Protective effects of a traditional herbal extract from Stellaria dichotoma var. lanceolata against Mycobacterium abscessus infections

Stellaria dichotoma var. lanceolata (SdLv), a member of the Caryophyllaceae, is a traditional herbal medicine that has been used to treat fever, night sweats, and malaria in East Asia. Inflammation plays an essential role in both host defense and pathogenesis during infection by diverse intracellular pathogens. Herein, we showed that an herbal extract from SdLv effectively attenuated inflammatory responses from infection of Mycobacterium abscessus (Mab), but not Toxoplasma gondii (T. gondii). In primary murine macrophages, Mab infection resulted in the rapid activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK), as well as in the generation of proinflammatory cytokines, such as tumor necrosis factor α and interleukin-6, which were all significantly inhibited by pretreatment with SdLv. However, herbal extracts from Bupleurum chinense DC. (Buch) or Bupleurum falcatum L. (Bufa) did not affect M. abs-induced activation of proinflammatory responses. Importantly, we demonstrated that generation of intracellular reactive oxygen species, which are important signaling intermediaries in the activation of NF-κB and the MAPK signaling pathway, was rapidly increased in Mab-infected macrophages, and this was effectively suppressed by pretreatment with SdLv, but not Buch and Bufa. We further found that the treatment of Buch and Bufa, but not SdLv, led to the activation of NF-κB and the MAPK signaling pathway and the generation of intracellular reactive oxygen species. Moreover, oral administration of SdLv significantly reduced lethality in Mab-infected mice. Collectively, these results suggest the possible use of SdLv as an effective treatment for Mab infection.


Introduction
Natural products from plants have been used both clinically and as folk medicines for the treatment of various diseasesand have also been demonstrated to be an important resource of novel lead compounds [1,2]. Stellaria dichotoma L. var. lanceolota Bunge (SdLv), generally called a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 In the present study, we examined the anti-inflammatory role and molecular mechanisms of traditional herbal medicines, including SdLv, Buch, and Bufa, in primary macrophages infected with intracellular Mab or T. gondii. Moreover, we further investigated the function of SdLv in the in vivo infection mouse model of Mab.

Preparation of the Buch, Bufa and SdLv
The roots of Bupleurum chinense DC., Bupleurum falcatum L. and Stellaria dichotoma var. lanceolata Bunge were purchased from Kwangmyeongdang Medicinal Herbs Co. (Ulsan, Korea). . Buch (779.31 g), Bufa (798.38 g) and SdLv (793.33 g) were extracted twice with 70% (v/v) ethanol using a 2 h reflux extraction at 80˚C and then concentrated under reduced pressure. The 70% ethanol extracts were filtered through a standard sieve, evaporated to dryness, and freeze-dried. The yields were 12.21% Buch, 20.63% Bufa and 41.86% SdLv. The percentage yield was calculated by dividing the mass of product obtained (g) by the mass of sample (g). Prior to use, the lyophilized powders were dissolved in 0.25% sodium carboxymethyl cellulose (CMC; for in vivo analysis) or 0.01% DMSO (for in vitro analysis).
Tachyzoites of T. gondii RH strain were maintained ARPE-19 cells for 2 or 3 days at 37˚C, 5% CO2 and biweekly passaged in DMEM with 10% FBS, nutrient mixture F-12, antibiotics. To collect T. gondii tachyzoites, cell debris including ARPE-19 cells and parasites were washed in cold phosphate-buffered saline (PBS) and then resuspended in cold culture medium. The suspension was passed through a 27-gauge needle and a 5 μm pore size polycarbonate membrane (Millipore, Bedford, MA, USA) to remove host cells.

Ethics statement
Animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Chungnam National University (CNU-00706) and at Chungnam National University Hospital (CNUH-017-A0012) and conformed to National Institutes of Health guidelines. Animal husbandry was provided by the staff of the IACUC under the guidance of supervisors who are certified Animal Technologists, and by the staff of the Animal Core Facility. Veterinary care was provided by IACUC faculty members and veterinary residents located on the Chungnam National University School of Medicine. The animals were fed standard rodent food and water ad libitum, and housed (maximum of 5 per cage) in sawdust-lined cages in an air-conditioned environment with 12-hour light/dark cycles. To minimize pain or illness of Mab-infected mice, the irreversible condition leading the inevitable death, such as loss of body condition, failure to drink, abnormal breathing, blood in the feces, was monitored daily and each mouse were euthanized with carbon dioxide [39].

