Paeoniflorin Inhibits LPS-Induced Activation of Splenic CD4+ T Lymphocytes and Relieves Pathological Symptoms in MRL/lpr Mice by Suppressing IRAK1 Signaling

Interleukin-1receptor-associated kinase 1 (IRAK1) plays a critical role in systemic lupus erythematosus (SLE). It was reported that SLE was associated with an inflammatory response mediated by defective immune tolerance, including overproduction of autoantibodies, chronic inflammation, and organ damage. Previous reports stated paeoniflorin (PF) had an immunosuppressive effect. The purpose of this study was to determine the anti-inflammatory effect of PF in SLE and its underlying mechanisms. Followed by induced with lipopolysaccharide (LPS), the splenocytes and the isolated CD4+ T lymphocytes of MRL/lpr mice were divided into three groups: control group, LPS group, and LPS + PF group, respectively. MRL/MP mice were used as the control group (treated with distilled water). The MRL/lpr mice were randomly divided into three groups: the model group (treated with distilled water), the prednisone group, and the PF group. The MRL/lpr mice were treated with prednisone acetate (5 mg/kg) and PF (25, 50, and 75 mg/kg) for eight weeks. Subsequently, ELISA, qRT-PCR, western blotting, HE, and Masson staining were performed to detect various indicators. The results of Cell Counting Kit-8 (CCK-8) showed that 10 μg/mL of LPS had the optimum effect on cell viability, and 50 μmol/L of PF had no obvious cytotoxicity to LPS-treated cells. PF reduced the expression level of IRAK1-nuclearfactor-κB (NF-κB) and its downstream inflammatory cytokines in the splenocytes and CD4+ T lymphocytes of MRL/lpr mice stimulated by LPS, especially in the latter. The serum antibody contents in the PF group mice were reduced, and the kidney damage was also alleviated accordingly. Moreover, the IRAK1/inhibitor of the nuclear factor-κB kinase (IKK)/NF-κB inhibitor (IκB)/NF-κB pathways was found to be involved in the anti-inflammation effect of PF in the kidney and spleen. In conclusion, it is thought that PF may have the potential to be used as a therapeutic agent to reduce the inflammatory activity of SLE. Inhibition of the IRAK1-NF-κB pathway may help formulate novel therapeutic tactics for SLE.


