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Journal of Pharmacological Sciences 133 (2017) 115e121

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Aconitine: A potential novel treatment for systemic lupuserythematosus

Xiaodong Li a, b, 1, Liwei Gu a, 1, Lan Yang a, Dong Zhang a, Jianying Shen a, *

a Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, Chinab Gansu Provincial Hospital of Traditional Chinese Medicine, Lanzhou 730050, China

a r t i c l e i n f o

Article history:Received 6 September 2016Received in revised form30 December 2016Accepted 21 January 2017Available online 24 February 2017

Keywords:AconitineSystemic lupus erythematosus (SLE)Pristane-induced lupus (PIL)AutoantibodiesTraditional Chinese medicine (TCM)

* Corresponding author. No. 16 Nanxiaojie, DongzhiChina.

E-mail address: [email protected] (J. Shen).Peer review under responsibility of Japanese Pha

1 Equal contribution.

http://dx.doi.org/10.1016/j.jphs.2017.01.0071347-8613/© 2017 The Authors. Production and hostinlicense (http://creativecommons.org/licenses/by-nc-n

a b s t r a c t

Background: Aconitum plants have been widely used in China for thousands of years. Recent evidencesindicate that aconitine, the main active ingredient of Aconitum, has immunomodulatory properties thatmight be useful for treating autoimmune diseases, such as rheumatoid arthritis. In this study, we con-ducted a pilot study to explore the effect and mechanisms of aconitine on the treatment of systemiclupus erythematosus.Methods: A pristane-induced murine model was used. The pristane-induced mice were treated withaconitine (25, 75 mg kg�1 d�1, po) for 9 weeks. Every three weeks, proteinuria was detected to monitorthe kidney damage and blood was collected to measure serum levels of autoantibodies, besides thekidney pathological examination. The major B cell activating factor and major pro-inflammatory medi-ators, PGE2, IL-17a and IL-6, were also detected.Results: We found that aconitine significantly improved the mouse health, decreased the elevated bloodleukocyte counts, reduced the serum level of anti-double-stranded DNA (anti-dsDNA) antibody, greatlyameliorated renal histopathologic damage and reduced IgG deposit in glomerular. Furtherly, the levels ofPGE2, IL-17a and IL-6, were found to have decreased in aconitine treated mice.Conclusion: We have demonstrated that aconitine can inhibit the progression of disease and amelioratethe pathologic lesion of systemic lupus erythematosus.

© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese PharmacologicalSociety. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/

licenses/by-nc-nd/4.0/).

1. Introduction

Systemic lupus erythematosus (SLE) is a chronic autoimmunedisorder affecting multiple organs and characterized by a variety ofautoantibodies, immune complex deposition in tissues and subse-quent development of glomerulonephritis (1). Several animalmodels, genetically engineered or chemical-induced, are widelyused for SLE studies. Pristane (2, 4, 10, 14-tetramethylpentadecane)administration in Balb/c mice, known as the environmentallyinduced model, mimics human idiopathic lupus syndrome and ischaracterized by lupus-specific autoantibody production alongwith arthritis, hemorrhagic pulmonary capillaritis, proteinuria and

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glomerulonephritis (2,3). In addition, the autoantibody level ofpristane-induced lupus in BALB/c is comparable to that found inMRL/lpr mice, such as antiribonucleoprotein (RNP) antibodies(anti-Su, anti-Sm, and anti-U1RNP), anti-double-stranded (ds)DNA, anti-single-stranded (ss) DNA, and anti-histone (4,5). Thismodel has been widely used for exploring the pathogenesis of theSLE, which is still poorly understood.

Standard clinical therapies for SLE are glucocorticoids combinedwith immunosuppressive agents, antimalarial drugs and non-steroidal anti-inflammatory drugs. They often lead immunotol-erance or exhibit various side-effects and disease relapses aftertherapy discontinuation or tapered doses (6). Belimumab (Ben-lysta®) is the only drug approved by the US Food and DrugAdministration (FDA) for 56 years. It's a fully humanized mono-clonal antibody that inhibits B cell activating factor, and used fortreatment of autoantibody-positive SLE in adults (7). But the price isunacceptable for most SLE patients. To search less costly and moreeffective novel treatments, we turned to traditional Chinese med-icine (TCM).

