Anti-inammatory effects of DhHP-6 in LPS-induced RAW264.7 macrophages and carrageenan-induced air pouch model in rat

DhHP-6 (Deuterohemin-Ala-His-Thr-Val-Glu-Lys) is a novel peptide mimic of peroxidases that was previously designed in our laboratory. Here, we explored the anti-inammatory potential of DhHP-6 against lipopolysaccharide(LPS)stimulated inammatory response in RAW264.7 cells and carrageenan-induced air pouch model rats. DhHP-6 treatment dramatically attenuated the production of nitric oxide (NO), IL-6, andTNF-α in LPS induced RAW264.7 cells. Also, it blocked phosphorylation and degradation of IκBα and suppressed the nuclear translocation of p65. DhHP-6 (0.2, 0.6, and 2.0 mg/kg) signicantly reduced the levels of total proteins and WBC counts in the exudates of the air pouch model rats. Moreover, MDA contents in the plasma of rats were reduced and SOD activities were enhanced in the DhHP-6-treatment group. Our results strongly show the effectiveness of DhHP-6 as an anti-inammatory agent. The mechanism could be related to the reduction of Reactive oxygen species(ROS), inhibition of NF-κB nuclear translocation, and reduction of pro-inammatory cytokines.

cytokines in APPswe/PSEN1dE9, a transgenic mouse model of Alzheimer's disease [36]. Although DhHP-6 was shown great therapeutic potential, its mechanism of alleviating in ammation remains elusive.
Accordingly, here, we systematically explored the ROS scavenging bioactivity of DhHP-6 and its protective effect in alleviating in ammatory diseases.
LPS induced in ammationin RAW264.7 cells was used to study the anti-in ammatory activity and molecular mechanism of DhHP-6. Furthermore, we used carrageenan as an inducer to set up a rat subcutaneous air pouch model to examine the anti-in ammatory effect of DhHP-6 in vivo.
2.3 Cell line and cell culture RAW264.7 (murine macrophage) cell line (P4), purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), was cultured in DMEM with 10% FBS, 100U/mL penicillin, and 100µg/ mL streptomycin at 37 ℃ with 5% CO 2 and 95% humidity. The medium was changed every day. Cells were passaged at 70-80% con uency.

Estimation of ROS
RAW264.7 cells (5×10 5 cells/well) were seeded into 6-well plates and incubated overnight. The cells were pretreated with various concentrations of DhHP-6(0.25, 0.5, and 1 μM) for 2 h and then incubated with or without LPS (1 µg/mL in PBS) for another 12 h. The cellular ROS levels were estimated by ROS assay kit and a ow cytometer.
2.6 Assessment of NO levels and cytokines release RAW264.7 cells (5×10 3 cells/well) were seeded into 96-well plates and incubated for 24 h. The cells were divided into ve groups, namely the control group, model group, and three treatment groups of low (0.25 μM), middle (0.5 μM), and high (1 μM) doses. The control group was not treated with LPS or DhHP-6. The model group was treated with LPS (1 µg/mL) for 24 h. The cells of the treatment groups were pretreated with different concentrations of DhHP-6 (0.25, 0.5, and 1 μM) for 2 h, and then treated with LPS (1 μg/mL) for 24 h. Culture media was assayed for NO and in ammatory cytokines (IL-6and TNF-α) using NO detection and ELISA kits, respectively.

Western blotting
RAW264.7 cells (5×10 5 cells/well) were seeded into 6-well plates and incubated overnight. These werepretreated with different concentrations of DhHP-6 (0.25, 0.5, and 1 μM) for 2 h. After LPS (1 µg/mL) stimulation of the RAW264.7 cells for 12 h, cytoplasmic proteins and nucleoprotein were extracted using a nucleoprotein extraction kit. Sample protein concentration was determined by the BCA kit. Protein samples (50 µg, 20 µL each) were separated by 10% or 12% SDS-PAGE gels and transferred onto PVDF membranes. After blocking the PVDF membrane with 5% nonfat milk for 1 h, the membrane was incubated with primary antibodies at 4 °C overnight. Then, another incubation was performed with a secondary antibody for 1 h at room temperature (RT), and the bands were detected by ECL reagent. Protein blot images were capturedusing an automatic chemiluminescence image analysis system.
Cell nucleiwere stained with DAPI. Fluorescence images were acquired with a uorescence microscope system.

