Xin-Liang Qu,Guang-Jing Duan,Bo Zhao,Feng Xie,Bin Wang,Feng Gao,Pei-Feng Wei,Min Li

College of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang 712046, China

Keywords:Scutellaria baicalensis stems Polygonum cuspidatum Acute lung injury Inflammation TRPV1 receptor

ABSTRAC T Objective:The protective effect of Scutellaria baicalensis Stems and Polygonum Cuspidatum compatibility on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats was studied by observing the expression of TRPV1 and inflammatory cytokines. Methods: 48 male SD rats were randomly divided into 6 groups: control group, model group, dexamethasone group(5mg / kg) and Scutellaria baicalensis Stems -Polygonum Cuspidatum (3.5, 7 and 14g / kg).The administration group was gavaged for 7 days, and the control group and model group were given the same amount of 0.9% sodium chloride. On the 8th day, except the control group, rats in other groups were injected with 8mg / kg LPS through caudal vein to induce Ali model. Take the rat lung tissue 6 hours after modeling, and calculate the wet/dry weight ratio(W/D) of the rat lung tissue; HE staining to observe the pathological changes of lung tissue;Determine the content of tumor necrosis factor-α (TNF-α) and interleukin-1β (1L-1β)in alveolar lavage fluid (BALF) and the activity of superoxide dismutase (SOD) in serum;Detect the mRNA and protein expression levels of TRPV1 receptor in rat lung tissue. Results:Compared with the model group, Scutellaria baicalensis Stems -Polygonum Cuspidatum can significantly reduce the damage of lung tissue structure and bleeding state, W/D value and TNF- α、 IL-1 β The content of TRPV1 decreased, the level of SOD increased, and the mRNA and protein expression of TRPV1 receptor decreased. Conclusion: The combination of Scutellaria baicalensis Stems -Polygonum has a protective effect on acute lung injury in rats,and its mechanism may be related to down-regulating the expression of TRPV1 and inhibiting the levels of TNF-α and IL-1β in inflammatory cells.

1. Introduction

AThe pathological process of acute lung injury (ALI) is complex and usually caused by various factors, such as infection, trauma, sepsis and so on. Patients mainly have chest tightness, shortness of breath,dyspnea and other clinical manifestations, and further evolution can lead to acute respiratory distress syndrome (ARDS), with a fatality rate of 30%-45% [1].The main pathogenesis of acute lung injury is related to the inflammatory response, which is manifested in the increased production of pro-inflammatory factors in the lung induced by various causes, the massive release and activation of inflammatory factors that make the inflammatory response in the lung out of control, and the damage of alveolar capillary endothelial cells and alveolar epithelial cells. , Extensive pulmonary edema,further lead to pulmonary inflammatory cell infiltration, edema, gas exchange disorders [2].

Scutellaria baicalensis and Polygonum cuspidatum are two traditional Chinese herbal medicines, both of which have anti-inflammatory, anti-tumor, anti-viral, antioxidant, and pharmacological effects [3-6].The two are often used in combination to clear away heat and detoxify [7].Scutellaria baicalensis stems and leaves have similar pharmacological effects to Scutellaria baicalensis roots, and also have anti-inflammatory effects [8].The interaction relationship between Scutellaria baicalensis stems and leaves -Polygonum cuspidatum has not been clarified, and the influence and mechanism of anti-inflammatory efficacy have not been deeply studied, which limits its modern research and clinical application to a certain extent. Therefore, it is of great significance to clarify its compatibility mechanism.

Transient receptor potential vanilloid 1 (TRPV1) is a member of the transient receptor potential (TRP) family. Studies have shown that TRPV1 plays an important role in reducing the secretion of inflammatory mediators and inhibiting inflammatory responses after being activated at the site of infection. regulation of lung injury.Therefore, this study will explore the effect and mechanism of Scutellaria baicalensis stem and leaf-polygonatum compatibility on the expression of inflammatory factors and TRPV1 in LPS-induced ALI in rats, and provide a theoretical basis for clinical application.