Introduction
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that afects multiple organ systems. It was reported that SLE was associated with an immune tolerance defciency-mediated infammatory response, including overproduction of autoantibodies, chronic infammation, and organ damage [1,2]. Glucocorticoids and immunosuppressive agents are efective in the treatment of SLE.
However, there are many serious side efects, such as metabolic disorders, susceptibility to infection, and withdrawal reactions [3]. Biotherapies, such as belimumab, limit their extensive clinical application due to antisingle subtype and expensive price [4]. Recently, increasing attention has focused on revealing SLE pathogenesis, especially its role in infammatory signals.
Interleukin-1receptor-associated kinase 1 (IRAK1) is a serine/threonine kinase that targets the downstream of the interleukin-1 receptor (IL-1R) and participates in the activation of the autoimmune Toll-like receptor (TLR) signaling pathway [5,6]. TLRs have been identifed as therapeutic targets for SLE, among which TLR4 is found to contribute to the auto-infammatory diseases [7,8]. IRAK1 is a crucial signal molecule involved in the TLR4 signal transduction pathway [3]. Te related connexin IRAK1 will be activated by TLR through the myeloid diferentiation primary response 88 (MyD88)-dependent signaling pathway, which leads to the activation of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and inhibitor of nuclear factor-κB (NF-κB) kinase (IKK) [9]. Eventually, NF-κB is activated to induce the synthesis and secretion of infammatory cytokines [10,11]. It was reported by multiple studies that the IRAK1 gene polymorphism was related to the susceptibility of human SLE, which might be a potential therapeutic target for SLE [12][13][14]. However, the exact functional outcome of the IRAK1 gene polymorphism on SLE susceptibility still needs further research.
It was found that the misfunction of the NF-κB pathway was engaged in autoimmune diseases and infammatory diseases [15,16]. NF-κB was reported to be involved in the pathogenesis of SLE, in which IRAK1 played a crucial role in the abnormal activation of NF-κB in infammatory diseases [9]. Consequently, inhibiting the activity of IRAK1-NF-κB may be an efective strategy for developing new therapies for SLE or other infammatory diseases. However, there are few studies on the function of the IRAK1-NF-κB signal in SLE.
At present, traditional Chinese medicine (TCM) for the clinical treatment of autoimmune infammatory diseases has shown excellent development potential [17]. Te Jieduquyuziyin Prescription (JP), containing ten traditional Chinese herbs, is clinically used to treat SLE. Our previous research confrmed that JP could inhibit infammation, alleviate the side efects of clinical drugs, and reduce the incidence of infection [18]. Recent clinical studies have found that JP could reduce the side efects and enhance the efcacy of glucocorticoids (GC) in the treatment of SLE [19,20]. Importantly, paeoniforin (PF) is the main active ingredient of JP [21]. PF was the chief biologically active ingredient in the herb Paeonia lactifora Pall (Chinese peony), which had good anti-infammatory and immune-regulating functions [22,23]. PF could regulate the activation and proliferation of T lymphocytes and participate in the infammation and immune process of autoimmune diseases through signal pathways such as the NF-κB pathway [24]. Published studies state that PF might prevent the depressive behavior caused by SLE through the high mobility group protein B1 (HMGB1)/TLR4/NF-κB pathway [25]. Due to the multiple roles of PF in anti-infammatory and immune regulation, it is speculated that PF may be an efcacious drug for treating SLE. Nevertheless, the defnite contribution of PF to the pathogenesis of SLE infammation has not been fully elucidated.
Previously, we only studied the related pathways of IRAK1-NF-κB in macrophages of MRL/lpr mice at the cellular level [26]. Studies have reported that IRAK1 inhibitors can inhibit infammatory signals in splenic monocytes of lupus mice [27]. However, questions like whether IRAK1 inhibitors could function on CD4 + T lymphocytes and whether PF has the same efect as IRAK1 inhibitors have not been answered yet. Tus, our study aimed to explore the function of PF on infection during the pathogenesis of SLE in vivo and in vitro. Assuming that inhibition of IRAK1 activity might relieve infammation in lupus mouse models, we studied the lipopolysaccharide (LPS)-induced splenocytes, CD4 + T lymphocytes, and pathological symptoms of MRL/lpr mice. Overall, we explored the anti-infammatory efect of PF on MRL/lpr mice through the IRAK1-NF-κB signaling pathway.

Isolation and Culture of Splenocytes and CD4+ T
Lymphocytes. Te spleens were obtained from MRL/MP and MRL/lpr mice and placed in RPMI1640 (Gibco, CA, USA) supplemented with penicillin (100 unit/mL) and streptomycin (100 μg). A single cell suspension of spleen in each group was prepared with a cell strainer (40 μm, Corning, NYC, USA). After centrifugation (1200 rpm, 5 min, 4°C), the supernatant was removed, and cell lysis bufer (Beyotime, Shanghai, China) was added. Next, cells were washed twice in RPMI1640 containing 10% fetal bovine serum (FBS, Gibco, CA, USA) and splenocytes were obtained after centrifugation [28]. Finally, splenocytes are a suspension of single cells from the spleen [29].
Te collected splenocytes were resuspended in a bufer (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the operating instructions of the CD4 + T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Te cells were incubated with Biotin-Antibody Cocktail and Antibiotin MicroBeads successively. Ultimately, the unlabeled cells collected by the LS Column (Miltenyi Biotec, Bergisch Gladbach, Germany) were CD4 + T cells.

Flow Cytometry.
Te collected CD4 + T cells were resuspended in 1640 medium containing 10% FBS and centrifuged to discard the supernatant. Ten, anti-mouse CD4 and PE and anti-mouse CD3 and FITC (Multi Sciences, Hangzhou, China) were added after the cells were washed with phosphate-bufered saline (PBS). Ten, the cells were mixed slowly and incubated in the dark. Finally, they were detected by a fow cytometer (BD, CA, USA).

