Anti-inflammatory properties and characterization of water extracts obtained from Callicarpa kwangtungensis Chun using in vitro and in vivo rat models

Callicarpa kwangtungensis Chun (CK) is a common remedy exhibits anti-inflammatory properties and has been used in Chinese herbal formulations, such as KangGongYan tablets. It is the main component of KangGongYan tablets, which has been used to treat chronic cervicitis caused by damp heat, red and white bands, cervical erosion, and bleeding. However, the anti-inflammatory effects of CK water extract remains unknown. This study assessed the anti-inflammatory effects of CK in vivo and in vitro, characterized its main components in the serum of rats and verified the anti-inflammatory effects of serum containing CK. Nitric oxide (NO), tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6) release by RAW264.7 cells was examined by ELISA and Griess reagents. Inflammation-related protein expression in LPS-stimulated RAW264.7 cells was measured by western blotting. Furthermore, rat model of foot swelling induced by λ-carrageenan and a collagen-induced arthritis (CIA) rat model were used to explore the anti-inflammatory effects of CK. The components of CK were characterized by LC–MS, and the effects of CK-containing serum on proinflammatory factors levels and the expression of inflammation-related proteins were examined by ELISA, Griess reagents and Western blotting. CK suppressed IL-6, TNF-α, and NO production, and iNOS protein expression in LPS-stimulated RAW264.7 cells. Mechanistic studies showed that CK inhibited the phosphorylation of ERK, P38 and JNK in the MAPK signaling pathway, promoted the expression of IκBα in the NF-κB signaling pathway, and subsequently inhibited the expression of iNOS, thereby exerting anti-inflammatory effects. Moreover, CK reduced the swelling rates with λ-carrageenan induced foot swelling, and reduced the arthritis score and incidence in the collagen-induced arthritis (CIA) rat model. A total of 68 compounds in CK water extract and 31 components in rat serum after intragastric administration of CK were characterized. Serum pharmacological analysis showed that CK-containing serum suppressed iNOS protein expression and NO, TNF-α, and IL-6 release. CK may be an anti-inflammatory agent with therapeutic potential for acute and chronic inflammatory diseases, especially inflammatory diseases associated with MAPK activation.


CK increased IκBα expression in LPS-stimulated RAW264.7 cells
NF-κB, which is an important nuclear transcription factor 23 , can regulate the inflammatory response and immune response 14 , and is related to rheumatoid arthritis 24 .To gain further insights into how CK inhibits inflammation, LPS-induced RAW264.7 cells were used to investigate the effect of CK on the NF-κB pathway.After 15 min of LPS stimulation, the phosphorylation of p65 increased and IκBα expression reduced.Compared with that in the LPS stimulation group, the phosphorylation of p65 in the DEX group reduced and IκBα expression in the DEX group increased.The decrease of IκBα expression by LPS stimulation was inhibited by CK.However, p65 phosphorylation was not significantly inhibited after treatment with the CK water extract (Fig. 3).C-E) Effect of CK on the expression of COX-2 and iNOS (Due to the blurring of marker, the representative image of iNOS is replaced after the editor's consent.All the complete strip images in this article are in the supplementary file.) in LPS-stimulated RAW264.7 cells.Cells were pretreated with CK (1, 10, or 100 µg/mL) or DEX for 1 h and then stimulated with LPS (1 µg/mL) for 18 h.The protein expression of iNOS and COX-2 was analysed by western blotting.The data are presented as the means ± SDs of three experiments.## P < 0.01, ### P < 0.001, versus the control group.* P < 0.05, ** P < 0.01, *** P < 0.001, versus the LPSstimulated group.