nese Pharmacological Society. This is an open access article under the CC BY-NC-ND

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X. Li et al. / Journal of Pharmacological Sciences 133 (2017) 115e121116

Aconitum plants (Ranunculaceae family) have been widely usedto treat various diseases, such as rheumatism, knee pain, wheezing,cough, cyanosis, chronic diarrhea, impotence, dense tinea, herpeszoster, scabies and other disorders in China for thousands of years(8). In TCM, the pharmacological effects of Aconitum plants includereviving Yang for resuscitation, dispelling Wind to eliminatedampness, warming Channels to expel coldness. Aconitine (AC,MW: 645.74, molecular formation: C34H47NO11, chemical structure:shown in Fig. 1A) is the main active component in Aconitum plants(9). It has been used for treatment of pain and inflammation (10). Inthis study, using pristane-induced murine model, we have studiedthe therapeutic effects of aconitine in lupus symptoms and ourresults for the first time have demonstrated that aconitine may be apotential novel treatment for SLE.

2. Materials and methods

2.1. Materials

Aconitine (C34H47NO11, MW, 645.74) was supplied by the Na-tional Institutes for Food and Drug Control (NIFDC), with a purity of99.5%. Prednisone acetate tablets were purchased from Topfondpharmaceutical (Zhumadian, Henan, China). Concanavalin A (ConA) was supplied by Solarbio (Beijing, China). The Prostaglandin E2(PGE2) levels were measured using an ELISA kit (Fanke industrialco., LTD, Shanghai, China). Other chemicals and reagents were ofanalytical grade.

2.2. Mice

Female BALB/c mice (aged 4e6 weeks) were purchased from theVital River experimental animal technical co. (Beijing, China). Themice were housed under specific pathogen-free conditions and fedstandard rodent chow and water ad libitum. Mice were injectedintraperitoneally (ip) with either 0.5ml of pristane (Sigma, St. Louis,Missouri, USA) or saline (controls) and monitor proteinuria every 4weeks to track the disease progression. Animal experiments wereconducted according to the institutional ethical guidelines for an-imal experiments and approved by the Institutional Animal Care

Fig. 1. A, Chemical structure of aconitine. B, The difference of body weight during the treatmgroup. C, Time course of clinical characteristics and levels of proteinuria in BALB/c mice.

and Use Committee (IACUC) at Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences.

2.3. Preparation of aconitine and treatment of animals

When proteinuria appeared obviously in disease model mice,mice (n ¼ 12 per group) were treated with aconitine 75 mg/kg or25 mg/kg body weight by oral gavage daily for nine weeks inexperimental groups and the positive control group were admin-istered only with prednisone acetate 6.3 mg/kg. The normal controland model control mice (n ¼ 14) received the same volume of 0.5%sodium carboxymethylcellulose.

2.4. Scoring of proteinuria

The assessment was performed every three weeks starting atthree weeks after injection of pristane (or saline in normal controlgroup). Proteinuria test stripes were supplied by Pearl Riverchemical reagent Co. (Guangzhou, China). Urine was collected bycatching stress and assessed by using semiquantitative scores (for0 ¼ �, 1 ¼ ±, 2 ¼ þ, 3 ¼ þþ, 4 ¼ þþþ). The same standard wasadopted in each test.

2.5. Detection of auto-antibodies

Serum anti-double-stranded DNA antibody was measured withenzyme-linked immunosorbent assay (ELISA) (Cusabio Biotech Co.Ltd, Wuhan, China). Results are presented as ng/ml employing thestandard curve provided by the manufacturer.

2.6. Routine blood tests

For laboratory measurements, 100 ml of mouse whole blood wascollected into tubes containing EDTA anticoagulant. Routine bloodtests were immediately performed using a SYSMEX-XS800i auto-mated hematology analyzer (Sysmex Corp., Hyogo, Japan).

ent. *, p < 0.05, **, p < 0.01, calculated versus Control group; ##, p < 0.01, versus model

X. Li et al. / Journal of Pharmacological Sciences 133 (2017) 115e121 117

2.7. Flow cytometry

Heparin anticoagulant blood was stained with fluorochrome-conjugated monoclonal antibodies against surface markers: APC-B220, PerCP-CD3e and PE-CD8a (eBioscience, San Diego, Califor-nia, USA), FITC-CD4 and appropriate isotypic antibodies (BD Phar-mingen, New Jersey, USA) were used as controls. After remove thered blood cells, the centrifugal pellet was washed with phosphatebuffer saline, and then resuspended in 0.3 ml of PBS contain 0.02%FBS. The cell surface markers were analyzed using a flow cytometer(Accuri C6, BD, New Jersey, USA).

