Anti-Inflammatory Effects of Clematis terniflora Leaf on Lipopolysaccharide-Induced Acute Lung Injury

For centuries, natural products are regarded as vital medicines for human survival. Clematis terniflora var. mandshurica (Rupr.) Ohwi is an ingredient of the herbal medicine, Wei Ling Xian, which has been used in Chinese medicine to alleviate pain, fever, and inflammation. In particular, C. terniflora leaves have been used to cure various inflammatory diseases, including tonsillitis, cholelithiasis, and conjunctivitis. Based on these properties, this study aimed to scientifically investigate the anti-inflammatory effect of an ethanol extract of leaves of C. terniflora (EELCT) using activated macrophages that play central roles in inflammatory response. In this study, EELCT inhibited the essential inflammatory mediators, such as nitric oxide, cyclooxygenase-2, tumor necrosis factor-α, interleukin- (IL-) 6, IL-1β, and inducible nitric oxide synthase, by suppressing the nuclear factor-κB and mitogen-activated protein kinase activation in macrophages. Acute lung injury (ALI) is a fatal respiratory disease accompanied by serious inflammation. With high mortality rate, the disease has no effective treatments. Therefore, new therapeutic agents must be developed for ALI. We expected that EELCT can be a promising therapeutic agent for ALI by reducing inflammatory responses and evaluated its action in a lipopolysaccharide- (LPS-) induced ALI model. EELCT alleviated histological changes, immune cell infiltration, inflammatory mediator production, and protein-rich pulmonary edema during ALI. Collectively, our results may explain the traditional usage of C. terniflora in inflammatory diseases and suggest the promising potential of EELCT as therapeutic candidate for ALI.


Introduction
Plants are essential elements for treating various diseases in oriental medicine.Clematis is a herb genus containing approximately 300 species in the family Ranunculaceae [1].Wei Ling Xian, which comes from the roots and rhizomes of several Clematis species, is prescribed as an analgesic, antipyretic, anticancer, and anti-infammatory agent in the ancient books of East Asia, including Chinese Pharmacopeia and Donguibogam (Korean traditional ancient book completed in 1613 by Jun Heo) [2,3].One particular plant, Clematis ternifora var.mandshurica (Rupr.)Ohwi, is mainly distributed in China, Japan, and Korea.It is known as "Earri" or "Wi Ryeong Seon" in Korea and also has been used to address pain and fever issues in oriental medicine [4,5].In particular, the leaf of C. ternifora is described as treatment for infammationrelated diseases, such as tonsillitis, cholelithiasis, and conjunctivitis in Traditional Chinese Medicine Dictionary [6].
ALI is a pulmonary disease characterized by excessive infammatory responses and dysfunction of the respiratory tract.ALI can be caused by both direct and indirect lung damage, including sepsis, aspiration pneumonia, inhalation injury, pancreatitis, and blood transfusions [14].Whatever etiology causes, the ALI pathogenesis is attributed to out-of-control infammatory response.As irreversible infammation and pulmonary damage can lead to death, it is recognized as a serious public health problem [15].ALI treatments can be divided into supportive care and pharmacological interventions (corticosteroids, antiinfammatory agents, and antioxidants); however, these therapies cannot efectively cure ALI and improve patients' quality of life [16,17].Terefore, it is urgent to develop new drugs for ALI.As Gram-negative bacterial infection is one of the most common causes of ALI, the intratracheal injection of lipopolysaccharide (LPS) into animals is typically considered as a well-established experimental model, which imitates human ALI [18,19].In this model, LPS induces several infammatory symptoms, including tissue damage, protein-rich edema, immune cell infltration, and increased infammatory mediators in the lung [20].
Te study of traditional medicinal plants is considered an attractive means for developing promising complementary and alternative medicine in current health problems.Accordingly, we examined the mechanism action of ethanol extract of leaves of C. ternifora (EELCT) in infammatory responses using LPS-stimulated macrophages.Moreover, we expected that EELCT has a therapeutic possibility against infammatory diseases and identifed the anti-infammatory activity in an LPS-induced ALI model.