Discussion
Inflammation is an adaptive response that restores tissue function and homeostasis 33 .However, this response generally occurs at the expense of many other physiological processes, indicating that excessive and chronic inflammatory responses cause further damage to the human body 34 .Many studies have indicated that regulating inflammatory responses can treat various diseases and forestall disease complications.However, the use of common anti-inflammatory drugs in the clinic is restricted by side effects.CK is a traditional Chinese medicine, that is thought to have anti-inflammatory properties.However, to our knowledge, no published studies have evaluated its anti-inflammatory effects in vivo or in vitro.This study investigated the anti-inflammatory effects of CK water extract by in vivo and in vitro for the first time.
The anti-inflammatory effects of many traditional Chinese medicine extracts have been confirmed in a classic LPS-induced inflammatory cell model 35 .Therefore, we selected LPS-stimulated RAW264.7 cells as an inflammatory model for in vitro experiment.The steroidal drug dexamethasone (DEX) is frequently used in the clinical treatment of various inflammatory diseases 36,37 .Therefore, we selected DEX as a positive control to evaluate the anti-inflammatory activity of CK.
The anti-inflammatory effect of plant extracts involves inhibiting the secretion of proinflammatory factors and reducing protein expression by affecting related signaling pathways, but not stimulating cells through toxic effects.Therefore, we performed an MTT experiment.iNOS and COX-2 are regulatory enzymes associated with NO release and PGE 2 production, respectively.Many studies have investigated the role of NO in acute and chronic inflammation.NO is also considered to be a mediator of rheumatoid arthritis 38 , and a previous study suggested that NO was involved in RA pathogenesis by boosting blood flow in the synovium and regulating synovial function and articular chondrocytes.PGEs, which are active substances containing unsaturated fatty acids, exist widely in animals and humans and include PGE 2 and thromboxane A 2 (TXA 2 ) 39 .Previous studies have shown that PGE 2 and TXA 2 are regulated by COX-2 protein expression.Therefore, suppressing the levels of NO and PGE 2 is important for the development of anti-inflammatory medicines.Our results showed that 0-100 µg/mL CK water extract had no effect on the viability of nonactivated cells or LPS-induced activated cells.
The CK water extract could inhibit NO release by suppressing iNOS expression, and this inhibitory effect was stronger than that of DEX.However, the CK water extract had no significant effect on the expression of COX-2, suggesting that CK might have a lower cardiovascular risk than DEX (Fig. 10).
In inflammatory responses, activated macrophages release large amounts of cytokines, further increasing inflammatory responses 40 .NO, TNF-α and IL-6 are produced in acute and chronic inflammatory diseases 41 .In this study, all of these inflammatory cytokines were increased by LPS stimulation and were reduced by CK (Fig. 10).These results showed that CK had strong anti-inflammatory activity.Thus, further investigation of its anti-inflammatory potential is necessary.
Typically, inflammation is controlled through the NF-κB and MAPK signaling pathways 42 .After cells are stimulated by LPS, NF-κB is released and quickly transferred to the nucleus, where it activates the transcription of its target genes 43 .MAPKs can regulate cytokine, iNOS and COX-2 gene expression 44 , and are activated by LPS-stimulation.Three parallel MAPK pathways (JNK, ERK and P38) are activated in mammalian cells 45 .Our results showed that CK strongly inhibited JNK, ERK and P38 phosphorylation in the MAPK pathway, and increased IκBα expression in the NF-κB signaling pathway.However, CK did not inhibit p65 phosphorylation in NF-κB signaling pathway.Based on these findings, it can be inferred that CK exerts an anti-inflammatory effect by suppressing activation of the MAPK and NF-κB signaling pathway (Fig. 10).
Moreover, for the first time, we assessed the anti-inflammatory effects of CK by experiment in vivo.The λ-carrageenan induced rat foot swelling model is a classic acute inflammatory model used for new drug development.The CIA rat model is a standard model used in preclinical studies 46 .Therefore, in this study, both of above these animal models were used to assess the therapeutic effects of CK on acute and chronic inflammation.Our results indicated that λ-carrageenan alleviated foot swelling in rat model of foot swelling and reduced the arthritis score and incidence of arthritis in a CIA rat model.These findings are consistent with the in vitro results described above, indicating that CK exerts therapeutic effects on a rat model and may have be used to treat acute and chronic inflammatory conditions, especially rheumatoid arthritis.In brief, CK has been shown to exert strong anti-inflammatory effects on acute and chronic inflammation, and as a new anti-inflammatory traditional Chinese medicine, it has the potential to be further investigated and developed.
The effective constituents of drugs are the foundation for therapeutic action 47 .Traditional Chinese medicine has many components, but only some compounds that migrate in serum are active components 48,49 .Although, the organic acids, phenylethanoid glycosides, and flavonoids of CK have been studied by several research groups 32 , this study examined the components of CK found in rat serum for the first time.Our results showed that a total of 31 components of CK were found in rat serum.We hypothesized that the anti-inflammatory effects of CK were attributable to these factors.
Serum pharmacology involves pharmacological observation of herbal drug-containing serum.We examined the anti-inflammatory effects of CK-containing serum for the first time.Our results showed that blank serum and serum containing 5-1% CK had no effect on cell viability.Consistent with the effect of CK water extract, CK-containing serum regulated iNOS but not COX-2 protein expression.CK-containing serum also regulated the release of NO, indicating that the anti-inflammatory effects of CK water extract were attributable to these components.Furthermore, we examined the effect of CK-containing serum on proinflammatory cytokines.The results showed that CK-containing serum reduced NO, TNF-α and IL-6 levels, which was consistent with the effect of CK water extract.These data further verified that the components of CK within serum had major anti-inflammatory effects.However, there are still limitations to this study.We have not been able to further identify all the anti-inflammatory components in CK, or effectively predict the targets of blood components in diseases of concern.We examined the key targets of the anti-inflammatory effects of CK through pathway analysis, functional analysis, interaction network analysis and other methods.In the future, further investigation and screening of anti-inflammatory components that enter the blood is necessary.