Animals
Male Sprague-Dawley (SD) rats (6-8 weeks old) were obtained from the Liaoning Changsheng biotechnology corporation (Certi cate of Conformity: SCXK (Liao) 2020-0001). All animal protocols were performed following the guidelines of Guide for the Care and Use of Laboratory Animals of the Jilin University (License No.: 20210417) and were approved by the Animal Care Committee of Jilin University.

Carrageenan-induced air pouch model in rats and experimental design
Forty-eight SD rats were randomly divided into six groups: control group, model group, dexamethasone treatment group, and three DhHP-6 treatment (low, medium, and high dose) groups.Solid DhHP-6 was dissolved in normal saline to prepare DhHP-6 solution. The rats were correspondingly injected intraperitoneally (IP) with normal saline (10 mL/kg, control and model groups), dexamethasone (1 mg/kg), and DhHP-6 (0.2, 0.6, and 2 mg/kg), once a day during the seven days of the challenge period. The air pouch model was established as described previously [5]. Brie y, on day 1, an airbag was formed by subcutaneous injection of 20 mL sterile air into the scapular region on the rat's back. Thereafter,the drugs were administered (IP) for seven consecutive days. On the third day, an additional 10 mL of sterile air was injected into the bag to prevent the cyst's closure. Subsequently, on the seventh day, 0.5 h after administration of the respective drugs, carrageenan (2 mL,0.25% solution in 0.9% w/v saline) was injected into the bag to induce in ammation in all the groups except the control group. The control group received the same volume of saline (0.9% w/v).

Assessment of WBC count and proteins in the exudate
Six hours after the administration of carrageenan or saline, all the rats were anesthetized with sodium pentobarbital (3 mg/mL, 0.1 mL/kg, IP). The airbag was washed with 4 mL of D-Hank's solution, and the entire volume of lavage solution was collected. From each group, 0.1 mL of lavage solution was diluted with 1 mL of red blood cell lysate buffer. The WBCs count in the mixture was estimated using Sysmex Microcell Counter. The lavage solutions were centrifuged for 10 min (1000g, 4 °C) to separate the supernatants. Protein concentrations in the supernatant were measured using the BCAkit.

Assessment of SOD activity and MDA content in the plasma
Blood samples were obtained from the abdominal aorta of anesthetized rats. These were centrifuged for 10 min(3000 g, 4 °C). From each group, 1 mL of the plasma was collected to analyze SOD activity and MDA content following the instructions of the kits.

Statistical analysis
All data were statistically evaluated using statistical software Origin 6.0, and the signi cance of the data was analyzed by single-factor analysis (one-way ANOVA). All the results are expressed as mean value ± standard deviation (SD). P values < 0.05 were considered statistically signi cant. All the charts were drawn using Origin 6.0.

DhHP-6 reduces LPS-induced pro-in ammatory cytokines
Pro-in ammatory cytokinesproduced by the in ltrated cells amplify acute in ammation [3,12]. Therefore, we explored the impact of DhHP-6 on the production of pro-in ammatory cytokines (TNF-α and IL-6). The release of pro-in ammatory cytokines in the culture medium was determined by corresponding ELISA kits. As shown in Fig. 3, LPS treatment enhanced the production ofTNF-α and IL-6 in RAW 264.7 cells. However, after DhHP-6 (0.25, 0.5, and 1 μM) addition, the secretion of TNF-α and IL-6 signi cantly decreased compared to the LPS treated group. Moreover, it was a dose-dependenteffect (Figs. 3A and B).
3.4 DhHP-6 reduces LPS-induced production of NO and expression of iNOS Cellular production of NO is stimulated by nitric oxide synthase (NOS) whichis namely divided into 3 types; endothelial (eNOS), neuronal (nNOS), and induced (iNOS) nitric oxide synthases. Under in ammatory conditions, the production of NO is regulated by iNOS while the expression of iNOS is in uenced by endotoxins, cytokines, etc. [22]. We measured the iNOS levels in the RAW264.7 cells by Western blotting and found that the protein level of iNOS was markedly increased after LPS-stimulation. However, DhHP-6 pretreatment reduced the level of iNOS in a dose-dependent manner (Fig. 4B).