EELCT Preparation and Identifcation
. Clematis ternifora var.mandshurica (Rupr.)Ohwi was obtained from the Korea National Arboretum in Pocheon, Republic of Korea (voucher specimen no.JMC15119).Material (30 kg) was ground and extracted in ethanol at 70 °C for 5 h twice in water bath.Ten, the extract was fltered, lyophilized, and then stored at 4 °C.Te dried extract yield was approximately 5.3% from starting crude materials.To identify individual components in EELCT, we used high-performance liquid chromatography (HPLC) as previously reported [21].Analyses were performed using Agilent HP 1260 Infnity Series liquid chromatograph (Agilent Technologies, Santa Clara, CA) with a Capcell Pak C18 column (5 μm × 4.6 mm × 250 mm; Shiseido, Japan) and Agilent 6120 single quadrupole.Chromatography was performed at room temperature at a fow rate of 1 mL/min, and a 10 μL sample was analyzed over 120 min.Te mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B) in a ratio specifed by the following binary gradient with linear interpolation: 0 min 20% B, 60 min 30% B, 70 min 60% B, 100 min 70% B, and 120 min 20% B. UV spectra were collected using a diode array detector system (DAD) (Agilent Technologies) every 0.4 s from 260 nm to 345 nm with a resolution of 2 nm.Mass spectrometry was carried out using Agilent 6120 single quadrupole (Agilent Technologies) and analyzed using Mass Hunter Qualitative Analysis Software Version B.06.00 (Agilent Technologies).Te analyses were performed under the following conditions: capillary voltage, 3.2 kV; sample cone voltage, 27 V; source temperature, 100 °C; gas fow, 600 L/h; and gas temperature, 40 °C.Te mass scan range was 50-1000 m/z.Raw data were processed based on retention time and characteristic behavior of MS, including the exact mass, quasi-molecular ions, and in-source fragmentation.Tese data were compared with those of known compounds from an in-house plant database and existing literature.Tese data are shown in Figure 1.

Cell Culture and Measurement of NO Production.
RAW 264.7 cells were grown in DMEM supplemented with heat-inactivated 10% FBS and antibiotic-antimycotic solution in an atmosphere of 5% CO 2 at 37 °C.In this study, Dex (3.925 μg/mL; 10 μM), a typical corticosteroid medicine for infammation, was used as a positive control [22].To 2 Evidence-Based Complementary and Alternative Medicine measure NO production, RAW 264.7 cells (1 × 10 5 cells/well in a 96-well plate) were pretreated with EELCT (1, 10, or 100 μg/mL) or Dex (10 μM) for 1 h and stimulated with LPS (100 ng/mL).After 24 h, supernatants were collected and centrifuged at 400 g for 5 min at 4 °C.NO concentration was measured using Griess reagent consisting of 1% sulfanilamide, 0.1% N-[1-naphthyl]-ethylenediamine dihydrochloride, and 2.5% phosphoric acid.In a 96-well plate, equal volumes of culture media and Griess reagent were incubated for 5 min at room temperature.Te absorbance was measured at 540 nm using a spectrophotometer (Molecular Devices).A standard curve using serial dilutions of sodium nitrite was constructed, and the amount of NO was calculated.
2.4.RNA Extraction and qPCR.RAW 264.7 cells (5 × 10 5 cells/well in a 12-well plate) were pretreated with EELCT (1, 10, or 100 μg/mL) or Dex (10 μM) for 1 h and stimulated with LPS (100 ng/mL) for 6 h.Before qPCR, total RNA was extracted using RNAiso Plus (Takara Bio Inc., Shiga, Japan) and quantifed using a NanoDrop 2000 spectrophotometer (Termo Fisher Scientifc, Wilmington, MA). cDNA was then synthesized from 1 μg of total RNA using a RevertAid First Strand cDNA Synthesis Kit (Termo Fisher Scientifc) according to the manufacturer's protocol.qPCR was conducted using an Applied Biosystems StepOnePlus ™ Real-time PCR System (Life Technologies Co., Kallang Avenue, Singapore) according to the manufacturer's protocol.In brief, 1 μL of cDNA, 1 μL of each sense Evidence-Based Complementary and Alternative Medicine and antisense primer solution (0.5 μM), 10 μL of QGreenBlue Master Mix High ROX (QBHR-05 2X, Termo Fisher Scientifc), and 7 μL of nuclease-free water were mixed in reaction tubes.Relative quantifcation of target gene was calculated using the 2− ∆∆Cq method in StepOne-Plus PCR system software (Termo Fisher Scientifc).Primer sequences used in this study are shown in Supplementary Table S1.First, 30 mice were randomly divided into six groups: control; ALI; ALI and 0.1, 1, and 10 mg/kg EELCT; and 2 mg/kg Dex.Te dosages were selected based on our preliminary experiments.Mice were intraperitoneally injected with a phosphate-bufered saline (PBS) : ketamine : Rompun (7 : 2 : 1) mixture anesthetic and intratracheally injected with 50 μL of LPS (5 mg/kg).An equal volume of PBS was injected into control group.EELCT and Dex were orally administrated 1 h before ALI induction.After 24 h, mice were humanely sacrifced by anesthetic overdose and lung tissue and bronchoalveolar lavage fuid (BALF) were collected for analysis.