Cell Viability Analysis.
Splenocytes and CD4 + T lymphocytes were cultured alone or with PF (10, 25, 50 μmol/L) for 24 h after induction with LPS (10 μg/mL). Cell viability was detected by counting kit-8 (CCK-8, Beyotime, Shanghai, China). Cells treated with vehicle (1640 supplemented with 10% FBS) were taken as the control group. Te absorbance at 562 nm was detected in a microplate reader (PerkinElmer, EnSpire, MA, USA). All mice were randomly divided into the following groups: control group, model group, prednisone group (PDN group, a positive control drug), and PF group, and were given drugs and diluted water by intragastric administration for eight weeks as described previously [21]. MRL/ MP mice taken as a control group and MRL/lpr mice taken as a model group were fed with distilled water. Prednisone tablets (Xainju Pharma, Zhejiang, China) suspension was prepared with distilled water and gavaged to MRL/lpr mice at a dose of 5 mg/kg for eight weeks [30,31]. Te mice in the PF group were given 25, 50, and 75 mg/kg of PF dissolved in distilled water, respectively [25]. In the end, the spleen, kidney, urine, and blood were harvested after all the mice were euthanized with carbon dioxide [18].
All the above animal studies were approved by the Animal Experiment Ethics Committee of Zhejiang Chinese Medical University. Te ethics approval/permit number for the use of animals in this study is IACUC-20181119-04/ SYXK2018-0012.
2.6. Enzyme-Linked Immunosorbent Assay (ELISA). Cells were grown in 6-well plates and treated with LPS alone or with paeoniforin. Te levels of TNF-α and IL-6 in cell supernatant were detected by ELISA kits (NOVUS biologicals, SLLC, USA) according to the manufacturer's instructions.
Te supernatant of mouse urine was collected by centrifugation, and the concentration of urine protein was determined according to the urine protein and urinary creatinine test kit (Jiancheng, Nanjing, China). Te venous blood of each mouse was kept at room temperature, and the serum was obtained after centrifugation. Afterwards, the concentration of anti-dsDNA and anti-nRNP/Sm in serum was detected according to the instructions of the anti-dsDNA ELISA Kit and anti-nsRNP/Sm ELISA Kit (Euroimmun, Lubeck, Germany).

Kidney Histopathology.
Te kidney specimens were fxed by using 4% paraformaldehyde and embedded in parafn wax. Parafn segments were cut and stained with hematoxylin-eosin (H&E) and Masson staining using standard procedures. Consequently, the digital pathological section scanning system (Hamamatsu Photonics K.K., Shizuoka Pref., Japan), NDP. View 2 Plus (Hamamatsu Photonics K.K., Shizuoka Pref., Japan), and light microscopy (Motic, Xiamen, China) were used to observe and analyze the pathologic change.

Western
Blotting. Te total proteins of splenocytes, CD4 + T lymphocytes, spleen, and kidney tissues from different groups were extracted with the Qproteome Mammalian Protein Prep Kit (Qiagen, Dusseldorf, Germany). Te BCA Protein Assay Kit (Biosharp, Hefei, China) was applied to determine the protein concentrations. Te protein membranes were blocked at room temperature, and then incubated with primary antibodies diluted in a blocking solution overnight. Afterwards, the membranes were incubated with IgG antibodies (1 : 1,000) and fnally developed the membranes with ECL Substrate (Millipore Corp., Billerica, MA, USA). Te signals were quantifed by an imager (Proteinsimple, CA, USA), and the ratio of protein band to α-tubulin was quantifed by FluorChem FC3 software (Proteinsimple, CA, USA).

Statistical Analysis.
Te data were presented as mean ± standard deviation (SD). One-way analysis of ANOVA and t-test were determined by the software GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA) and SPSS 22.0 (IBM SPSS, Chicago, IL, USA). p < 0.05 was statistically signifcant.

