Bullatine a Exerted Anti-Inflammatory Effects by Inhibiting JNK/ROS/NF-κB Pathway and Attenuates Systemic Inflammatory Response in LPS-Challenged Mice


 Background: The genus Aconitum has rich pharmacological characteristics. Aconiti brachypodi Radix (Xue-shang-yi-zhi-hao) is a dried root of aconitum, which is considered to be analgesic and anti-inflammatory in modern medical and pharmaceutical clinical studies. Bullatine A (BA), a major active ingredient of this plant, has been reported for its significant anti-analgesic effect in previous studies. However, the role of BA in inflammation is unknown. In the current study, we aimed to explore the effect of BA on lipopolysaccharide (LPS)-induced inflammatory response both in vitro and in vivo and its potential anti-inflammatory mechanism.Materials and Methods: The anti-inflammatory effect of BA was evaluated in two different types of LPS-induced macrophages, including BV-2 microglial cells and immortalized murine bone marrow-derived macrophages (iBMDMs), and in acute inflammation mouse models induced by LPS. Immunofluorescence, flow cytometry, quantitative RT-PCR, western blot and Hematoxylin-Eosin staining were used to determine the anti-inflammatory properties of BA.Results: The results showed that BA significantly reduced the mRNA levels of several pro-inflammatory cytokines induced by LPS both in BV-2 cells and iBMDMs. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in response to LPS were also decreased by BA. Further investigations indicated BA significantly blocked the phosphorylation of IκB kinase, degradation of the inhibitor IκBa and the nuclear translocation of nuclear factor-κB (NF-κB) p65. BA also reduced c-Jun N-terminal kinases (JNK) phosphorylation and ROS generation in iBMDMs activated with LPS, but had no effect on other mitogen-activated protein kinases (MAPKs) family proteins such as extracellular signal-regulated kinase (ERK) or p38. Furthermore, BA treatment alleviate liver and lung tissue damage, reduce inflammatory cell infiltration, and inhibit the expression of inflammatory cytokines in LPS-challenged mice.Conclusions: This study illustrated that BA has obvious anti-inflammatory effects both in vitro and in vivo, and its underlying anti-inflammatory mechanism may be via inactivating JNK/ROS/NF-κB pathway. Therefore, BA may have a certain therapeutic potential for inflammatory-related diseases.

Bullatine A (BA, C22H33NO2, shown in Figure. 1A), an alkaloid, is one of the major bioactive compounds isolated from Aconiti brachypodi Radix. In recent years, the alkaloids of aconitum has been proved to have therapeutic analgesic effects 6 . Evidence from Ren et al. 3 systematically administered the ethanol extract of Aconiti brachypodi Radix (including BA) and found that it effectively attenuated pain response in mice tests with hot-plate, acetic acid, and formalin. Further study reveals that BA is the active compound, which attenuates the pain hypersensitivity in a variety of rat pain models 7 . Recently, BA is found to inhibit ATP-induced BV-2 microglial cell death and P2X receptor-mediated in ammatory responses 2 . These studies suggest BA might have the anti-in ammatory activity. Not only that, BA has great potential to be a candidate drug for the prevention or treatment of in ammation-mediated diseases, since studies has shown that it is less toxic than most other alkaloids isolated from aconitum 7,8 .
However, up to now, few studies have investigated whether BA has a potential effect in lipopolysaccharide (LPS) -induced in ammatory responses in vivo, and its underling mechanism remains unclear.
Accordingly, the aimed of present study was to explore the effect of BA on LPS-induced in ammation in macrophages and microglia, with a protective effect of BA pretreatment on LPS inducing systemic in ammation in model mice. Furthermore, the study also sought to elucidated the underlying molecular mechanism during these processes.

Measurement of intracellular ROS
The intracellular ROS was measured by staining cells with 2',7'-Dichloro uorescin diacetate (DCFH-DA, Beyotime). Brie y, cells were treated as indicated above, and incubated with 10 μM DCFH-DA at 37 °C for 20 min. Cells were then washed twice with serum-free medium and analyzed by FACS Vantage (BD Biosciences) ow cytometer.

Immuno uorescence Assay
Cells grown on coverslips were treated with BA for 1h and were given LPS stimulation for the next 2h.
In vivo LPS challenge LPS (5 mg/kg) was injected into the peritoneal cavity of mice after pretreatment with 10 mg/kg BA (diluted with saline) i.p. injection for two times. Four hours later, all mice were killed, liver and lung tissue were collected and stored -80 ℃.