Cell counting kit (CCK) 8 assay
The cytotoxicity effects of herbal extracts on BMDMs were determined using CCK 8 assay (Dojindo Molecular Technologies, CK04-11) according to manufacturer's protocol. Briefly, BMDMs were seeded in 96-well plates and differentiated with M-CSF for 5 days as described in cell preparation. Cells were replaced with serum-free media and then incubated with various herbal extracts for 24 hours. Then, 10 μl of the CCK-8 solution was added and incubated for 1 hours at 37˚C. Absorbance was measured at 450 nm using a microplate reader (SPECTRO star Nano, BMG Labtech, Ortenberg, Germany).

Experimental in vivo infection models
For in vivo experiments, we designed two model based on the infection rate of Mab. 1) Mice were orally administrated with SdLv (200 mg/kg) for 4 days, consecutively and then intravenously (i.v.) infected with Mab (5 × 10 8 CFU/mouse). On the 1 day after infection of Mab, mice were further administrated with SdLv for 3 days, consecutively. On the 4 day after infection of Mab, mice were euthanized, and bacterial burden and mRNA expression of Tnf were analyzed in the lungs and spleens, respectively. (2) Mice were orally administrated with SdLv (200 mg/kg) for 4 days, consecutively and then intravenously (i.v.) infected with Mab (5 × 10 9 CFU/mouse). On the 1 day after infection of Mab, mice were further administrated with SdLv for 7 days, consecutively. The body weight and survival of each mouse were monitored for 12 days. For a negative control, 0.25% CMC was orally administered instead of SdLv.
For Western blot analysis, cell lysates were collected and lysed in PRO-PREP (iNtRON BIOTECHNOLOGY, Korea) containing additional set of phosphatase inhibitors. Protein concentration was determined using a BCA assay kit. Proteins (30 μg/each conditions) were immediately heated for 5 min at 100˚C. Each sample was subjected to SDS-PAGE on gel containing 12% (w/v) acrylamide under reducing conditions. Separated proteins were transferred to PVDF membranes (Millipore Corp., Billerica, MA, USA), and then the membranes were blocked with 5% skim milk. Membranes were developed using chemiluminescence assay kit (ECL; Millipore Corp., Billerica, MA, USA) and subsequently analyzed using Chemiluminescence Imaging System (Davinch-K, Seoul, Korea). Data were analyzed using Alliance Mini HD6 (UVitec Cambridge, MA, USA).
In the sandwich ELISA, cell culture supernatants were analyzed using DuoSet antibody pairs (BD Pharmingen) for the detection of mouse tumor necrosis factor (TNF)-α and interleukin (IL)-6.

Statistical analyses
Differences between averages were analyzed by a two-tails paired Student's t-test with Bonferroni adjustment or log-rank test in survival and are presented as the means ± SD. Statistical comparisons were carried out using GraphPad Prism software (GraphPad Software, Inc. La Jolla, CA, USA). Differences were considered significant at p <0.05.

SdLv has no cytotoxic effects on primary macrophages
To investigate the host protective roles of herbal extracts against intracellular pathogens, we first determined the cytotoxic effects of three candidates, including SdLv, Bufa, and Buch, in BMDMs. As shown in Fig 1, the stimulation of BMDMs with SdLv had no significant effect on either cell viability at various concentrations (5-200 μg/mL) over 24 hours (Fig 1C) or on solvent control-stimulated cells. However, cell viability was significantly decreased after treatment with 20 μg/mL Buch (Fig 1A) or 50 μg/mL Bufa (Fig 1B). Based on these results, we used concentrations of SdLv (5-200 μg/ml), Bufa (5-20 μg/ml), and Buch (5-10 μg/ml) that did not exert a cytotoxic effect.