2. Materials and methods

2.1Test drug

Scutellaria baicalensis stems and leaves (Daxinganling,Heilongjiang Province); Polygonum cuspidatum pieces (Haozhou Yonggang Pieces Factory Co., Ltd.). Take 100g each of Scutellaria baicalensis stems and leaves and Polygonum cuspidatum medicinal materials, add 15 times the amount of water, soak for 30 minutes,boil with high heat, simmer for 50 minutes, filter with double-layer gauze; add 10 times the amount of water for second decocting, boil with high heat, simmer for 40 minutes, filter , the two filtrates were combined, concentrated to 1 mL of medicinal liquid containing 1 g of crude drug, and stored below 4 ℃.

2.2Animals

48 SPF-grade healthy male SD rats (Sichuan Laboratory Animal Quality Inspection Center), 150±10g, 6 weeks old, production license number: SCXK (Chuan) 2020-030, rearing environment:temperature (25±1℃) , relative humidity (50% to 60%), free food and water, animal experiments and methods were approved by the Ethics Committee of Shaanxi University of Traditional Chinese Medicine.

2.3 Reagents

LPS (lipopolysaccharide, sigma, product number: L8880); TNF-α,1L-1β ELISA kit (Shanghai ELISA, product number: ml002859,ml003057); SOD activity detection kit (Nanjing Jiancheng, product number: A001-3 -1); TRPV1 antibody (Wuhan Boster, product number: CX0125); SYBR Green PCR kit (QIAGEN, Germany,product number: 208052); RNA extraction kit TRI reagent(Invitrogen, USA, product number: 15596018).

2.4 Instruments

ELx808 automatic microplate reader (Bio-Tek), ICX40 inverted microscope (Sunny Optical Technology Co., Ltd.), high-speed refrigerated centrifuge (Thermo), Bole electrophoresis instrument(BIO RAD), fluorescence quantitative PCR instrument (Shanghai Roche Pharmaceutical).

2.5 Animal modeling and grouping

Forty-eight SD male rats were randomly divided into 6 groups:control group, model group, dexamethasone positive group,Scutellaria baicalensis stem-leaf-polygonatum compatibility lowdose, middle-dose and high-dose groups. 8 per group.Scutellaria baicalensis stem-leaf-polygonatum compatibility low-dose,medium-dose, and high-dose groups were given 3.5, 7, and 14 g/kg by gavage, respectively, and the control group and model group were given the same amount of 0.9% sodium chloride.Rats in the dexamethasone group were intraperitoneally injected with 5 mg/kg dexamethasone. Administered once a day for 7 consecutive days.On the 8th day, except the control group, the other groups used a 1 mL syringe to draw LPS solution (prepared to 1 mg/mL) and injected 8 mg/kg LPS into the tail vein of rats to induce ALI model.After 6 hours of modeling, the animals were anesthetized, 5 mL of abdominal aortic blood was collected, and the serum was separated for use and stored at -80℃.

2.6 Specimen collection and testing

2.6.1 Determination of wet/dry mass ratio of rat lung tissue

Take fresh right lung tissue, wash it in ultrapure water, absorb the water, and weigh the wet mass. The lung tissue was placed in a 60℃ incubator to dry, and after 72 h, the lung dry weight was weighed,and the wet/dry mass (W/D) ratio was calculated according to formula (1).

W/D = wet weight of lung tissue/dry weight of lung tissue × 100%.(1)

2.6.2 Pathological changes of rat lung tissue

Lung tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sliced into thin sections (about 4 μm), deparaffinized in xylene, dehydrated with graded ethanol, deparaffinized, stained with hematoxylin-eosin staining solution (HE), and used for optical The pathological changes of lung tissue were observed under a microscope at a magnification of 100 times.

2.6.3 Detection of related inflammatory factors in ratsThe contents of TNF-α and 1L-1β in BALF supernatant in rat lung tissue were determined. The right lung was anesthetized and ligated, the trachea was exposed for tracheal intubation, the right main bronchus was separated and ligated, cold saline was slowly injected through the trachea, BALF was collected, centrifuged, and stored at -80℃.Strictly follow the instructions of the ELISA kit, use a microplate reader (450nm) to read the absorbance OD value, and finally calculate the concentration of the sample.

