Effect of compound Tinglizi Decoction on non-classical pathway of pyroptosis in rats with chronic obstructive pulmonary disease related pulmonary hypertension

Objective To observe the effect of compound Tinglizi Decoction on non-classical pathway of cell death in rats with pulmonary hypertension, and to explore its mechanism of action in the treatment of COPD-related pulmonary hypertension. Methods 60 male SD rats were randomly divided into Western group, normal group, model group, high-dose group, medium dose group and low-dose group, with 10 rats per group. We detected the protein expression levels of caspase-11, GSDMD and IL-6 by Western blot, detected serum TNF-α by ELISA, and observed the morphology of lung tissue by hematoxylin and eosin (HE) staining. Results In the expression of caspase11, GSDMD, IL-6 mRNA and protein in lung tissue, the model group was significantly higher than the normal group (P < 0.01), and the high-dose group, middledose group, low-dose group and western medicine group were significantly lower than the model group (P < 0.01). The levels of serum TNF-α and serum creatinine in the model group were significantly higher than those in the normal group (P < 0.01); and those in the high-dose group, middle-dose group, low-dose group and western medicine group were significantly lower than those in the model group (P < 0.05). Conclusion The mechanism of compound Tinglizi Decoction in the intervention of COPD-related pulmonary hypertension rats is related to the inhibition of non-classical pathway of cell death.


Introduction
Chronic obstructive pulmonary disease (COPD) is a chronic, progressive respiratory disease with high morbidity and is characterized by persistent airflow limitation. In 3 recent years, with the in-depth research on molecular biology of pulmonary hypertension, pyroptosis plays an important role in respiratory diseases (1,2). COPDrelated pulmonary hypertension (PAH) is a disease or pathophysiological syndrome of abnormally elevated pulmonary arterial pressure caused by respiratory diseases and/or hypoxia. It has pulmonary circulation disorders and a high load on the right heart, which eventually leads to right heart failure and even death. The hemodynamic criteria for pulmonary hypertension are: sea level, resting, mean pulmonary artery pressure (mPAP) 25mmHg (1mmhg = 0.133kpa) (3). The gold standard of COPD related pulmonary hypertension is right heart catheterization. Currently, the treatment method is oxygen therapy for patients with primary disease and hypoxia.
Pyroptosis is dependent on the activation of Caspase-1 and is a newly discovered form of programmed cell death in recent years. Its characteristic is that the cell continues to expand until the cell membrane ruptures, causing the release of intracellular material, which then activates a strong inflammatory response (4). When pathogens invade, the body promotes the formation of inflammasome in a certain way. This in turn activates the non-canonical caspase-4/5/11-dependent pathway and/or the canonical caspase-1-dependent pathway. In the nonclassical pathway, bacterial lipopolysaccharide (LPS) can activate caspase-4 / 5 / 11 and combine with it to induce Pyroptosis. For one thing, activated caspase-4 / 5 / 11 can activate NLRP3 inflammasome to activate caspase-1 by exposing the N-terminal domain of GSDMD protein, and then induce cell death. On the other hand, activated caspase-4/5/11 can activate the pannexin-1 channel and open the purine P2X7 channel. This promotes the release of inflammatory mediators and the efflux of intracellular K+, which activates the NLRP3 inflammasome and promotes the outward release of IL-1β. LPS can also bind to the card domain of caspase-1 to further regulate NLRP3 expression and IL-1 4 secretion and activation (5). In the previous experimental research of the research group, it was found that compound Tingli Zi caspule can significantly relieve the clinical symptoms and the number of acute attacks in patients with COPD-PAH. And further animal experiments confirmed that it can reduce pulmonary hypertension, improved pulmonary vascular remodeling (6)(7)(8).However, the anti-inflammatory mechanism of compound Zhixiezi decoction on COPD-PAH and whether the non-classical cell death pathway mediates its anti-inflammatory mechanism are unclear. Therefore, the purpose of this study was to observe the effect of compound Tinglizi Decoction on COPD-PAH and to explore the mechanism of its intervention in COPD-PAH.

Drugs
Compound Tinglizi decoction is composed of lepidium seed 15g, peach kernel 10g, carthamus tinctorious 10g, leech 6g, ligusticum wallichii 15g, poria cocos 15g, 5 cassia twig 15g, bighead atractylodes rhizome 15g, ardisia 10g, Scutellaria baicalensis 10g, liquorice 6g and licorice 6g. All the pieces of traditional Chinese medicine were purchased from the Chinese pharmacy of Hunan Academy of traditional Chinese medicine. These medicines were identified by Tian Qixue, chief pharmacist of Hunan Academy of traditional Chinese medicine Affiliated Hospital. and meet the relevant standards of Pharmacopoeia of the people's Republic of China. Decocting method: compound Tinglizi decoction was added 10 times of distilled water and soaked in cold water for 30 min. , then heated to boiling and decocted for 40 min. Then filter the decoction, add 8 times of distilled water, decoct as before, and filter out the decoction.
The filtrate was combined twice and precipitated. After cooling, the filtrate was stored at 4 ℃ and used up within 1 week.