BALF Analysis.
To obtain BALF, an 18-gauge catheter was inserted into the trachea and perfused twice with 800 µL of autoclaved PBS through the tracheal cannula as described previously [14].Collected BALF was then centrifuged at 400 g for 10 min at 4 °C, supernatant was transferred to fresh tubes, and pellets were resuspended in PBS.Total protein content in BALF was determined using a Bradford assay kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol.In brief, 10 µL of BALF and 200 µL of Bradford agent were added to a 96-well plate and incubated for 5 min at room temperature.Te absorbance was measured at 595 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA).To count diferential cells, pellets were resuspended in PBS, fxed onto slides, and stained using a Dif-Quik staining kit (Sysmex Co., Kobe, Japan).Total cell numbers in BALF were determined using a hemacytometer.

Statistical Analysis.
Statistical analyses were performed in GraphPad 7 software (GraphPad Software Inc., San Diego, CA) using one-way analysis of variance followed by Dunnett's multiple comparisons test.p values <0.05 were considered statistically signifcant.Te results were expressed as mean ± standard deviation (SD) for in vitro experiments and mean ± standard error of the mean (SEM) for in vivo experiments.

Efects of EELCT on Infammatory Mediators in Macrophages.
To investigate the mechanism action of traditional usage of C. ternifora on infammation, in vitro studies were conducted using LPS-stimulated macrophages.First, to rule out the cytotoxicity of EELCT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays were performed.Cell viability was not afected up to 1,000 μg/mL; therefore, we used below 4 Evidence-Based Complementary and Alternative Medicine 100 μg/mL EELCT in our study (Supplementary Figure S1).Te various mediators from activated macrophages are closely involved in development of infammation.From our data, EELCT signifcantly inhibited LPS-induced NO production in macrophages (Figure 2(a)).In this regard, expression of iNOS was also investigated.Our data showed that EELCT suppressed the gene and protein expression of COX-2, iNOS, TNF-α, IL-6, and IL-1β in a concentration-dependent manner (Figures 2(b)-2(d) and Figure 3).

Efects of EELCT on NF-κB and MAPK Activation in Macrophages.
To identify signaling mechanisms of the EELCT action, NF-κB and MAPK activation was assessed by determining phosphorylation of subfamilies.We observed that LPS-induced phosphorylation of p65-NF-κB, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 was reduced by EELCT (Figure 4).Collectively, EELCT appeared to inhibit the expression of major infammatory mediators, such as NO, COX-2, TNF-α, IL-6, IL-1β, and iNOS, through suppression of NF-κB and MAPK activation in macrophages.

Efects of EELCT on Histology and Immune Cell Infltration in ALI.
To investigate the pharmacological efects of EELCT in the ALI model, mice were administrated EELCT or Dex and intratracheally injected with LPS (5 mg/ kg).After 24 h, lung tissue and BALF were collected.Lung tissue was stained with H&E for histological analysis.In the ALI group, lung destruction, including immune cell infltration, alveolar collapse, hemorrhage in the stroma, and airspace edema were observed, but these were alleviated in the EELCT-administrated group (Figure 5(a)).Te immune cell infltration was investigated by counting cell numbers in BALF.In our data, increased total cell numbers during ALI were reduced by oral administration of EELCT (Figure 5(b)).In addition, the infltration of macrophages and neutrophils, which are important efector cells in ALI pathogenesis, was determined using Dif-Quik staining.As a result, both macrophage and neutrophil numbers were increased by LPS and decreased by EELCT in a dose-dependent manner (Figures 5(c) and 5(d)).

Efects of EELCT on Infammatory Mediators and Pulmonary Edema in ALI.
To investigate the anti-infammatory efect of EELCT during ALI, we evaluated TNF-α, IL-6, IL-1β, and MPO levels in BALF.Oral administration of EELCTdose-dependently suppressed the LPS-induced TNFα, IL-6, IL-1β, and MPO production (Figures 6(a)-6(c) and Supplementary Figure S2).In addition, we confrmed the expression of important proteins in lung tissues.As a result, LPS increased the expression of NF-κB, COX-2, and iNOS that reduced by the oral administration of EELCT.In addition, EELCT inhibited the phosphorylation of NF-κB and MAPKs (ERK) induced by LPS (Figure 6(d)).Protein-rich pulmonary edema occurs from alveolar barrier dysfunction and is a major symptom of ALI [25].To determine the extent of protein accumulation, total protein concentration in BALF was measured.Te mouse body and lung were also weighed using precision balance (Ohaus, Parsippany, NJ) to investigate the efects of EELCT in edema.As a result, total protein levels and lung/body weight ratios were decreased by EELCT in the LPS-induced ALI model (Figures 6(e) and 6(f )).Te body weight and spleen weight changes are important resulting factors of ALI.Our data showed that the body weight was reduced by approximately 30% in LPStreated group compared with normal state, and it was alleviated by oral administration of EELCT (Figure 6(g)).
EELCT also inhibited the increased spleen/body weight ratio during ALI (Figure 6(h)).Tese results indicated that oral administration of EELCT can attenuate the infammatory symptoms and systemic damage during ALI.

