung Resistance (RL) and the respiratory resistance (Re) of an LPS group are remarkably increased (P <0.05), the dynamic lung compliance (Cydn) is remarkably reduced (P <0.05), and compared with the LPS group, the RL and Re of a Hedera aponin C group mouse are remarkably reduced (P <0.05), and the Cydn is remarkably increased (P < 0.05); (3) modeling with 4mg/kg LPS, routinely detecting the change trend of White Blood Cells (WBC), neutrophils (Neu) and lymphocytes (Lym) by blood, observing the pathological histological change of the lung by an H & E staining method, detecting the expression levels of TNF-alpha, IL-6 and IL-1 beta in serum, alveolar lavage fluid and lung tissue by ELISA, and detecting the expression of proteins related to the NF-kappa B/PIP2/NLRP3 signal channel of the lung tissue by a Western blotting method. Compared with the control group, the mice in the LPS group have obviously increased WBC, Neu and Lym in whole blood (p <0.05), and have obviously increased TNF-alpha, IL-6 and IL-1 beta contents in serum, alveolar lavage fluid and lung tissues (p < 0.05). The pathological changes of lung tissues are mainly manifested by obvious consolidation, damage and collapse of alveoli with obvious extrusion, unclear boundaries and obvious inflammatory cell infiltration. Compared with LPS group, the WBC, Neu and Lym level in whole blood of Hedera aponin C group is obviously reduced (P <0.05), the pathological damage degree of serum, alveolar lavage fluid and lung tissue TNF-alpha, IL-6 and IL-1 beta water lung tissue is obviously reduced compared with LPS group, and the P-TAK1, P-PKC alpha, P-P65, NLRP3, cleared-caspase-1, cleared-IL-1 beta, PIP2, DAG and IP3 protein expression of lung tissue is also obviously reduced, while the protein expression of PLC gamma 2 is obviously increased, which shows that Hedera aponin C obviously inhibits NF-kappa B/PIP2/NLRP3 signal channel activation. Therefore, Hedera aponin C has a certain protective effect on LPS-induced acute lung injury of mice, and the mechanism of Hedera aponin C is probably related to the inhibition of proinflammatory cytokine release in the lung and the inhibition of NF-kB/PIP 2/NLRP3 signal channel.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

Use of Hedera aponin C in the preparation of a medicament for the treatment and prevention of acute lung injury, wherein Hedera aponin C has the structural formula (I):
2. the use of claim 1, wherein the acute lung injury is lung tissue damage, inflammation and infection caused by upper respiratory tract infection, chronic bronchitis, pulmonary edema, pneumonia, lung abscess, and cardiac and cerebral ischemia and organ transplantation.
3. The use according to claim 1, wherein the acute lung injury is caused by an influenza virus infection, a bacterial infection and/or a fungal infection.
4. The use of claim 1, wherein the medicament comprises a therapeutically effective amount of Hedera aponinC and a pharmaceutically acceptable carrier.
5. The use of claim 1, wherein the medicament is formulated into a pharmaceutically acceptable dosage form.
6. The use of claim 1, wherein the medicament down-regulates the levels of inflammatory factors in a subject with acute lung injury, inhibits activation of the PIP2 signaling pathway, the NF- κ B signaling pathway, and the NLRP3 inflammasome.
7. The use according to claim 1, wherein Hederaponin C is administered at a dose of not less than 5 mg/kg-d.