Potential Cytotoxicity and Cytoprotection of PF in LPS-Induced CD4 + T Lymphocytes of MRL/lpr Mice. It has been
reported that lupus-susceptible mice carry a great quantity of activated T cells, and CD4 + T cells in SLE have signal defects [32,33]. To further elucidate the mechanism of PF on CD4 + T lymphocytes in MRL/lpr mice, we adopted the LPS induction method. Firstly, we purifed and identifed the extracted CD4 + T lymphocytes. As shown in Figure 1(a), the ratio of CD3ε before sorting was 34.5%, the ratio of CD4 was 27.2%, and the ratio of CD3ε+CD4 was 24.8%. After sorting, the ratio of CD3ε was 98.7%, the ratio of CD4 was 97.4%, and the ratio of CD3ε+CD4 was 97.6%. Te obtained CD4 + T lymphocytes could be used for the following experiment.
Additionally, the functions of LPS and PF on the cell viability of splenocytes and CD4 + T lymphocytes from MRL/ MP mice and MRL/lpr mice were studied. First, we observed the efects of LPS and PF drug concentrations on splenocytes and CD4 + T lymphocytes for further exploration. It was found that LPS could dramatically increase the viability of splenocytes and CD4 + T lymphocytes at concentrations of 0.001-10 μg/mL (Figure 1(b)). We discovered that 10 μg/mL of LPS had the most apparent efect, as previously reported [29]. Paeoniforin was one of the active ingredients in Paeoniae Radix. When the concentration of PF was 10, 25, or 50 μmol/L, it would not have a toxic inhibitory efect on splenocytes and CD4 + T lymphocytes (Figure 1(b)). Subsequently, it was verifed whether PF had an efect on the cell viability of splenocytes and CD4 + T lymphocytes. As shown in Figure 1(c), PF reduced the cell viability of LPS-induced cells in a concentration-dependent manner. However, after 24 h of drug intervention, the concentration of PF as high as 50 μmol/L showed no signifcant cytotoxicity to the LPStreated cells, which indicated that this concentration could be used for further experiments.

PF Inhibits Infammatory Cytokines Release In Vitro and
In Vivo. To determine the role of PF in the expression of infammatory cytokines in MRL/lpr and MRL/MP mice, the splenocytes and CD4 + T lymphocytes were treated with LPS alone or in combination with PF, and the expressions of TNF-α, IL-6, and IL-1β were detected by Rt-qPCR and ELISA (Figures 2(a) and 2(b)). It was reported that the expressions of TNF-α and IL-6 were generally higher in SLE patients [34]. TNF-α and IL-1β stimulate innate immune response and expand infammatory cascade by simulating the secretion of other infammatory cytokines [25,35]. After LPS stimulation, the expressions of TNF-α, IL-6, and IL-1β in the cells of MRL/lpr mice were up-regulated, especially in CD4 + T lymphocytes. Tis demonstrated that CD4 + T lymphocytes might be involved in the pathogenesis of infammation in MRL/lpr mice. After PF intervention, the infammatory cytokines in splenocytes and CD4 + T lymphocytes of MRL/lpr mice decreased signifcantly, which was consistent with our previous study of PF on peritoneal macrophages in lupus mice [26]. So, we speculated that PF might inhibit the secretion of infammatory cytokines in diferent immune cells of lupus mice. Notably, PF restricted infammatory cytokines more signifcantly in CD4 + T lymphocytes from MRL/lpr mice. Tese data illustrated that PF markedly diminished the release of infammatory cytokine in lupus cells induced by LPS.
Te above results were based on in vitro experiments in MRL/lpr mice. To better understand the intervention efect of PF on SLE, we detected infammatory cytokines mRNA in the spleen and kidney of mice. It was revealed that the expressions of TNF-α, IL-6, and IL-1β in the spleen and kidney were dramatically increased in the model group (Figure 2(c)). Of note, the TNF-α, IL-6, and IL-1β mRNA expressions in the PF group and PDN group were signifcantly decreased. Taken together, PF could suppress the secretion of infammatory cytokines in MRL/lpr mice.