Discussion
As mentioned above, C. ternifora leaf is a traditional herbal medicine to treat diverse infammatory diseases.Tis study aimed to provide the scientifc evidence for antiinfammatory efect of C. ternifora leaf using activated macrophages.Macrophages are closely involved in infammatory response by releasing various mediators, such as NO, COX-2, and infammatory cytokines.Excessive NO production induces the overexpression of infammatory cytokines that can conversely promote the NO production, forming a positive feedback cycle that causes infammation.In addition, COX-2 has been implicated in several infammatory diseases, such as asthma, arthritis, and cancer.In in vitro study, EELCT markedly inhibited LPS-induced high levels of NO, COX-2, TNF-α, IL-6, IL-1β, and iNOS in macrophages.LPS stimulates macrophages via toll-like receptor 4 leading to activation of NF-κB, MAPKs, and other intracellular signaling pathways [26].Upon macrophage stimulation, NF-κB is phosphorylated and translocated to the nucleus following degradation of IκB.In the nucleus, NF-κB binds (as a transcription factor) to promoter region in target genes [27][28][29].MAPKs are well-characterized signaling pathways related to infammatory reaction in activated macrophages [30].Indeed, it is well known that expression of infammatory mediators mentioned above is regulated by NF-κB and MAPKs.We confrmed that EELCT inhibited p65-NF-κB, ERK, JNK, and p38 phosphorylation in LPS-stimulated macrophages.Taken together, these fndings indicate that EELCT exerted anti-infammatory efects by suppressing the expression of NO, COX-2, TNF-α, IL-6, IL-1β, and iNOS through regulation of NF-κB and MAPK activation in activated macrophages.
ALI and its more severe form (ARDS) are lifethreatening infammatory diseases with high fatality rates [31].However, there is no efective pharmacotherapy, and the ALI management remains in supportive care [32].In recent years, considerable research has been conducted to develop new ALI treatment from natural products [16].On the basis of the anti-infammatory efect of EELCT, we investigated the therapeutic activity of EELCT against ALI.Multiple immune cells regulate the immune responses and Evidence-Based Complementary and Alternative Medicine the microenvironment in the lungs by releasing diferent infammatory mediators [33,34].Notably, resident or recruited macrophages and neutrophils mainly contribute to pathogenesis of ALI.Alveolar macrophages have essential roles in initiating and maintaining the infammatory responses.Additionally, activated neutrophils release several toxic mediators, which enhance tissue damage and increase vascular permeability and alveolar edema [35,36].Tese immune cells mediate infammatory responses by releasing various infammatory cytokines, such as TNF-α, IL-6, and IL-1β [37,38].Tese cytokines can promote other signaling cascade and directly lead to lung injury.TNF-α and IL-6 are major cytokines, which stimulate other cytokine and chemokine secretion and contribute to severity of ALI [39,40].Furthermore, IL-1β is an important early mediator in ALI and infammatory conditions [41].MPO is one of the main toxic mediators from neutrophils and leads to tissue disruption during pulmonary infammation [42].As mentioned earlier, NF-κB is an important signaling molecule that regulates various infammatory factors, ultimately participating in lung damage [43].Accordingly, inhibition of this system can be a critical strategy for therapeutic capabilities in infammation during ALI.In this study, oral administration of EELCT reduced the macrophage and neutrophil numbers in BALF in addition to the expression of infammatory mediators during ALI.Terefore, our results suggest that EELCT suppressed infammatory responses during ALI.
Infltrated immune cells induce alveolar barrier dysfunction and microvascular permeability increase, which causes protein-rich pulmonary edema [25].Once pulmonary edema fuid accumulates, it causes increased breathing and impairs gas exchange leading to hypoxemia and ultimately acute respiratory failure [44].Our data showed that the accumulated protein amounts in lung and mouse lung/ body weight ratios were increased in ALI group and decreased in EELCT-administrated group.Terefore, oral administration of EELCT reduced protein-rich pulmonary edema, suggesting that EELCT alleviated alveolar barrier dysfunction during ALI.In addition, ALI induces a reduction of body weight, which is known to be involved in ARDS clinical outcomes [45].Te spleen is the largest peripheral lymphoid organ and performs a wide range of immunological functions as the frst line of defense against bacterial endotoxins [46].In this regard, the increased spleen/body weight ratio is regarded as a resulting factor of ALI [47,48].In our results, the oral administration of   Evidence-Based Complementary and Alternative Medicine EELCT attenuated the reduction of body weight and increased spleen/body weight ratio during ALI.Collectively, EELCT suppressed typical ALI symptoms, such as tissue damage, immune cell infltration, production of infammatory mediators, and pulmonary edema.Tus, this study suggested that EELCT has therapeutic efect against ALI.