2.6.4 Detection of SOD in rat lung tissueThe rat serum was collected, and the SOD level in the lung tissue of the rats in each group was determined according to the kit instructions.

2.6.5 Detection of TRPV1 mRNA expression in lung tissue by qRT-PCR

Total RNA was extracted by Trizol method, and the RNA was reverse transcribed into cDNA according to the kit instructions.Using cDNA as template and β-actin as internal reference, qRTPCR kit and Roche 480 II qRT-PCR instrument were used to detect the mRNA level of TRPV1 .The relative expression of TRPV1 was calculated by the 2-ΔΔCTmethod. The primer sequences are shown in Table 1. The reaction conditions were 95℃ for 30s to activate the hot-start enzyme, 95℃ for 5s denaturation and 60℃ for 30s extension,A total of 45 cycles.

Table 1 Primer sequence of TRPV1

2.6.6 Detection of protein expression in lung tissue by Western blot

The RIPA lysis solution was added to the lung tissue and ground in a homogenizer. After fully lysed, the supernatant was collected by centrifugation, and part of the supernatant protein was taken to measure the protein concentration with a BCA kit.The adjusted protein concentration was mixed with an equal volume of 5 loading buffer, boiled in boiling water for 5 min and loaded, and subjected to SDS-PAGE gel electrophoresis, 200 mA constant flow transfer membrane for 70/90 min; after blocking, TRPV1 (1:1000) and B-actin (1:2000), incubated overnight at 4 C.The next day, the membrane was washed, and Ⅱ antibody (1:2000) was added for incubation at room temperature for 2 h.The ECL chemiluminescence method was used for development, and ImageJ statistical processing image analysis method was used for analysis, and the grayscale ratio of the target protein and the internal reference protein B-actin was used to indicate the level of the corresponding protein expression level.

2.7 Statistical analysis

SPSS 21.0 software was used for statistical analysis, and the data results were expressed as±s. One-way analysis of variance(ANOVA) was used for comparison between groups, and P＜0.05 was considered statistically significant.

3. Results

3.1 Comparison of the W/D values of lung tissue in each group of rats

Compared with the control group, the W/D of the lung tissue of the rats in the model group was significantly increased (P＜0.01),indicating that the pulmonary edema was more serious; , high dose significantly decreased the W/D value of rat lung tissue (P＜0.05),suggesting that the drug can prevent pulmonary edema. Among them, the middle-dose effect of Scutellaria baicalensis stem-leafpolygonatum compatibility group was more significant (P＜0.05).See Table 2.

Table 2 Comparison of W/D of Lung Tissue of Rats in Each Group(n＝8, ±s)

Table 2 Comparison of W/D of Lung Tissue of Rats in Each Group(n＝8, ±s)

Note: *P ＜ 0.05, **P ＜ 0.01 compared with the control group; #P ＜ 0.05, ##P ＜0.01 compared with the model group.

Group Dosage (g/kg) W/D Control - 2.06±0.21 Model - 6.92±0.35**Positive group 5 5.74±0.7**#SLPC 3.5 3.5 4.99±0.53**#SLPC 7 7 3.84±0.20*##SLPC 14 14 4.51±0.76**##F-31.62 P-＜0.0001

3.2 Comparison of pathological changes of lung tissue in each group of rats

The results showed that the alveolar structure of the rats in the control group was intact, without inflammatory cell infiltration, and no pathological changes were observed; compared with the control group, the alveolar structure of the rats in the model group was destroyed, with flaky blood foci and a large number of inflammatory cell infiltration; Compared with the model group, the damage of alveolar structure in the middle and high dose groups of Scutellaria baicalensis stem and leaf-polygonatum was significantly improved,and the inflammatory infiltration was alleviated. see picture 1.