PF Relieves the Progression of Lupus Symptoms in MRL/lpr
Mice. Previous experiments have shown that MRL/lpr mice show obvious symptoms of lupus, the most serious of which is kidney disorders [21]. Meanwhile, serious changes in urine protein, urinary creatinine, blood indicators, and kidney function could also be found. To further assess the efect of PF on the improvement of pathological symptoms in MRL/lpr mice, ELISA was used to determine the concentration of urine albumin, urinary creatinine, serum anti-dsDNA, and anti-nRNP/ Sm (Figures 3(a)-3(d)). Te data demonstrated that urinary    Evidence-Based Complementary and Alternative Medicine albumin and urinary creatinine markedly decreased in MRL/lpr mice after PF intervention (Figures 3(a) and 3(b)). Meanwhile, the anti-nRNP/Sm and anti-dsDNA levels of serum were dramatically reduced in the PF group (Figures 3(c) and 3(d)).
Te increased size and swollen state of glomerulus were found in the model group, which lead to severer infammatory cell infltration, proteinuria, and glomerular lesions (Figure 3(e)). However, such pathological changes were alleviated by PF intervention, and obvious congestion and massive infammatory cell infltration in the renal interstium could not be found. Te renal glomerular lesions of the mice in the PDN group were less obvious, while the kidney structure of the control group was relatively complete, with normal glomerular morphology and no prominent pathological changes. Meanwhile, Masson staining showed noticeable glomerular swelling and interstitial fbrosis in the kidney tissue of the model group. After PDN and PF treatment, the above performance was greatly improved (Figure 3(f)). Tese results elucidated that PF alleviated the progression of lupus symptoms in MRL/lpr mice.

IRAK1 and Its Phosphorylation Are Suppressed by PF in MRL/lpr Mice.
As a critical regulatory molecule in TLR, IRAK1 is also one of the potential therapeutic targets in SLE. And it is valuable to inhibit the IRAK1 signaling pathway to reduce the infammatory cascade-mediated tissue damage [27]. In the previous study, we used lentiviral transfection technology to overexpress shRNA with IRAK1 to prove that TCM reduced IRAK1 expression in peritoneal macrophages of MRL/lpr mice [36]. Tus, we evaluated IRAK1 expression levels in vivo and in vitro to further understand its role in the infammatory pathway through gene and protein detection experiments. As shown in Figure 4 (Figure 4(b)). Tis proved that PF had a particular regulatory efect on the expression of IRAK1 in MRL/lpr mice.
To further confrm the therapeutic efect of PF on lupus mice, cDNA and protein were extracted from the spleen and kidney tissues, and the expression of IRAK1 was determined ( Figures 5(a)-5(c)). Additionally, we evaluated the phosphorylated expression of IRAK1. As we observed, the expression of IRAK1 and p-IRAK1 in the spleen and kidney was dramatically increased in the model group, indicating that the IRAK1 molecule was activated (Figures 5(b) and 5(c)). Nevertheless, PF and PDN could reduce the expression of IRAK1. Tese results proved that PF suppressed the activation of IRAK1 molecules in MRL/lpr mice.

PF Down-Regulates IRAK1 Downstream NF-κB Pathway in MRL/Lpr
Mice. NF-κB has been confrmed to have an abnormal active expression in SLE. IRAK1 is a crucial regulator of the NF-κB pathway, so it is speculated that if the upstream IRAK1 molecule is abnormally expressed, the downstream NF-κB signaling pathway can be activated. Te expressions of NF-κB in CD4 + T lymphocytes of MRL/lpr mice were increased dramatically after LPS stimulation, and binding to TLR4 [37]. Te IKK complex can phosphorylate IκBα, and then NF-κB protein can be transferred to the nucleus to mediate infammation. Regarding this pathway, we performed western blotting to observe the expression of IKKα/β and IκBα and their phosphorylation. Te results illustrated that the phosphorylation of IKKα/β in the spleen and kidney was signifcantly increased in the model group and decreased after intervention with PF (Figures 7(b) and 7(c)). As the phosphorylation of IKKα/β directly afected the expression of IκBα directly, the phosphorylation level of IκBα in the spleen and kidney was dramatically reduced in the PF group, consistent with the results of IKKα/β. Te protein data indicated that PF decreased the expression of IκB in the splenocytes and CD4 + T lymphocytes of MRL/lpr mice pretreated with LPS, but it was not evident in the splenocytes of MRL/MP mice (Figure 6(b)). Taken together, PF suppressed the activation of the NF-κB infammatory signaling pathway in MRL/lpr mice.