2.8. Lymphoproliferation

The spleens of the mice were harvested after nine weeks oftreatment. The spleens were mashed and passed through a 200-mesh sterile sieve to prepare a single cell suspension. The cellswere suspended in RPMI-1640 (Invitrogen, Paisley, Scotland, UK)medium with 10% fetal bovine serum (FBS) and centrifuged. Thepelleted cells were resuspended in Red Blood Cell Lysis BufferLysing Buffer to lyse erythrocytes. After being washed in PhosphateBuffer Solution (PBS), the total number of cells was calculated and2 � 106 cells were seeded in 96-well plates, then stimulated withConA (5 mg/ml) for 68 h. Lymphoproliferation was determined byMTT assay.

The culture supernatant were collected after 68 h and keptat �80 �C for detecting PGE2 and cytokines.

2.9. Cytokine detection

Interleukin-6 (IL-6), IL-4, IL-10, IFN-g, TNF-a, IL-17a and IL-2cytokines was detected using a mouse Th1/Th2/Th17 cytometricbead array kit (CBA; BD Biosciences, San Diego, California, USA) andwas analyzed on a FACSCalibur flow cytometer. Standard curveswere determined for each cytokine from a range of 10e5000 pg/ml.

2.10. Histopathology and immunohistochemistry assay

Kidney and spleen tissue samples were collected at the time ofharvest and fixed in 10% buffered formalin, embedded in paraffin.Hematoxylin and eosin (H&E), periodic acid-Schiff stain (PAS) andMasson stain were to used check and define the pathological stageof fibrosis of the specimen tissue.

For immunohistochemical staining, the kidney tissues (3 mmthick) were stained with fluorescein-conjugated anti-mouse IgGand IgM (Abcam, Cambridge, UK). To observe the distribution of

Fig. 2. Auto-ds-DNA levels in BALB/c mice. *p < 0.05, **p < 0.01, calculate

collagen under and immunoglobulins deposition used a lightmicroscope.

2.11. Statistical analysis

All group results are expressed as mean ± standard deviation(SD), if not stated otherwise. ANOVA, Student's t test or Fisher'sexact test (two tailed) were used for the comparison of groupvalues. For comparing group values that did not follow Gaussiandistribution, ManneWhitney's test (two tailed) was used.

3. Results

3.1. Gross observation and body weight measurement

In the positive control group, prednisone induced deleteriouseffects in this experiment, and two mice died at the end of treat-ment. During the treatment, mice manifested emaciation, listless-ness and dry and dull hair in model and prednisone groups.However, aconitine-treated mice demonstrated glossy hair (datanot shown) and less body weight loss (Fig. 1B).

3.2. Time course of proteinuria

Proteinuria levels were measured for detecting renal injurydegree. The proteinuria levels in aconitine groups were decreasedmore significantly than control group and prednisone group. Aco-nitine has displayed positive effects after three weeks administra-tion (p < 0.05) (Fig. 1C).

3.3. Time course of autoantibodies in BALB/c PIL

Serum levels of anti-dsDNA, anti-nuclear antibody (ANA) andproliferating cell nuclear antigen (PCNA) antibodies were measuredfor detecting autoantibodies production. It is found that aconitinestrongly reduced the production of anti-dsDNA antibodies in thesera of lupus mice after administration for three to six weeks(Fig. 2). In this study, the used dosages of aconitinewere nontoxic tomice. Because the weight changed in aconitine group is less thancontrol group and all of aconitine group mice in good shape bothphysically and psychologically during the experiment. These resultsdemonstrated that aconitine significantly ameliorated the symp-toms of SLE. However, ANA and PCNA levers were under thedetection limits in our ELISA kits.

d versus control group; #, p < 0.05, ##, p < 0.01, versus model group.