Conclusions
Overall, the anti-inflammatory effect of CK was investigated in vivo and in vitro for the first time.Our results showed that CK exerted notable anti-inflammatory effects and exhibited unique advantages, including (1) inhibiting NO production, leading to anti-inflammatory effects; (2) downregulating the expression of inflammatory cytokines to inhibit the progression of the inflammatory response; (3) suppressing iNOS protein expression

Preparation of CK water extracts
The impurities were removed, and the samples (dry stems and leaves) were moistened thoroughly, cut into small segments or tablets, and dried (60-70 °C), resulting in an 86-98% yield.The sample was mixed at a 1:3 ratio of medicinal materials to water, and heated at 60-80 °C.One sample was heated for 3 h and the other was heated 2 h, after which the decoction was filtered, and the two decoctions were combined.www.nature.com/scientificreports/ the decoction tank, and the filtrate was concentrated into an extract with a relative density of approximately 1.28-1.32(55-60 °C).The sample was placed into the material tray of the microwave vacuum dryer and dried into powder 75-80 °C.

Cell culture
RAW264.7 cells (American Type Culture Collection) were incubated in complete medium consisting of DMEM, 10% FBS and 1% PS in an atmosphere containing 5% CO 2 at 37 °C.After being dissolved by DMSO, the CK water extract was filtered through a 0.22 µm filter membrane, and further diluted with DMEM.

Cell viability assay
RAW264.7 cells (1 × 10 5 cells/mL) were evenly seeded and treated with different concentrations of CK water extract/serum for 24 h.LPS (1 µg/mL) was added one hour later.After 18 h, 10 µL of MTT (Bio Basic Inc) solution (1 mL of medium containing 5 mg of MTT) was added to each well and incubated for 4 h.The crystals were carefully dissolved in DMSO (100 µL/well), and examined using a microplate reader (BMG LABTECH, Germany).