Discussion
Our research demonstrated that PF signifcantly inhibited the activation of the IRAK1 and its downstream NF-κB infammatory signaling pathway in CD4 + T lymphocytes of MRL/lpr mice. Tese results provide experimental evidence for the relationship between CD4 + T lymphocytes and the pathogenesis of SLE infammation. Notably, we observed that MRL/lpr mice treated with PF exhibited slighter kidney damage and fewer autoantibodies and infammatory factors. Taken together, these data verifed our hypothesis that PF might inhibit the activation of the IRAK1-NF-κB infammatory signals to treat infammation in SLE mouse models.
In recent years, new anti-infammatory drugs have attracted more and more attention due to their efective properties and fewer side efects [38]. Innovative Chinese medicines, such as artemisinin, ligustrazine, and berberine, have become common clinical drugs for their stable quality and strong efcacy. PF had the advantages of a wide range of sources, simple extraction, and pleiotropic efects [39]. Moreover, PF reduced infammation and tissue damage in autoimmune diseases because it could balance immune cell subpopulations and regulate abnormally activated signal pathways [23,24]. Besides, total glucosides of paeony (TGP) is a drug approved by the China Food and Drug Administration for the treatment of rheumatoid arthritis (RA) and other autoimmune diseases [24,40]. PF, the main active ingredient of TGP, has been reported as a promising drug for autoimmune diseases and infammatory diseases [23]. Nevertheless, the mechanism of PF on SLE has not yet been determined, and further specifc functional studies are still needed. Existing studies proved that PF had a protective efect on liver injury by inhibiting TLR4/MyD88/NF-κB signaling in MRL/lpr mice [41]. Furthermore, JP, a commonly used prescription for the treatment of SLE in Zhejiang Provincial Hospital of TCM, was analyzed by HPLC and LC-MS. Interestingly, we found that PF was the most important active component in JP [36]. Terefore, we tested the possible efective inhibitory efect of PF on the infammatory response of SLE. Studies have suggested that IRAK1 antagonism can reduce the production of TNF-α and IL-6 [42]. However, it was not confrmed whether PF had a similar antagonistic efect on IRAK1. Hence, this study aimed to explore the possible anti-infection-related mechanisms of PF in treating SLE through more in-depth in vivo and in vitro experiments.
At present, MRL/lpr mice are internationally recognized as the most classical animal models of SLE, and their autoimmune disease symptoms are similar to those of SLE patients, including obvious serum autoantibodies and kidney damage [16]. In this study, the kidney pathological sections of the model group showed typical nephritis features such as glomerular cell swelling, infammatory cell infltration, mesangial matrix proliferation, and interstitial fbrosis. Meanwhile, the model group showed signifcantly elevated proteinuria content and higher levels of autoantibodies in the serum, such as anti-dsDNA and anti-nRNP/ Sm.
T cells played an essential role in the pathogenesis of SLE and could amplify the autoimmune response when its tolerance was impaired [43]. Published studies demonstrated that overactive autoreactive T cells in SLE produced a large number of autoantibodies to form immune complexes and secreted various infammatory cytokines, resulting in cell signaling, tissue infammation, and organ damage [44][45][46]. As one of the therapeutic targets of SLE, TLR4 is also the primary source of infammatory cytokines and participates in various infammatory responses [47,48]. Due to the TLR activation of the adaptor protein MyD88, NF-κB is activated, which leads to the secretion of infammatory cytokines [49]. LPS is reported to play an important role in the pathogenesis of SLE according to previous experiments. As the ligand of TLR4, LPS is an important component of the outer membrane of Gram-negative bacteria and can induce the expression of infammatory cytokines in splenocytes in vivo. Indeed, we found that LPS promoted the secretion of infammatory cytokines in the splenocytes and CD4 + T lymphocytes of MRL/lpr mice. In addition, recent research reported that IRAK1 was overexpressed in the PBMC of SLE patients [27]. Dihydroartemisinin (DHA) could inhibit TLR4 expression in splenocytes of MRL/lpr mice by reducing