3.4. Effects of aconitine on blood cell subsets

This is an objective data from complete blood count as per-formed on an automated instrument, including an automatedwhite blood cell (WBC) differential count. It is found that aconi-tine could significantly reduce the WBC count, neutrophil (NEUT)and lymphocyte number (LYMPH). In addition, the average redblood cell hemoglobin concentration (MCHC) also decreased inaconitine groups (Supplementary Table 1). Prednison significantlyreduce the CD4 T and B lymphocyte number, and aconitine has adirect inhibitory trend in both lymphocyte subtypes by flowcytometry analysis (Table 1, and FACS results not shown). Theseresults demonstrated aconitine could suppress the immuneresponse mildly. Which would effect on staved off SLE symptom inmice.

3.5. Effect of aconitine on PGE2 and cytokines production in thecultured supernatant of splenocyte

To investigate further the role of PGE2 and Th1/Th2 cytokinesin disease pathogenesis and the potential of aconitine as thera-peutic agents in SLE, the effect of aconitine on inflammatoryfactor PGE2 production of splenocyte in aconitine treated micewere assayed. The production of cytokine was measured at 68 hin ConA-stimulated splenocytes. It was found that there was asignificant reduction of inflammatory factor, particularly treatedmice with aconite (25 mg/kg) compared with model controls.Mixed culture supernatant in each group was detected by FCM.IL-6, IL-2, IL-4, IL-10, IL-17a, IFN-g and TNF-a were analyzed.After treatment with aconitine, IL-6 and IL-17a reduced signifi-cantly (Fig. 3).

Table 1The number of lymphocyte in blood.

Group Mice number Dose CD4-T (109

Control 14 e 2.56 ± 0.65Model 14 e 4.49 ± 1.61Prednison 10 6.3 mg/kg 2.77 ± 0.85Aconitine 12 25 mg/kg 3.43 ± 1.57

12 75 mg/kg 3.66 ± 2

The results shown are representative of all animals in each group. Data represent the meaversus Model group.

Fig. 3. Effect of AC on the content of pro-inflammatory mediators in the mixed cultured ssplenocytes were prepared and co-cultured with ConA 5 mg/ml for 68 h, then the cytokinessignificantly suppress the IL-6 and IL-17a level. Values are the mean ± SD of animals in eacgroup; #, p < 0.05, ##, p < 0.01, versus model group.

3.6. Effects of aconitine treatment on histopathology of spleen andkidney

Tissues of spleen and kidney were collected at the end of nine-week administration of aconitine. Histological examinationexhibited by H&E, PAS and Masson staining (Fig. 4). Pristane in-duces a lupus-like syndrome in nonautoimmune mice whoseglomerular basementmembrane thickening withmesangial matrixexpansion and mesangial cell proliferation. Moreover, the thick-ening of the glomerular capillary basement membrane were alsonoted. However, aconitine can completely correct these seriouskidney injuries. PAS and Masson staining reached a similarconclusion.

3.7. Aconitine reduced the production of lupus-associated IgG butnot IgM autoantibodies

IgG accumulation were found within the kidney tissue in pris-tane induced lupus mice. It was found two of ten mice in 6.3 mg/kgprednisone treated group. Unlike mice in model group (8/14), therewere only few (3/12) obvious abnormal changes were evidencedafter completion of aconitine administration (Fig. 5AeE). Further-more, the deposition of IgM autoantibodies was not detected inkidney in our experiment (data not shown). It is suggest that aco-nitine could relieve immune globulin deposition in kidney.

4. Discussion

Systemic lupus erythematosus (SLE) is an autoimmune diseasewhich is potentially fatal. Over the past several decades, tremen-dous enthusiasm and efforts have been devoted to tackling the

/l) CD8-T (109/l) B cell (109/l) CD4/CD8

0.62 ± 0.19 1.5 ± 0.66 4.21 ± 0.61** 1.19 ± 0.46** 2.51 ± 0.9** 3.82 ± 0.57## 0.92 ± 0.29** 1.75 ± 0.57# 3.05 ± 0.53

0.9 ± 0.33* 1.92 ± 0.71 3.75 ± 0.680.92 ± 0.48* 1.93 ± 0.99 4 ± 0.71

n ± SD.*p < 0.05, **p < 0.01, calculated versus Control group; #, p < 0.05, ##, p < 0.01,

upernatant of splenocytes from BALB/c mice in vitro. A. IL-6, B. IL-17a, C. PGE2. Miceproduction were determined. Values are the mixed of animals in each group. Aconitineh group, each performed in triplicate. *, p < 0.05, **, p < 0.01, calculated versus control