Figure 1 HE staining of lung tissue of rats in each group(HE,100×)

3.3 Comparison of inflammatory factors in BALF of rats in each group

Compared with the control group, the levels of TNF-α and 1L-1β inflammatory cytokines in the lung tissue of the rats in the model group were significantly increased (P＜0.01), indicating that there were more inflammatory infiltration; The levels of TNF-α and 1L-1β in the BALF of mice were significantly decreased(P＜0.05), especially in the high and high doses, and the differences were statistically significant (P＜0.05). The levels of TNF-α and 1L-1β in the BALF of the positive drug dexamethasone group were significantly decreased (P＜0.05, P＜0.01). See Table 3.

Table 3 Comparison of the concentrations of TNF-α and 1L-1β in the BALF of rats in each group(n＝8, ±s)

Table 3 Comparison of the concentrations of TNF-α and 1L-1β in the BALF of rats in each group(n＝8, ±s)

Note: *P ＜ 0.05, **P ＜ 0.01 compared with the control group; #P ＜ 0.05, ##P ＜0.01 compared with the model group.

Group TNF-α(pg/ml) 1L-1β(pg/ml)Control 213.36±39.21 10.50±0.64 Model 250.52±23.32** 16.07±0.38**Positive group 175.95±24.83*# 13.15±0.34**##SLPC 3.5 190.92±29.68## 12.12±0.92**##SLPC 7 162.18±19.40**## 11.60±0.49*##SLPC 14 165.41±11.25**## 12.08±0.16**##s F 11.13 36.68 P＜0.0001 ＜0.0001

3.4 Comparison of SOD activity in serum of rats in each group

Compared with the control group, the SOD activity in the oxidative stress level of the lung tissue of the rats in the model group decreased (P＜0.01);compared with the model group, the SOD level in the serum of the lung tissue of the rats in the positive group and the SLPC group increased significantly(P＜0.05). . See Table 4.

Table 4 Effects of SOD activity in serum of rats in each group(n＝8, ±s)

Table 4 Effects of SOD activity in serum of rats in each group(n＝8, ±s)

Note: *P ＜ 0.05, **P ＜ 0.01 compared with the control group; #P ＜ 0.05, ##P ＜0.01 compared with the model group.

Group SOD(U/ml)Control 107.76±3.89 Model 62.46±6.03**Positive group 86.1±11.26*##SLPC 3.5 81.06±8.40**#SLPC 7 83.94±11.80**#SLPC 14 93.90±8.11##F 8.87 P 0.001

3.5 Comparison of TRPV1 mRNA expression in lung tissue of rats in each group

Compared with the control group, the relative expression of TRPV1 mRNA in the lung tissue of the rats in the model group was significantly up-regulated (P＜0.01); The relative expression was significantly down-regulated, and the differences were statistically significant (P＜0.05), and the relative expression of TRPV1 mRNA in the high-dose Scutellaria baicalensis stem-leaf-polygonatum group was lower than that in the Scutellaria baicalensis stem-leafpolygonatum low-dose group (P＜0.05). See Table 5.

Table 5 Comparison of TRPV1mRNA expression in lung tissues of rats in each

3.6 Effect of TRPV1 protein expression in lung tissue of rats in each group

Compared with the control group, the expression level of TRPV1 protein in the lung tissue of rats in the model group was significantly up-regulated (P＜0.01). 0.01). See Figure 2 and Table 6.

Figure 2 The expression bands of TRPV1 protein in the lung tissues of rats in each group

Table 6 Comparison of TRPV1 protein expression in lung tissues of rats in each group(n＝8, ±s)

Table 6 Comparison of TRPV1 protein expression in lung tissues of rats in each group(n＝8, ±s)

Note: *P ＜ 0.05, **P ＜ 0.01 compared with the control group; #P ＜ 0.05, ##P ＜0.01 compared with the model group.