Grouping and preparation of animal models
A rat model of COPD associated pulmonary hypertension was established by 7 cigarette smoke exposure and lipopolysaccharide infusion (9). Sixty rats were randomly divided into normal group, western medicine group, model group, high-dose group, middle-dose group, and low-dose group, with 10 rats in each group. On the 1st and 14th day of the experiment, after pentobarbital sodium anesthesia, the model group and each drug group were instilled with LPS (1 mg/kg) intratracheally, and the normal group was not treated. No smoking was given on the same day. Except for the first day and the 14th day, the rats in each administration group and model group were placed in the selfmade animal plexiglass fumigation box. Close the lid and seal the gap with plastic wrap to prevent fumes from escaping. Huangguoshu brand cigarette (10 mg flue-cured tar; Guizhou Zhongyan Industry Co., Ltd.) was inserted into the self-made tray, ignited it and put it into the fumigation box. 20 cigarettes were lit each time, once in the morning and afternoon. 2 hours each time (10 minutes apart). 6 days a week, a total of 60 days.

Test index
Detection of the expression of caspase-11, GSDMD and IL-6 in lung tissue by real time PCR Take 0.25mg lung tissue, add 1ml Trizol, grind thoroughly, mix well, and split in the chamber for 5min. After adding 200ul of chloroform, shake vigorously for 15s, and let stand for 3min at room temperature. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was removed. And 1 ml of 75% sterile DEPC-treated water was added to wash the precipitate. Centrifuge at 12,000 rpm and 4°C for 3 min, discard the  Table 1. TBST was washed 4 times for 10 min each, then horseradish peroxidase-conjugated II antibody was added and incubated for 90 min at room temperature. After incubation, TBST was washed 3 times for 10 min each. GAPDH was used as internal reference, ECL was used to develop and take pictures in the imaging system. The results were analyzed by quantity one software.

Detection of TNF-α in serum through ELISA Level detection
A certain amount of lung tissue was collected and ELISA kit was used to determine TNF-α.The content of the product was determined.

The morphology of lung tissue was observed by hematoxylin and eosin (HE) staining and the degree of pulmonary vascular muscle was determined by immunohistochemistry
After the experiment, the rat was killed, and the extracted lung tissue was fixed with 10% formaldehyde. After 1 week, the paraffin-embedded lung tissue was cut into 4 μm thick, baked at 60 ℃ and perform hematoxylin eosin (HE) staining. Lung tissue from each rat was prepared and placed under the microscope. Five high-power microscope fields were randomly selected for observation. Nikon ti-s inverted microscopes were used to analyze and process images. The expression of α-SMA in pulmonary blood vessels was detected by immunohistochemistry to evaluate the degree of pulmonary vascularization.

Statistical analysis
The data were analyzed and processed by IBMS PSS Statistical 20.0 statistical software. The mean value was used in the experimental data ± Standard deviation (X±S). The LSD test was used when the variance was normally distributed, and Dunnett's T3 test was used when the variance was not uniform. ANOVA was used to compare the data. The skew distribution was tested by rank sum test. Test level was set at both sides α =0. 05, P < 0.05 was considered statistically significant.

The effect of compound tanglizi Decoction on caspase-11, GSDMD, IL-6mRNA expression in lung tissue of COPD PH rats
Compared with the normal group, caspase11, the expression of caspase11, GSDMD and IL-6 mRNA in the lung tissue of the model group was significantly increased (P<0.01); Compared with model group, the expression of caspase-11, GSDMD and IL-6mRNA in lung tissue of rats in the high-dose group, middle-dose group, low-dose group and western medicine group were significantly decreased. The results were shown in Table 2.   Figure 1.

Compound decoction of triplizi in serum TNF-α of COPD PAH rats Level impact
The results showed that compared with the normal group, the TNF-α in the model group was significantly higher (P ＜ 0.01); the level of serum creatinine was significantly increased (P < 0.1); Compared with the model group, the levels of the low dose group, the middle dose group, the high dose group and Wetern group were significantly decreased (P < 0.05), and the results were shown in Table 3 and Figure2.

Degree of vascularization
Immunohistochemistry showed that the rats in the blank control group were mainly non myogenic and partially myogenic vessels, and the fully myogenic vessels in the model group increased significantly. Compared with the model group, the vascularization of low-dose group, medium dose group, high-dose group and Western medicine group decreased in varying degrees. The composition of the prescription is: lepidium seed 15g, peach kerne l10g, carthamus tinctorious10g, leech 6g, ligusticum wallichii15g, poria cocos15g, cassia twig 15g, bighead atractylodes rhizome 15g, ardisia 10g, scutellaria baicalensis 10g, liquorice 6g and licorice 6g. The whole prescription is compatible with dispelling lung, resolving phlegm, relieving asthma. And in clinical verification (13), Compound TingliZi Decoction has the effects of reducing pulmonary arteriovenous pressure, improving blood circulation and reducing pulmonary congestion. However, the mechanism of how compound Tinglizi decoction can reduce inflammatory reaction and inhibit pulmonary vascular remodeling by inhibiting cell death is still unclear.
In the experiment, we found that the activation of non-classical pyrolytic pathway,

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the key proteins of pyrolytic, GSDMD, IL-6 and TNF-α, existed in COPD related pulmonary hypertension rats induced by cigarette smoke exposure combined with lipopolysaccharide. Compound Tinglizi decoction can inhibit the activation of nonclassical cell death pathway and reduce the level of proinflammatory factors in COPD related pulmonary hypertension rats. This suggests that compound Tinglizi decoction may intervene COPD related pulmonary hypertension through non-classical way of regulating cell death.

Conclusions
The mechanism of compound Tinglizi Decoction in the treatment of COPDrelated pulmonary hypertension may be associated with the inhibition of non-classical pathway of cell death and the reduction of the level of pro-inflammatory factors, which provides a certain theoretical basis for the clinical treatment of COPD-related pulmonary hypertension.