the TLR4-mediated infammatory signal pathway triggered by LPS [29]. All the above fndings confrm our results. After LPS stimulation, the expression of the IRAK1-NF-κB infammatory signaling pathway in splenocytes and CD4 + T lymphocytes of MRL/lpr mice was higher than that of MRL/MP mice. Interestingly, we found that CD4 + T lymphocytes had more obvious indicators of infammation compared with the splenocytes of MRL/lpr mice. Studies have found that CD4 + T cells are dysfunctional in SLE [32,33,50], which may explain the active role of CD4 + T lymphocytes in the pathogenesis of SLE. As a conclusion, it was speculated that targeting CD4 + T lymphocytes might be a new strategy for the treatment of SLE.
SLE is a severe systemic autoimmune disease in which immune complexes are deposited on blood vessels, leading to the recruitment of complement factors and severe infammation and tissue destruction of the organs such as the Evidence-Based Complementary and Alternative Medicine spleen and kidney [51][52][53]. Much evidence indicated that the spleen of MRL/lpr mice showed an enlarged pathological state, but the pathogenesis of SLE splenomegaly was still unknown [54,55]. Although the spleen is not a common target organ of SLE, it plays a vital role in antibody production. However, it is essential to understand the pathogenesis of lupus splenomegaly and the infammation of the spleen [54]. In the present study, we found that the level of infammatory factors and the expression of the IRAK1-NF-κB pathway in the spleen of the model group were increased, and the spleen infammation was alleviated after PF intervention. Almost certainly, kidney involvement is a serious complication of SLE, and its severity is closely related to the prognosis of SLE. More seriously, the replacement of fbrotic tissue and the destruction of normal renal parenchyma could be found [56]. As we found in the model group, the urine protein was markedly increased in the model group, and the pathological section of the kidney showed severe nephritis. However, these symptoms in mice in the PF group were appropriately relieved. Similar to the spleen results, PF could also inhibit the secretion of infammatory factors and the expression of infammatory signals in the kidney of MRL/lpr mice. Generally, interventions on the kidney were more obvious than those on the spleen. According to reports, CD4 + T cells mediated the induction of autoantibodies to aggravate lupus nephritis [57]. Terefore, we tried to extract immune cells (including T cells, macrophages, etc.) from the kidney to further observe the anti-infammatory mechanism of PF.
Studies have shown that IRAK1-defcient mice have sufcient immune responses and are less sensitive to systemic autoimmunity [12,58]. Consequently, we hypothesized that the reduction of infammation in the SLE mouse model might be the result of inhibiting the activity of IRAK1. IRAK1 regulates the expression of infammatory genes through TLR ligands or IL-1 family members [59]. And further signal transduction leads to transcriptional activators, including NF-κB regulators [60,61]. In this study, a consistent conclusion that the expressions of NF-κB decreased in the spleen and kidney of MRL/lpr mic after PF intervention were reached. Furthermore, the combination of LPS and TLR4 could lead to the activation of the IKK/IκB/ NF-κB infammation pathway [62]. We found that the expressions of IKKα/β and IκBα in MRL/lpr mic were signifcantly reduced after PF intervention. Notably, clinical studies have elucidated that IRAK1 is overexpressed and can be highly activated in CD4 + T cells in SLE patients [63]. Our data support that CD4 + T lymphocytes induced by LPS showed a higher level of IκBα expression, and its expression was markedly reduced after PF intervention.

Conclusion
PF could better inhibit the infammatory activity in CD4 + T lymphocytes than that in the splenocytes of MRL/lpr mice induced by LPS. Meanwhile, PF could also alleviate the infammation in the kidney and spleen of MRL/lpr mice. Te anti-infammatory efect of PF was related to the regulation of IRAK1-NF-κB signaling (Figure 8). Our research showed that PF had an infuential role in the anti-infammatory mechanism of MRL/lpr mice, and inhibiting the IRAK1-NF-κB pathway might be a unique and promising treatment strategy for lupus infammation.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.