SdLv attenuates Mab-induced generation of proinflammatory cytokines in primary macrophages
Based on the previous studies showing that alkaloid constituents from SdLv and exhibited anti-inflammatory [4] and anti-allergic effects [41], we examined the effects of herbal extracts, including SdLv, Bufa, and Buch, on the Mab-mediated activation of inflammatory responses. As shown in Fig 2A and 2B, we infected BMDMs with Mab for various time periods and then assessed the mRNA (Fig 2A) and protein ( Fig 2B) expressions of inflammatory cytokines, such as TNF-α and IL-6. We also assessed the mRNA expression levels of the Tnf and Il6 genes at 18 hours after the infection of Mab in a MOI-dependent manner ( Fig 2C).
To determine whether herbal extracts, including SdLv, Bufa, and Buch, inhibited the Mabmediated generation of inflammatory cytokines, BMDMs were infected with Mab in the presence or absence of each herbal extract, and the expressions of TNF-α and IL-6 were evaluated by RT-PCR, qPCR, or an ELISA (Fig 2D-2F and Fig 3A-3D). Mab-induced mRNA expression of the Tnf ( Fig 2D) and Il6 ( Fig 2E) genes were significantly attenuated by pretreatment with SdLv in a concentration-dependent manner. Moreover, the secretions of TNF-α and IL-6 in The anti-inflammatory roles of S. dichotoma var. lanceolata in response to M. abscessus culture supernatants after Mab infection were also decreased by pretreatment with SdLv in a concentration-dependent manner ( Fig 2F). However, these inhibitory effects were not observed in the presence of Buch (Fig 3A and 3B) or Bufa (Fig 3C and 3D); rather, Mabinduced mRNA expressions of the Tnf and Il6 genes were significantly increased under some The anti-inflammatory roles of S. dichotoma var. lanceolata in response to M. abscessus conditions when pretreated with Buch and Bufa. These results indicated that SdLv, but not Bufa and Buch, regulated Mab-mediated inflammatory responses.

T. gondii-mediated inflammatory responses are not altered by pretreatment of primary macrophages with SdLv
Because innate immune recognition of T. gondii, a well-known intracellular parasite, promotes host immunity and inflammatory responses similar to those of mycobacterial infection [42,43], we determined the effects of SdLv in regulating the T. gondii-induced activation of proinflammatory responses in primary macrophages. We first evaluated the mRNA (S1A  Because SdLv has anti-inflammatory properties during Mab infection, we identified its roles in T. gondii infection. BMDMs were infected with T. gondii in the presence or absence of SdLv, and the expressions of TNF-α and IL-6 were evaluated by RT-PCR, qPCR, or ELISA (S1D-S1F Fig). As shown in S1D and S1E Fig, pretreatment of SdLv did not affect T. gondiiinduced mRNA expression of the Tnf and Il6 genes compared with T. gondii-infected BMDMs pretreated with the solvent control. Moreover, the secretions of TNF-α and IL-6 in culture supernatants after T. gondii infection were also not altered by pretreatment with SdLv (S1F Fig). Together, these results indicated that SdLv, unlike infection with Mab, had no effects on the regulation of the inflammatory responses of T. gondii infection.

SdLv modulates the activation of c-Jun N-terminal kinase (JNK) and the p38 MAPK pathway in response to Mab but not to T. gondii
MAPKs are known to be essential kinases for the formation of transcription factor complex AP-1, which is involved in the activation of inflammatory responses upon diverse microbial infections [30,44]. We therefore examined whether SdLv affected the MAPK signaling pathways activated by the infection of Mab or T. gondii. Primary macrophages infected with Mab ( Fig 4A) or T. gondii (S2A Fig) showed rapid activation of all three MAPK subfamilies, including p38, extracellular signal-regulated kinases (ERK) 1/2, and JNK, within 30 minutes. Notably, Mab-mediated phosphorylation of p38 and JNK, but not ERK 1/2, were attenuated by pretreatment with SdLv in a concentration-dependent manner (Fig 4B). We next examined whether SdLv regulated the activation of three MAPK subfamilies in response to T. gondii infection in a similar manner as Mab infection. As shown in S2B Fig, T. gondii-mediated phosphorylation of p38, ERK 1/2, and JNK were not modulated by pretreatment with SdLv in BMDMs. We also found that Mab-mediated activations of these kinases were not attenuated by pretreatment with Bufa and Buch, but was rather enhanced in Mab-infected BMDMs pretreated with Buch (10 μg/ml) and Buch (5 and 10 ug/ml) (Fig 4C). Furthermore, we found that BMDMs treated with Buch or Buch showed the enhanced phosphorylation of ERK 1/2, p38, and JNK in a dose-dependent manner, whereas SdLv has no effects ( Fig 4D). Taken together, these findings indicated that SdLv may selectively inhibit the Mab-mediated activation of inflammatory responses through regulation of the p38 and JNK signaling pathways.