Histological assessment
The liver and lung tissues were xed in 4% paraformaldehyde and embedded in para n. After that, the sections of various organs were stained with hematoxylin & eosin (H&E). The pathological scores for the liver and lung were determined according to previous studies with minor modi cations 9 . The score was mainly determined as the degree of immune cell in ltration and structure disruption as follows: 0 = none, 1 = mild, 2 = moderate, and 3 = severe.
Quantitative real-time PCR Total RNA was collected from tissues and cultured cells by extraction with TRIzol TM Reagent (#15596026, Life Technologies) according to the manufacturer's instructions. Tissues were nely chopped in TRIzol and immediately homogenized using a homogenizer. Then, mRNA from each sample was converted to cDNA according to cDNA synthesis kit (AE311-03, TransGen Biotech, Beijing, China). Quantitative PCR was performed using SYBR Green master mix (#A304-10, GenStar, Beijing, China) with ABI studio Q3 Real Time PCR system (Life techonologies, USA). The primer sequences used were listed in Table 1.

Western blotting
Western blot analyses were conducted as previously described 10 , In brie y, tissues or cells were lysed with Radioimmunoprecipitation assay (PIRA) buffer, which contains a cocktail of protease and phosphatase inhibitors. The protein was isolated by SDS-PAGE and then transferred to polyvinylidene uoride membrane (PVDF) membrane (#ISEQ00010, Millipore, Darmstadt, Hessen, Germany). The membrane was blocked with 5% nonfat milk in Tris-buffered saline and incubated overnight with primary antibodies at 4 °C. Finally, the protein was detected by horseradish peroxidase-conjugated secondary antibodies.

Statistical Analysis
We used GraphPad Prism software (version 8) for statistical analyses. All data were presented as means ± SD unless otherwise noted. The signi cance of differences was assessed by unpaired Student's t-test or one-way ANOVA analysis of variance. p < 0.05 was considered statistically signi cant.

Bullatine A inhibits transcription levels of pro-in ammatory cytokines in LPS-activated microglia and macrophages
To evaluate the potential cytotoxicity of BA ( Figure 1A), BV-2 microglial cell lines were incubated with or without BA for 24 h. Cell viability assay con rmed that incubation with BA for 24 hours had no signi cant cytotoxicity on BV-2 cells at concentrations below 80 μM ( Figure 1B). Therefore, this concentration range was used to investigate the following anti-in ammation effect of BA in vitro. To further investigate con rm the anti-in ammation effect of BA, BV-2 cells were pretreated with BA (40 and 80 μM) for 1 h and then incubated with 1 ug/mL LPS for 6 h. Results showed mRNA levels of several pro-in ammatory cytokines, including IL-6, iNOS and TNF-α, were signi cantly upregulated after LPS stimulation. However, this effect was signi cantly weakened when BA concentration reached 80 μM ( Figure 1C to E). Furthermore, we also examined the effect of BA on iBMDMs, macrophages that play critical roles in host innate immunity and are therefore commonly used for in vitro cell-based assays examining the mechanisms of innate immune activation 11 . Consistent with the results in BV-2 cells, BA inhibited IL-1β, IL-6 and iNOS mRNA expression occurred in a dose-dependent manner in LPS-stimulated iBMDMs ( Figure 1F, G, and H). Together, these results indicate that BA inhibited LPS-induced in ammation effectively in microglia and macrophages.
Bullatine A reduced the high expression of iNOS and COX-2 proteins induced by LPS We further determined the effects of BA on the protein levels of iNOS and COX2 induced by LPS. The experimental results were shown in Figure 2A, the protein levels of iNOS and COX-2 proteins were signi cantly increased after LPS stimulation. However, 80 μM of BA signi cantly inhibited the high expression of iNOS and COX-2 protein and the density ratio of iNOS and COX-2 proteins to the corresponding GAPDH bands was shown in the Figure 2B and C. These results further con rmed the antiin ammation activity of BA.