DhHP-6 inhibits LPS-stimulated nuclear translocation of NF-κB
The expression of in ammatory cytokines and iNOS is regulated byNuclear Factor Kappa B(NF-κB) [15]. To understand the in ammation alleviating mechanism of DhHP-6, we examined its effect on the NF-κB signaling pathway. As shown in Fig. 5A, RAW264.7 cells treated with LPS alone (for 12 h) showed a decreased level of IκB-α and increased cytosolic level of phosphorylatedIκB-α. Meanwhile, the cytoplasmic levels of NF-κB p65 also decreased in RAW264.7 cells, and it got accumulated in the nucleus. Interestingly, cellular pretreatment with DhHP-6 reversed these changes in a dose-dependent manner. Furthermore, immuno uorescence experiments were carried out to study the effect of DhHP-6 on the NF-κB pathway. After LPS treatment, cytoplasmic p65 was translocated to the nucleus (Fig. 5B). However, the DhHP-6 treatment could inhibit the nuclear translocation of p65. These results suggest that DhHP-6 could attenuate in ammation by inhibiting the activation of the NF-κB pathway.

DhHP-6 inhibits the exudation of WBCs and proteins in air pouch model rats
We found that compared to the sham group, the population of WBCs and protein contents in the exudates were signi cantly higher in the diseased model group (p < 0.01, Figs. 6A andB). At a high dose, DhHP-6 (2 mg/kg) signi cantly reduced the WBCs count and protein contents in the exudates (both having p < 0.05). A similar anti-in ammatory response was also observed in the dexamethasone-treated group (p< 0.05). In the low (0.2 mg/kg) and medium (0.6 mg/kg) dose DhHP-6 treated groups(0.2 and 0.6 mg/kg), the WBCs count and protein contents were not signi cantly different, compared to the model group (both p> 0.05). All these results indicate that high doses (2 mg/kg) of DhHP-6 may potently inhibit the leucocytes and protein extravasation in response to in ammation.

DhHP-6 enhanced SOD activity and reduced MDA content in the plasma of air pouch model rats
The results (Figs. 7A and B) showed that compared to the sham group, SOD activities were signi cantly decreased, whereas MDA contents were increased in the rat's plasma of the diseased model group (both p< 0.05). However, compared to the model group, SOD activities increasedsigni cantlyafter treatment with a high (0.6 mg/kg) and medium (2.0 mg/kg) dose of DhHP-6 (both p< 0.01). However, low-dose DhHP-6 (0.2 mg/kg) showed no statistical difference (p> 0.05). Furthermore, compared with the model group, different doses of DhHP-6 (2.0, 0.6, and 0.2 mg/kg) signi cantly decreased the MDA contents in the plasma of air pouch model rats (compared with the model group, p <0.01, p <0.01, and p <0.05, respectively). All these results suggest a signi cant anti-oxidant effect of DhHP-6.