SdLv attenuates the activation of the Mab-induced NF-κB signaling pathway in macrophages
NF-κB signaling is activated by the stimulation of diverse pathogens, including bacterial, viral, fungal, and parasitic agents, and is closely associated with the activation of inflammatory responses [44]. We showed that infection with Mab ( Fig 5A) and T. gondii (S2C Fig) resulted in the rapid activation of the kinases, IKKα and IKKβ, leading to the degradation of the NF-κB inhibitor, IκB-α. To examine the inhibitory roles of SdLv in Mab-induced activation of NF-κB signaling, BMDMs were infected with Mab for 30 minutes in the presence of SdLv, and phosphorylation of IKKα/β and degradation of IκB-α were then evaluated using immunoblot assays. As shown in Fig 5B, pretreatment with SdLv in Mab-infected BMDMs effectively attenuated the activation of NF-κB signaling. However, these inhibitory effects were not only observed in Bufa-and Buch-pretreated cells, but were also activated in some conditions pretreated with Bufa and Buch (Fig 5C). Similar to those shown in MAPK signaling pathways ( Fig  4D), the stimulation of Bufa or Buch, but not SdLv, resulted in the degradation of IκB-α and the phosphorylation of IKKαβ in primary murine macrophages (Fig 5D). We next examined the effects of SdLv in T. gondii-induced activation of NF-κB signaling expression. Similar to MAPKs signaling, T. gondii-mediated phosphorylation of IKKαβ and degradation of IκB-α were not attenuated by SdLv pretreatment of BMDMs (S2D Fig). Taken together, these results showed that SdLv effectively attenuated Mab-mediated activation of the NF-κB signaling pathway, which may be crucial for the regulation of inflammation.

SdLv regulates Mab-induced generation of intracellular reactive oxygen species (ROS) in macrophages
Previous studies have reported that intracellular ROS are required for the activation of inflammatory responses as a signaling intermediate [35,38,45,46]. Moreover, Lim et al., reported that enzyme extracts from Stellaria dichotoma had antioxidant properties [6]. We thus assessed the generation of intracellular ROS using oxidized DCFDA (for hydrogen peroxide), DHE (for superoxide anion), and CellROX (for detection of general oxidative stress) using flow cytometry, fluorescence microscopy, or laser-based confocal microscopy, respectively. Exposure of BMDMs to Mab resulted in rapid generation of intracellular superoxide within 10-30 minutes, The anti-inflammatory roles of S. dichotoma var. lanceolata in response to M. abscessus with peak activation at 1 hour after infection (Fig 6A), with an increase of cellular oxidative stress ( Fig 6B). Additionally, pretreatment with SdLv effectively attenuated the Mab-mediated activation of cellular oxidative stress (Fig 6C), intracellular superoxide production (Fig 6D), and intracellular hydrogen peroxide production ( Fig 6E). However, the stimulation of Buch and Bufa mediated the significant increase of cellular oxidative stress in BMDMs. In addition, Mab-induced the activation of cellular oxidative stress was slightly increased in the presence of Buch or Bufa (S3 Fig). Together, these results indicated that Mab-induced activation of oxidative stress was attenuated by SdLv.