Bullatine A inhibits the activation of NF-κB pathway
It is well known that NF-κB signaling pathway plays a critical role in cellular in ammatory response, which is mainly manifested by the NF-kB transcription factor regulating the expression of proin ammatory cytokines, iNOS and COX-2 proteins and involved in oxidative stress and in ammation 12 .
To further explore the anti-in ammatory mechanisms of BA, we evaluated changes in several key components of NF-κB signaling. As displayed in the Figure 2D, LPS stimulation markedly increased the protein levels of p-IKKα/β and p-IκBα. These effects, however, were signi cantly inhibited after BA pretreatment, especially after 1h of LPS stimulation, and the band density corresponding to the phosphorylation levels of IKKα/β and IκBα proteins were normalized on the basis of their total proteins ( Figure 2E and F).
In response to LPS stimulation, the translocation of NF-κB p65 subunit to the nucleus was dramatically increased, which is considered to be an important feature in the detection of NF-κB signaling pathway. Our immuno uorescence assay results showed that BA preconditioning inhibited the NF-κB p65 subunit transport to the nucleus after 2 hours of LPS stimulation ( Figure 2G and H). Western blot also supported this phenomenon, as shown in the Figure 2I, LPS exposure resulted to the translocation of the NF-κB p65 from the cytosol to the nucleus, whereas a signi cant reduction of nuclear p65 protein and a signi cant increment of cytoplasmic p65 protein was observed after pretreatment with BA ( Figure 2J and K). Taken together, these results indicate that BA inhibits the release of pro-in ammatory factors induced by LPS probably through the inhibiting activation of NF-κB signaling pathway.
The effect of Bullatine A on the activation of MAPKs signaling pathway Mitogen-activated protein kinases (MAPKs) also play an important role in extracellular signal transduction into cellular responses 12 . Therefore, we next to explored the effect of BA on the MAPKs signaling pathway by Western blot. As shown in Figure 3A, all phosphorylation pathways of MAPKs, such as p38, JNK and ERK1/2, were signi cantly increased after 0.5 h or 1.0 h stimulated by LPS. The results showed that pretreatment BA signi cantly inhibited the phosphorylation of JNK, while did not show remarkable inhibitory effect on p38 and ERK1/2 ( Figure 3B to D). These results indicated that BA inhibits the activation of NF-κB pathway by impairing the phosphorylation of JNK.

Bullatine A reduced LPS-induced stimulation of reactive oxygen species (ROS)
It's reported that JNK could be activated by ROS, and ROS is a pro-in ammatory factor in LPS-induced in ammation. To investigate whether BA could inhibit the ROS production, we measured intracellular ROS levels was assayed using DCFH-DA. As shown in Figure 3E, LPS induced ROS rise was signi cantly as compared with control group. However, pretreatment with BA (80μM) markedly attenuated LPS induced ROS rise ( Figure 3F). These results con rmed that BA could obviously restrain intracellular ROS production, which is consistent with the effect on the activation of JNK pathway.

Bullatine A protected liver and lung injuries in LPS-challenged mice
The above studies proved that BA had an anti-in ammatory activity in vitro, we then intend to study its activity in vivo. To investigated whether BA pretreatment could suppress in ammatory responses in vivo, we set out induced production of systemic in ammatory disturbance in mice via intraperitoneal injection LPS. The effect of BA on LPS-induced tissue damage was demonstrated by H&E staining. As shown in Figure 4A, after LPS administration, neutrophils, characterized by dark nuclei (indicated by arrows) were in ltrate the liver wall. However, pretreatment with BA inhibited neutrophil in ltration. Similarly, LPS administration caused disruption of alveolar space compared with lung of saline-treated mice. However, pretreatment with BA improved LPS-induced disruption of alveolar space structure ( Figure 4A). All the above pathological changes were statistically signi cant by histological assessment ( Figure 4B).

Bullatine A reduced in ammation gene expression in LPS-challenged mice
Next, we investigated whether BA pretreatment inhibited the LPS-induced gene expression of the proin ammatory cytokines in liver and lung. Our results demonstrated that intraperitoneal injection of LPS induced the increased the mRNA levels of in ammation-related genes (IL-1β, IL-6 and iNOS) in the liver of model mice, consistent with the induction of acute in ammation. However, pretreatment with BA signi cantly reduced the expression of the above indicators ( Figure 4C). A similar effect was observed in the lungs, but the effect was not signi cant ( Figure 4D). Taken together, these results indicating that BA pretreatment also exerted favorable anti-in ammatory effects in vivo.