Fig. 4. Aconitine ameliorates kidney injury in pristane induced mice. (A) Control, (H&E � 400). (B) Model, showing glomerular basement membrane thickening with mesangialmatrix expansion and mesangial cell proliferation. The glomerular capillary basement membranes were thickening (H&E � 400). (C) 6.3 mg/kg prednisone treated mice, beside theinjuries in model mice, protein cast even appeared (H&E� 400). (D) 25 mg/kg aconitine (H&E� 400) and (E) 75 mg/kg aconitine treated mice completely correct these serious kidneyinjuries (H&E � 400). (F) Control, (PAS � 400). (G) Model, glomerular basement membrane thickening with mesangial matrix expansion and mesangial cell proliferation(PAS � 400). (H) 75 mg/kg aconitine treated group (PAS � 400). (I) Control, showing normal kidney structure (Masson � 400). (J) Model (Masson � 400), showing glomerularbasement membrane thickening with mesangial matrix expansion and mesangial cell proliferation. Blue-staining are increased collagen fibers. The glomerular capillary basementmembranes were thickening. (K) 25 mg/kg aconitine (Masson � 400) and (L) 75 mg/kg aconitine treated mice completely correct these serious kidney injuries. The pathologicalsections did not show abnormal kidney structures (Masson � 400).


numerous challenges to understand the etiopathogenesis of SLE.However, the precise mechanism of this disease is still unclear.

In this pilot study, pristane induced mice displayed the symp-toms of SLE. The IgG induced by pristane, targeted to a variety ofnuclear components including dsDNA, ssDNA and nucleoproteins.Anti-dsDNA antibodies are highly specific for lupus and are impli-cated in the pathogenesis of lupus nephritis (11). The prevalence ofanti-dsDNA antibody is 70e80% in SLE patients, so we chose it forthe important indicator in our study. The anti-dsDNA antibodies inthe sera of normal mice, measured before pristane injection, and inthe sera of SLE model mice, measured before treatment, were6.9 ± 1.77 ng/ml and 14.39 ± 4.44 ng/ml, respectively (Data notshown), indicating the establishment of SLE model induced bypristane.

In Traditional Chinese medicine, Aconitum plants have beenwidely used to treat diseases such as rheumatism and knee pain.Aconitine, which is useful for treatment of pain and inflammation(10), is the main active component in Aconitum plants (9). However,the clinic application of aconitine is limited by its high toxicity. Itslethal dose 50% (LD50) for mice is 1.8 mg/kg (orally) and 0.308 mg/kg (intraperitoneally) (12). In a long-term administration study thatan oral dose of 1 mg/kg per day was administered to mice for 22days, the frequency of arrhythmias remarkably decreasedwith timeand repeated administration of aconitine (13). To minimize thetoxicity of aconitine, we used oral dose of 75 mg/kg or 25 mg/kg bodyweight for 9 weeks. No death or toxicity was detected during andafter the treatment. During the treatment, mice manifested ema-ciation, listlessness and dry and dull hair in model and prednisone

Fig. 5. Kidney accumulation of IgG. (A) Control, (B) Model, (C) 6.3 mg/kg prednisone treated mice, (D) 25 mg/kg aconitine and (E) 75 mg/kg aconitine treated mice. The positive cellsdisplayed brownish yellow granules on the cell surface and/or cytoplasm (original magnification, �400).


groups. However, aconitine treatment ameliorated the diseasesymptoms, and decreased anti-dsDNA levels (Fig. 2). Aconitine-treated mice demonstrated glossy hair (data not shown) and lessbody weight loss (Fig. 1B).

Unexpectedly, during the treatment, the control mice treatedwith 0.5% sodium carboxymethylcellucose were found to loss theweight about 2 g. It may be caused by the age. As to Berlin FatMouse Inbred line 860 (BFMI860) mice, they reach the growth peakat the age of day 300 and begin to loss the body weight (14). In ourstudy, the BABL/c mice were at the age of day 240 when wemeasured the body weight last time, and they may have passed thegrowth peak and begin to loss the body weight. Compared withmice in other three groups, aconitine-treated mice demonstratedless body weight loss (Fig. 1B).