SdLv inhibits proinflammatory responses and increases survival of an in vivo mouse infection model of Mab
To investigate the in vivo efficacy of SdLv, we used an Mab mouse intravenous infection model [47,48]. As described in the Material and Methods section and Fig 7A and 7D, the mice that received SdLv (200 mg/kg) for 4 days were intravenously injected with Mab [5 × 10 8 CFU/ mouse (Fig 7A-7C) or 5 × 10 9 CFU/mouse (Fig 7D-7F)] and then further treated with SdLv for the indicated time periods. As shown in Fig 7B, the bacterial burden in the spleen and lung tissues were not significantly altered in SdLv-administered mice. However, the Tnf mRNA expressions in the spleen and lung tissue of each mouse were significantly attenuated by the administration of SdLv (Fig 7C). Based on the anti-inflammatory roles of SdLv in vivo, we further determined the body weight ( Fig 7E) and survival rate (Fig 7F) in each mouse infected by a high dose of Mab (5 × 10 9 CFU/mouse), as described in Fig 7D. As shown in Fig 7E and 7F, there was no change in the body weight between each experimental group ( Fig 7E); however, the lethality associated with the infection of Mab was dramatically decreased in SdLv-administered mice compared with in solvent control-treated mice. Together, these in vivo findings indicated that SdLv improved the survival of mice through the regulation of Mab-induced inflammatory responses but not through bacterial clearance.