Discussion
In recent decades, a large number of studies have sought natural compounds isolated from plants, which often have advantages in treating diseases due to their relatively low toxicity and side effects. BA, one of the major components extracted from Aconiti brachypodi Radix, has various pharmacological activities. It has been reported that BA can selectively inhibit P2X7 receptor mediated in ammatory response, suggesting its anti-in ammatory potential 2 . However, until now, its effect on LPS-induced in ammatory response whatever in vitro or in vivo and the potential mechanism of its anti-in ammatory activities have not been investigated in detail. Herein, we con rmed that BA exerts anti-in ammatory effects by inactivating the transcriptional activity of NF-κB, which provides a theoretical basis for the development of new drugs that targeting NF-κB signaling pathway in the clinical application of in ammatory diseases.
Macrophages are not only an important innate immune cell in the body's defense system, but also a major cell involved in the in ammatory response 13 . The reason why macrophages play an important role in the process of in ammatory response is that it involved in the acute phase of in ammatory diseases by releasing pro-in ammatory cytokines (TNF-α, IL-1β and IL-6, etc) 14 . If the activated macrophages continue to release superabundant proin ammatory factors and in ammatory mediators, an excessive or uncontrolled in ammatory response can result, leading to severe tissue damage and septic shock 15 . Therefore, therapies aimed at inhibiting proin ammatory cytokines is of great signi cance in in ammatory-related diseases. As the main component of endotoxins and the main molecular basis of pathogenesis, LPS is often used in experiments to activated macrophages to evaluate the antiin ammatory activity of some plant extracts 16 . To investigate the anti-in ammatory effects of BA in vitro, the effects of BA on the production of LPS-induced pro-in ammatory cytokines were examined in two different types of macrophages. The results of our study showed BA reduced the RNA and protein levels of in ammatory factors in LPS activated macrophages. Especially, the inhibitory effect of BA on the excessive production of pro-in ammatory factors in iBMDMs is much more pronounced, suggesting that it has a better medicinal effect on peripheral in ammation.
Next, we explored the mechanisms underlying the anti-in ammatory effects of BA. It is well known that NF-κB is a dimer formed by the p50 and p65 subunits, which plays an important role in the regulation of pro-in ammatory mediators. Its dysfunction has been associated with many choric diseases, including asthma, cancer, diabetes, rheumatoid arthritis, in ammation, and neurological disorders, and is therefore considered as a potential drug intervention target for the treatment of in ammation [17][18][19] . The nuclear translocation of p65 is thought to key signal for NF-κB activation 20 . While in the resting condition, NF-κB combined with its inhibitor protein IκBα and co-locates in the cytoplasm 21 . Once stimulated, IκB kinase engages in phosphorylation of NF-kB P65 and ubiquitin-mediated degradation of this product via the proteasome pathway, then translocated p65 into the nucleus where it triggers the transcription of speci c target genes such as TNF-α, IL-1β, and IL-6 22,23 . The protective effects of BA on LPS-induced in ammatory response may be attributed to the inhibition of NF-κB pathway. Results in the current study showed that BA inhibited the degradation of IκB-α and also inhibited the translocation of NF-κB p65 to the nucleus. In addition, iNOS and COX-2, the downstream targets of NF-κB 24 , which are believed to be closely related to the occurrence and development of a variety of in ammatory diseases 25 , and also suppressed by BA. These ndings suggest that BA may inhibit transcriptional activity of NF-κB, thereby could normalize the expression of pro-in ammatory cytokine and in ammatory mediator.
MAPKs are also involved in LPS-induced in ammation. Activated MAP kinases trigger the expression of target genes in controlling the synthesis and release of cytokines during the in ammatory response, leading to biological responses, including the expression of pro-in ammatory mediators 26 . Here, we found that BA signi cantly inhibited the phosphorylation of JNK and the production of ROS. ROS levels that exceed the antioxidant capacity of cells are thought to be involved in the pathogenesis and development of various pathological conditions, including cardiovascular disease, cancer, and aging 27 .
Moreover, it has been reported that ROS serves as an upstream molecule that directly activates the NF-κB pathway 28,29 . Previous studies suggested some natural components of plants can exert their pharmacological effects by inhibiting ROS/NF-κB pathway. For example, Yao et al. 30 reported that Marein protected human nucleus pulposus cells against high glucose-induced injury and extracellular matrix degradation by inhibiting the ROS/NF-κB pathway. Barbaloin was also reported possesses a protective effect on LPS-induced acute lung injury by inhibiting the ROS-mediated PI3K/AKT/NF-κB pathway 31 . In the present study, we found that LPS exposure led to ROS levels rise in iBMDMs, which was attenuated by BA, suggesting that BA exert anti-in ammatory activity may be through regulating the JNK/ROS/NF-κB pathway (Fig. 5).
In conclusion, we demonstrated that BA effectively inhibits the expression pro-in ammatory cytokines by inactivating JNK/ROS/NF-κB pathway in vitro. More importantly, BA preconditioning attenuates the in ammation and tissue injury of liver and lung in LPS-induced acute in ammation model mice. These ndings provide a theoretical basis for the clinical application of BA in the treatment of in ammatory diseases, reveal the pharmacological activities of different active components of Aconiti brachypodi Radix.

Figure 4
Bullatine A attenuated liver and lung in ammation and damage in LPS-challenged mice. BA (i.p., 10 mg/kg) was administrated to mice two times (once every 2h) prior to treatment with LPS (i.p., 5 mg/kg).
(A) Representative pictures of H&E staining of the sections of liver and lung after administration of saline, LPS, and/or BA (scale bars 100 μm). (B) The pathological score of liver and lung (n = 7). The mRNA level of IL-1β, IL-6 and iNOS of liver (C) and lung (D) was measured by quantitative RT-PCR (n = 7). Data are presented as mean ± S.E.M. *P < 0.05 vs. LPS treatment group.

Figure 5
Schematic diagram of our study.