Group TRPV1 protein Control 0.58±0.09 Model 1.45±0.05**Positive group 0.82±0.04**##SLPC 3.5 1.16±0.08**##SLPC 7 0.55±±0.03##SLPC 14 0.66±0.06##F 109.16 P＜0.0001

4. Discussion

ALI is a common clinical disease that leads to alveolar capillary membrane damage, extensive neutrophil infiltration and release of inflammatory mediators [9].Inflammatory response plays an important role in the occurrence and maintenance of ALI [10-11].Therefore, focusing on potential targets of inflammatory processes will provide new therapeutic strategies for the prevention and treatment of ALI.The stems and leaves of Scutellaria baicalensis contain various chemical components such as flavonoids and phenolic acids [12], and have significant pharmacological activities such as anti-inflammatory, antiviral, antioxidant and protection of myocardial ischemia [8].During the harvest of Scutellaria baicalensis,the aerial parts of Scutellaria baicalensis such as stems, leaves and flowers were discarded. Therefore, this study aimed to make full use of the stems and leaves of Scutellaria baicalensis and increase the development and utilization of medicinal resources of Scutellaria baicalensis [13].Polygonum cuspidatum mainly has anti-inflammatory,antioxidant, antibacterial, and antiviral effects [3]. Modern studies have found that [14] the main component of Polygonum cuspidatum,resveratrol glycosides, can reduce serum TNF-αand IL-6 levels,increase SOD levels, and improve acute lung injury caused by paraquat.The synergistic compatibility of the two drugs exerts the effect of clearing away heat and detoxification, but the specific effect and mechanism have not been elucidated. This study explored whether Scutellaria baicalensis stems and leaves combined with Polygonum cuspidatum had a synergistic protective effect on ALI and the specific mechanism of action.

In lung injury caused by various pathogenic factors, transient receptor potential vanilloid 1 (TRPV1) has been shown to play an important role in the inflammatory response [15-16].Most TRPV1 is expressed in the epithelial and subepithelial parts of the airway in the lung, around the blood vessels, in the smooth muscle of the airway,and in the alveoli [17].It can be activated by temperature (＞43°C),capsaicin, etc., so TRPV1 is also called "thermosensitive channel".It has been reported in the literature that the activation of TRPV1 channel can significantly reduce the inflammatory injury in mice with airway inflammation, and the process may be related to the reduction of pro-inflammatory cytokines such as TNF-α and IL-6 in vivo [18].TNF-α, IL-6 and IL-1β are key inflammatory mediators involved in the inflammatory response and play an important role in the occurrence and development of ALI [19-22].TNF-α and IL-1β increase the permeability of lung epithelial cells, which in turn induces lung tissue damage and neutrophil accumulation, leading to pulmonary edema [23].

The results of this study showed that compared with the model group, the alveolar structure of the rat lung tissue was severely damaged, with flake blood foci, significantly increased W/D, pulmonary edema and a large number of inflammatory cell infiltration; Significantly improved damaged alveolar structure,increased SOD activity, decreased rat lung tissue, and reduced pulmonary edema and inflammatory infiltration.

In this study, compared with the model group, Scutellaria baicalensis stems and leaves - Polygonum cuspidatum effectively reduced the levels of TNF-α and 1L-1β in BALF, indicating that the improvement of the drug on ALI is related to the inhibition of inflammatory response.In addition, the results of Western blot and qRT-PCR showed that compared with the model group,the Scutellaria baicalensis stem and leaf-polygonatum group significantly down-regulated the relative expression of TRPV1 protein and mRNA, suggesting that the drug may inhibit TRPV1 on lipopolysaccharide-induced acute lung injury. Rat have obvious protective effect.

To sum up, Scutellaria baicalensis stems and leaves-polygonatum has a protective effect on LPS-induced ALI in rats, and its mechanism may be related to promoting the activity of SOD,inhibiting the levels of TNF-α and IL-1β, and down-regulating the expression of TRPV1 to play an anti-inflammatory effect. .

Author Conflict of Interest Statement:

All authors declare no conflict of interest.

Author’s contribution Qu Xinliang: Experiment writing and animal model construction;Duan Guangjing: determination of W/D value of lung tissue; Zhao Bo, ELISA experiment; Xie Feng: animal feeding and grouping;Wang Bin: observation of pathological changes in lung tissue; Gao Feng: qRT-PCR experiment; Wei Peifeng: Western blot experiment;Li Min: experimental design and proofreading.