Discussion
To date, various efforts have been made to identify the function of major components and to characterize the underlying mechanisms to improve treatments for inflammatory disorders and infectious diseases. In East Asia, a variety of herbal extracts, including SdLv, Bufa, and Buch, have been used to treat fever-related illnesses, such as influenza and malaria [3,8,10]. Notably, diverse compositions including saikosaponin, fatty acids and essential oil in Bupleurum species and flavonoids, sterols, cyclic peptides, and β-carboline-type alkaloids in Stellaria dichotoma are important for their biological activities [6,49]. Although recent studies have shown that these extracts possess diverse biological and physiological properties, the functions of these herbal extracts in mycobacterial or toxoplasma infection and their mechanisms of action have yet to be fully elucidated. In this study, we identified the effects of herbal medicines on the innate immune response to Mab infection in primary macrophages and in vivo in a mouse model. The anti-inflammatory roles of S. dichotoma var. lanceolata in response to M. abscessus Increasing evidence has shown that the interaction between innate immune cells and diverse pathogens rapidly trigger inflammation, thereby playing a crucial role in the activation of host protective immunity. Furthermore, an aberrant activation of inflammation is responsible for immunopathogenesis in various infectious or non-infectious diseases [44]. The Mab complex causes pulmonary disease in patients with cystic fibrosis, which is closely associated with the disruption of balance between inflammation and tissue remodeling during repeated bacterial infections [23,50,51]. Although TNF-α is an essential cytokine that controls mortality and bacterial growth during mycobacterial infection [52,53], excessive production of TNFα results in the impaired survival of mice despite sufficient bacterial clearance [54]. These studies suggested that the optimal regulation of inflammatory responses may be responsible for the improvement of mycobacteria-associated diseases. In this study, we found that SdLv effectively attenuated the Mab-induced mRNA and protein expression of proinflammatory cytokines in primary murine macrophages. Consistent with this finding, in vivo administration of SdLv significantly reduced the mRNA expression of TNF-α in the spleen and lung tissues of Mabinfected mice, although the bacterial burden in each tissue was not altered by SdLv treatment. Moreover, the lethality in Mab-infected mice was significantly improved by treatment with SdLv. These findings indicated that SdLv contributes to host protection against Mab infection by the regulation of inflammatory responses rather than by mycobacterial killing.
During mycobacterial infection, different PRRs recognize bacteria and/or bacterial-derived components and then activate intracellular signals leading to the generation of inflammatory cytokines and the initiation of adaptive immune responses [55]. Previous studies showed that MAPKs signaling pathways are crucial for the activation of inflammatory responses through toll like receptor (TLR) 2 in response to Mab [32,33]. Additionally, during myeloid differentiation primary response gene 88 (MyD88) is also required for the production of TNF-α in BMDMs [33]. Consistent with these findings, M. abscessus subsp. massiliense (M. mas), which belongs to the Mab complex, induced the generation of TNF-α and IL-6 through MyD88-and JNK-dependent signaling pathways in murine macrophages [56]. In the present study, we found that SdLv specifically attenuated Mab-mediated activation of the JNK and p38 MAPK pathways but not of the ERK 1/2 pathways. However, SdLv did not inhibit T. gondii-induced production of inflammatory cytokines or activation of the three MAPK subfamilies. It would have been of interest if the inhibitory roles of SdLv had differed depending on the type of bacteria and protozoa. In acute toxoplasmosis, the hyperactivation of the inflammatory responses is mediated by extensive liver damage and lymphoid degeneration, which are closely related to lethality in mice [57]. The infection of the high virulence type 1 RH strain of T. gondii induced the activation of p38 MAPK and IL-12 production via a MyD88-independent manner in BMDMs [58]. Although we could not extend our findings to the role of MyD88-dependent inflammatory responses, our findings indicated the crucial role of SdLv in the regulation of inflammatory responses in response to Mab but not to T. gondii, which was mediated by the inhibition of the JNK and p38 MAPK signaling pathways in macrophages.
Stimulation of mycobacteria and their components induces the production of inflammatory cytokines through the NF-κB signaling pathway as well as the MAPK signaling pathway, which are crucial for the activation of innate and adaptive immunities [59]. Previous studies reported that Mab infection resulted in the translocation of NF-κB p65 into the nuclei [33,48]. Moreover, the M. mas-induced NF-κB signaling pathway was required for the production of TNF-α and IL-6 in BMDMs [56]. Here, we found that SdLv inhibited the degradation of IκBα and the phosphorylation of IKKα/β in Mab-infected BMDMs.
Numerous studies have reported that intracellular ROS act as signaling molecules and play essential roles in regulating a broad range of biological responses [60]. Previous studies also reported that intracellular ROS generation was required for the production of proinflammatory cytokines via TRAF6-mediated activation of the ASK1-p38 pathway in response to TLR4 [61]. Moreover, ROS regulated TLR4-mediated IL-8 expression via a NF-κB signaling pathway in the human monocyte/macrophage cell line, THP-1 [62]. In a similar manner, intracellular ROS are also involved in the activation of the inflammatory response in M. bovis bacille Calmette-Guerin-or M. tuberculosis-infected macrophages [37,38]. Moreover, Dectin-1-dependent activation of spleen tyrosine kinase is crucial for Mab-induced intracellular ROS generation and the production of inflammatory cytokines in BMDMs [32]. In response to M. mas, NADPH oxidase 2-induced ROS modulated the activation of the inflammatory response via the JNK-dependent signaling pathway [56]. Together, our data strongly support that SdLv plays an important role in the regulation of Mab-mediated intracellular ROS generation and it is thought to be effective against the infection of various atypical mycobacteria.
In the present study, we found that Mab infection induced the generation of inflammatory cytokines in primary murine macrophages, which was significantly attenuated by pretreatment with SdLv, but not Bufa or Buch. To elucidate the reasons for this disparity in the roles of these herbal extracts in Mab infection, we evaluated their cytotoxicity and effect on the activity of the intracellular signaling pathways that regulate the inflammatory response. The Bufa and Buch extracts had stronger cytotoxic effects at lower concentrations compared to the SdLv extract. Moreover, treatment for 1 h with Bufa or Buch, but not SdLv, led to activation of the NF-κB and MAPK signaling pathways and the generation of intracellular ROS. In future studies we plan to identify the active components that mediate cytotoxic and inflammatory effects in these compounds.
In summary, we identified novel functions of SdLv in regulating Mab-induced inflammatory responses by targeting the generation of ROS and the subsequent activation of MAPKs and NF-κB signaling pathways in macrophages. Additionally, SdLv had protective effects against Mab through the regulation of excessive inflammation in the spleen and lung tissues of mice. Our findings suggested the efficacy of SdLv not only against other mycobacterial infections but also for a variety of inflammatory diseases, including sepsis and inflammatory bowel disease, which are closely associated with uncontrolled oxidative stress. Because crude extracts isolated from SdLv contain diverse bioactive compounds, further studies should therefore identify the most effective compounds purified from herbal extracts, which can be used in the regulation of inflammatory responses and to evaluate possible clinical applications for the control of mycobacterial infections.