Conclusion
In summary, EELCT inhibited several critical infammatory mediators, such as NO, COX-2, TNF-α, IL-6, IL-1β, and iNOS, by suppressing the NF-κB and MAPK activation in LPS-stimulated macrophages.Additionally, EELCT suppressed infammatory response, including tissue damage, immune cell infltration, production of infammatory mediators, and protein-rich edema in an LPS-induced ALI model.Terefore, we propose that EELCT may function as a therapeutic candidate for ALI.However, more comprehensive research is needed for the practical use of EELCT as a pharmacotherapy.performed the statistical analysis.LS and CJM performed additional experiments.KSY, JGS, and KSH participated in study design and coordination and helped to draft the manuscript.All authors have read and approved the fnal manuscript.

Figure 2 :Figure 3 :Figure 4 :Figure 5 :
Figure 2: Efects of EELCT on NO production and COX-2 and iNOS expression in RAW 264.7 cells.(a) Cells were pretreated with/without drugs (EELCT or Dex) for 1 h and stimulated with LPS (100 ng/mL).After 24 h, NO production in culture media was determined using Griess reagent.(b, c) After 6 h, relative gene expression was verifed by qPCR.(d) After 12 h, protein expression was determined by Western blot.Graph data represent mean ± SD. * p < 0.05 compared with the LPS-stimulated group.β-Actin was the loading control.

Figure 6 :
Figure 6: Efects of EELCT on infammatory mediator production and pulmonary edema in ALI.Mice were intratracheally injected with LPS (5 mg/kg) before oral administration of EELCT or Dex.After 24 h, lung tissue and BALF were collected.(a-c) TNF-α, IL-6, and IL-1β levels in BALF were measured by ELISA.(d) NF-κB, ERK, COX-2, and iNOS expressions in lung tissues were investigated using Western blot.(e) Total protein concentrations in BALF were determined using the Bradford assay kit.(f ) Graph shows lung/body weight ratios.(g) Mice were weighed before EELCT or Dex administration and before sacrifce.Te graph reveals the body weight change.(h) Te graph represents spleen/body weight ratios.Graph data represent mean ± SEM. * p < 0.05 compared with the ALI group.β-Actin was the loading control.
5. ELISA.Infammatory cytokine levels in BALF and culture media were measured by ELISA.RAW 264.7 cells (5 × 10 5 cells/well in a 12-well plate) were pretreated with EELCT (1, 10, and 100 μg/mL) and Dex (10 μM) for 1 h and stimulated with LPS (100 ng/mL) for 12 h or 24 h.ELISA was performed on a 96-well immune plate (Nunc, Rochester, NY) using specifc kits according to the manufacturer's instructions.Te following kits were used: TNF-α and IL-6 (BD Biosciences, San Diego, CA); IL-1β (Invitrogen, Carlsbad, CA); and myeloperoxidase (MPO) (R&D Systems, Minneapolis, MN).Te absorbance was measured at 450 nm using a spectrophotometer (Molecular Devices).2.6.Western Blot.Te protein levels in lung tissues and macrophages were determined using Western blot.RAW 264.7 cells (1.5 × 10 6 cells/well in a 6-well plate) were pretreated with EELCT (1, 10, and 100 μg/mL) and Dex (10 μM) for 1 h and stimulated with LPS (100 ng/mL) for 15 min or 12 h.To obtain total protein extract, cells and tissues °C ± 1 °C, humidity of 55% ± 5%, light/dark cycle of 12 h, and air exchange rate of 15 min/h.Feed and water were supplied ad libitum.Animal experiments were conducted in accordance with Public Health Service Policy on the Humane Care and Use of Laboratory Animals Guidelines and approved by the Institutional Animal Care and Use Committee of Kyungpook National University (IRB #2022-0389).