Nitric oxide (NO) assay
The experimental cells (2 × 10 5 cells/mL) were cultured for 24 h and pretreated with CK water extract/ CK-containing serum or DEX for 1 h.Then, the culture solution was stimulated with LPS for 18 h.Next, the supernatant was collected and examined by a nitrite assay kit.

Enzyme-linked immunosorbent assay (ELISA)
ELISA was used to measure IL-6 and TNF-α levels in LPS-induced RAW264.7 cells with or without pretreatment with CK water extract/CK-containing serum or DEX.The supernatants were collected and measured at 450 nm.

Western blot assay
Cells (2 × 10 5 cells/mL) were seeded onto a 12-well plate and incubated for 24 h and pretreated with CK water extract/ CK-containing serum or DEX for 1 h.Then the cells were stimulated with1 µg/mL LPS for 0.25 h (for MAPK-and NF-κB-associated proteins) or 18 h (for iNOS and COX-2).After the total proteins were separated www.nature.com/scientificreports/

Preparation of serum samples
Twelve SD rats (220 ± 20 g) were stochastically divided into two groups (the control and CK groups).In the CK group, the rats were perfused with CK (1000 mg/kg) three times daily.After the seventh administration, the serum was collected, inactivated at 56 °C in a water bath for 15 min, and stored at − 80 °C.Methyl alcohol (four times the volume) was added, and the mixture was vortexed for 2 min.The supernatant was collected after centrifugation at 4 °C and 13,000 rpm for 10 min and then was dried with N2 at 40 °C.Then, methanol (150 µL) was added.The solution was mixed in an ultrasonic bath for 15 min.After centrifugation for 15 min at 13,000 rpm and being filtered through a 0.22 µm pore membrane, the samples were analyzed by LC-MS.

Statistical analysis
The data were statistically analysed with GraphPad Prism 8.0 software.Differences were determined by Student's t-test after one-way ANOVA and the log-rank (Mantel-Cox) test were used (P < 0.05).Activation of the MAPK signaling pathway leads to the activation of JNK, p38, and ERK, which increase the levels of proinflammatory mediators (iNOS) and cytokines (TNF-α and IL-6).CK can inhibit the decrease of IκBα expression and the activation of JNK, p38, and ERK, thereby decreasing the levels of proinflammatory mediators (iNOS) and cytokines (TNF-α and IL-6).In addition, CK-mediated inhibition of the expression of proinflammatory mediators (iNOS) and cytokines (TNF-α and IL-6) can ultimately suppress the release of NO.

Figure 1 .
Figure 1.CK suppressed the expression of iNOS in LPS-stimulated RAW264.7 cells.(A and B) Effect of CK on the viability of RAW264.7 cells.(C-E) Effect of CK on the expression of COX-2 and iNOS (Due to the blurring of marker, the representative image of iNOS is replaced after the editor's consent.All the complete strip images in this article are in the supplementary file.) in LPS-stimulated RAW264.7 cells.Cells were pretreated with CK (1, 10, or 100 µg/mL) or DEX for 1 h and then stimulated with LPS (1 µg/mL) for 18 h.The protein expression of iNOS and COX-2 was analysed by western blotting.The data are presented as the means ± SDs of three experiments.## P < 0.01, ### P < 0.001, versus the control group.* P < 0.05, ** P < 0.01, *** P < 0.001, versus the LPSstimulated group.

Figure 2 .
Figure 2. Effect of CK on NO and inflammatory cytokine production in LPS-stimulated RAW264.7 cells.The release of NO into the culture supernatant was examined with a Griess reagent kit (A).The levels of TNF-α (B) and IL-6 (C) were determined using ELISA kits.The values are expressed as means ± SDs (n = 3).### P < 0.001, versus the control group.* P < 0.05, ** P < 0.01, *** P < 0.001, versus the LPS-stimulated group.