Discussion
The in ammatory response is a kind of host defense mechanism; however, an uncontrolled in ammatory response can lead to several chronic diseases [17,18,25,19,28]. Therefore, effective control of in ammation is essential to treat such diseases. Excessive accumulation of ROS can damage normal tissues further aggravating in ammatory reactions [10,20,14]. DhHP-6, a novel peroxidase mimetic peptide, can effectively scavenge ROS [32]. Previously, we showed the therapeutic effects of DhHP-6 in in ammation-related diseases;however, the mechanism remained unknown. Here, we examined the antiin ammatory mechanism of DhHP-6.
Macrophages, one of the immune cells, produce major pro-in ammatory factors and play an indispensable role in in ammation. RAW264.7, a monocyte/macrophage-like cell line, is an established in vitro model for in ammation-related studies [8]. Endotoxins, such as LPS,are the key components of the cell wall of gram-negative bacteria [24]. LPS, from Escherichia coli O111:B4, is commonly used as an inducer to immune cells [4,30], which binds to the toll-like receptors (TLRs) and initiates in ammation [34].
LPS stimulates the release of a variety of pro-in ammatory cytokines, such as TNF-α, IL-6, and NO [1]. We measured TNF-α, IL-6, and NO in the cell supernatant after LPS treatment with or without pretreatment with DhHP-6 and found that DhHP-6 reduced their levels. The production of NO is mainly regulated by iNOS [22], an inducible and Ca 2+ -independent isoform of NOS [29]. We tested the effect of DhHP-6 on iNOS expression in LPS-treated RAW264.7 cells and found that DhHP-6 could reduceiNOS levels.
LPS binds to the TLR4 receptor of macrophages, and thereby activates many intracellular in ammatory pathways. NF-κB, an important nuclear transcription factor, promotes the transcription of in ammationassociated mediators, including iNOS, and pro-in ammatory factors such as TNF-α and IL-6; which contain NF-κB binding motifs κB DNA elements [33,31,9]. The prominent NF-κB family members are p50, p52, Rel (p65), c-Rel, and RelB [9]. The p65-p50 heterodimer is the most common form of NF-κB. Normally, the IκB protein binds to the NF-κB subunit to form a trimeric complex [13]. It keeps NF-κB in the cytoplasm and inhibits its transport to the nucleus. However, under in ammation, IκB kinase is activated to phosphorylate IκB-α, triggering IκB-α dissociation from NF-κB. Furthermore, phosphorylation of IκB-α leads to its ubiquitination at Lys 48 for proteasomal degradation. The inhibitory effect of IκB on NF-κB is relieved, and the cytoplasmic NF-κB gets translocated to the nucleus to initiate the transcription of target genes [7]. NF-κB signaling is modulated by posttranslational modi cations, such as acetylation [26], phosphorylation [23], and methylation [2]. Oxidative stress is involved in the non-classical activation pathway of NF-κB. ROS activate phosphatidylinositol 3-kinase (PI3K) which phosphorylates tyrosine 42 on IκB [27]. Here, we showed that DhHP-6 pretreatment reduced the level of ROS and phosphorylation of IκB, and promoted the level of IκB in LPS stimulated RAW264.7 cells. Also, the nuclear transfer of NF-κB and activation of NF-κB signal pathway by LPS were inhibited.
To further verify the in vivo anti-in ammatory effect of DhHP-6, we established a murine in ammation model. Some animal in ammatory models are simulated by exogenous substances to activate immune cells. The subcutaneous air pouch is one of such models, which involves the subcutaneous injection of sterile air into the intra-scapular area of the back of the animal. This is thenfollowed by an injection of irritants like carrageenan.Carrageenan, an acidic mucopolysaccharide, increases vascular permeability [5]. This in ammation model is convenient and the pathological changes include pouch lining of macrophage and broblast-like cells, increase in exudate. The cellular in ltration is extremely similar to human acute intra-articular synovitis. Therefore, we chose this model to explore the anti-in ammatory effects of DhHP-6. We showed that DhHP-6 exhibited anti-in ammatory activity, and inhibited the exudation of WBCs and protein in the air pouch mice model. SOD is an important anti-oxidase that clears the superoxide anion (O2 -· ) into hydrogen peroxide and molecular oxygen. The cellular level of MDA, the end product of lipid peroxidation, signi es in vivo oxidation status and is considered as an oxidative stress marker. Here, we showed that the peroxidase mimetic DhHP-6 (2.0 and 0.6 mg/kg) signi cantly increased the SOD activity (p < 0.01) while decreased the plasma content of MDA (p < 0.01). Overall, these results showed that DhHP-6 exerts a good antioxidant effect in the carrageenan-induced gasbag-synovitis model in rats.
In conclusion, we showed that DhHP-6 exhibited anti-in ammatory effects in both LPS induced RAW264.7 cells and the air pouch rat model. DhHP-6 may serve as a potential therapeutic agent for the treatment of acute and chronic in ammatory diseases.

ETHICS APPROVAL
The study was approved by the Animal Care Committee of Jilin University and conformed to the Animal Ethical Standards and Use Committee at Jilin University.

CONSENT TO PARTICIPATE
Not Applicable

CONSENT FOR PUBLICATION
The participants have consented to the submission of this research to the journal.

AVAILABILITY OF DATA AND MATERIAL
We make sure that all data and materials support the published claims and comply with eld standards.
All data and materials are fully available. Effect of various concentrations of DhHP-6 on the viability of RAW264.7 cells. The cells were exposed to different concentrations of DhHP-6. The cytotoxicity of DhHP-6 was determined by WST1 assay after 24 h of treatment. P < 0.05 vs. NC group,**p < 0.01 vs. NC group.  DhHP-6 reduces LPS-induced expression of pro-in ammatory cytokines in RAW264.7 cells. After different treatments, cell-secreted TNF-α, and IL-6 were measured by ELISA. TNF-α (A), and IL-6 (B) in the LPS group were signi cantly higher than in control groups; however, DhHP-6 could reduce them. All data are mean ± standard deviation (n=8). ##p< 0.01 vs. Control group, *p< 0.05 vs. LPS group, **p< 0.01 vs. LPS group.   DhHP-6 inhibited the exudation of WBCs and protein in gasbag-synovitis-induced rats. The exudates were collected as described in the method section. WBCs count was measured in the lavage solutions (A) and total protein was measured by BCA protein kit (B).All data are mean ± standard deviation (n=8). ##p< 0.01 vs. Control group, *p< 0.05 vs. LPS group, **p< 0.01 vs. LPS group.