Figure 3 .
Figure 3.Effect of CK on the NF-κB signaling pathway.Total cell proteins were prepared, the expression (Due to the blurring of marker, the representative image of p65 is replaced after the editor's consent.All the complete strip images in this article are in the supplementary file.) and phosphorylation levels of p65 (A, B), and IκBα expression (C) were examined by western blotting.The results are presented as an average of ± SD from three independent experiments.### P < 0.001, versus the control group.** P < 0.01, versus the LPS-stimulated group.

Figure 4 .
Figure 4. Effect of CK on the MAPK signaling pathway.The cells were plated in 12-well plates, incubated for 24 h, incubated with CK or DEX for 1 h, and then stimulated for 15 min with LPS (1 µg/mL).Total cell proteins were prepared and the expression (A) and phosphorylation levels of p38 (B), ERK (C) and JNK (D) were examined by western blotting.The results are presented as the average ± SD from three independent experiments.### P < 0.001, versus the control group.* P < 0.05, ** P < 0.01, *** P < 0.001, versus the LPS-stimulated group.

Figure 5 .
Figure 5.Effect of CK on foot swelling induced by λ-carrageenan in rats.(A) The foot swelling rates (%) of rats (n = 10) treated with carrageenan at 1 h, 2 h, 3 h and 4 h showed the difference in paw volume between the model group and the treatment group.** P < 0.01, *** P < 0.001, versus the model group.

Figure 6 .
Figure 6.Effect of CK on a collagen-induced arthritis (CIA) rat model.(A) Representative images of the effect of CK on CIA rats.(B) Effect of CK on the arthritis score.(C) Effect of CK on the incidence.The results are presented as the average ± SD (n = 9).# P < 0.05, ## P < 0.01, #### P < 0.0001 versus the control group.* P < 0.05, ** P < 0.01, *** P < 0.001, versus the model group.

Figure 7 .
Figure 7.The components of CK were verified by UHPLC-Q-Exactive Orbitrap MS. (A) Positive ionizationmode analysis of the CK water extract.(B) Negative ionization mode-analysis of CK water extract.(C) Positive ionization-mode analysis of CK-containing serum.(D) Negative ionisation mode-analysis of CK-containing serum.The number indicates the chemical composition.The arrow points to the peak time of each chemical component.

Figure 8 .
Figure 8. CK-containing serum decreased iNOS but not COX-2 protein levels in LPS-stimulated RAW264.7 cells.(A) Effect of CK-containing serum and blank serum on the viability of RAW264.7 cells.(B-D) Effect of CK-containing serum on the protein expressions of COX-2 and iNOS in LPS-stimulated RAW264.7 cells.Cells were pretreated with CK-containing serum (1%, 2.5% and 5%) or DEX for 1 h, and then stimulated with LPS (1 µg/mL) for 18 h.The protein expression of iNOS and COX-2 was analysed by western blotting.The data are presented as the means ± SDs of three experiments.## P < 0.01, ### P < 0.001, versus the control group.* P < 0.05, ** P < 0.01, *** P < 0.001 versus the LPS-stimulated group.

Figure 10 .
Figure 10.Proposed molecular mechanism by which CK inhibits LPS-stimulated macrophages.LPS, which is a common macrophage activator, can bind to TLR4, thereby activating macrophages through the NF-κB and MAPK signaling pathway.Activation of the NF-κB signaling pathway leads to the decrease of IκBα expression, which increase the levels of proinflammatory mediators (iNOS) and cytokines (TNF-α and IL-6).Activation of the MAPK signaling pathway leads to the activation of JNK, p38, and ERK, which increase the levels of proinflammatory mediators (iNOS) and cytokines (TNF-α and IL-6).CK can inhibit the decrease of IκBα expression and the activation of JNK, p38, and ERK, thereby decreasing the levels of proinflammatory mediators (iNOS) and cytokines (TNF-α and IL-6).In addition, CK-mediated inhibition of the expression of proinflammatory mediators (iNOS) and cytokines (TNF-α and IL-6) can ultimately suppress the release of NO.