One of the most common clinical feature of SLE is lupusnephritis (glomerulonephritis), which can be seen in up to 60% ofall SLE patients and this complications require hemodialysis andcause higher lethality (15). Lupus nephritis is thought to involveglomerular inflammation induced by immune complexes andcomplement deposition (16). This type of nephritis is diagnosed bythe presence of biomarkers for kidney damage such as proteinuria,creatinine and blood urea nitrogen. Recently, new urinary bio-markers, such as the inflammatory mediators IL-6, vascular celladhesion molecule-1 and urinary immune cells, have been gradu-ally accepted (17,18).

Present study has shown that aconitine could significantlysuppress the proliferation of glomerular mesangial cells andmaintain the glomerular capillary basement membranes. It hasgreatly ameliorated renal histopathologic damage and reduced IgGdeposit in glomerular. Overall, these data suggest that aconitinetreatment could inhibit the activity and progress of SLE and therebyimprove their quality of life.

Many reports suggested that cytokines may act as key players inthe immunopathogenesis of SLE (19,20). IL-17 is a potent pro-inflammatory cytokine produced by activated T lymphocytes. Itplays a critical role in recruit monocytes and neutrophils to the siteof inflammation, inducing production of proinflammatory IL-6 (21),and driving B-cell differentiation into plasma cells and productionof autoantibodies (22). IL-6 has a range of biological activities on

various target cells that plays an important role in immune regu-lation and inflammation. Data from several studies suggest that IL-6 plays a critical role in the B cell hyperactivity and immunopa-thology of human or murine models of SLE, induction of IgG pro-duction (23), and may even have a direct role in mediating tissuedamage (24). Meanwhile, IL-6 induces naïve CD4þ Tcells to developinto Th17 cells, the IL-17-producing cells (25). Consistent withprevious report, we have observed amarked increase in levels of IL-6 in the pristane induced BALB/c mice (26). PGE2 is a bioactive lipidwhich can elicit a wide range of biological effects, includinginflammation, cell proliferation, apoptosis and angiogenesis (27).Recent reports have elaborated PGE2 could promote the activationof Th17 cells and production of IL-17 (28). In accordance with manyother studies, we have demonstrated directly that PGE2 couldenhance IL-17a expression (29,30). Aconitine could inhibit IL-17aand IL-6 secretion, and thereby suppress B-cell hyperactivity andautoantibodies production. However, there is little influence inother Th1/Th2 cytokines, such as IL-2, TNF-a and IFN-g. So wepropose the influence of PGE2, IL-17a and IL-6 may be amechanismof aconitine action on SLE.

In conclusion, this pilot study for the first time has demon-strated that aconitine is effective for the treatment of SLE. Aco-nitine raised the body weight, improved health status, decreasedthe number of blood leucocytes, decreased the serum level ofanti-dsDNA antibody, ameliorated the renal pathology, andthereby relieved bodies suffering damage. These effects of aco-nitine might be achieved in part by decreasing the inflammationin kidney and inhibiting B cells activation through suppressingthe major pro-inflammation mediators, IL-17a, IL-6, and PGE2.Further studies will be required to confirm the efficacy andmechanism of action of aconitine, as to provide evidence for itspotential clinical application for human systemic lupus erythe-matosus in the future.

Author contribution

Conceived and designed the experiments: Xiaodong Li, Liwei Guand Jianying Shen. Contributed reagents/materials/analysis tools:Lan YANG. Performed the experiments: Xiaodong Li, Liwei Gu and


Dong Zhang. Analyzed the data: Jianying Shen, Liwei Gu andXiaodong Li. Wrote the paper: Jianying Shen and Liwei Gu.

Conflict of interest

There is no conflict of interest.

Acknowledgments

This work was supported by National Science and TechnologyMajor Projects for “Major NewDrugs Innovation and Development”(Project No. 2013ZX09301307001002) and National Natural ScienceFoundation of China (Grant No. 81402075) and China PostdoctoralScience Foundation funded project (2014M561160). The authorsthank Lanfang Li, Guihua Yu, Canghai Lee and Shuying Guo for theirtechnical help.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jphs.2017.01.007.

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Aconitine: A potential novel treatment for systemic lupus erythematosus1. Introduction2. Materials and methods2.1. Materials2.2. Mice2.3. Preparation of aconitine and treatment of animals2.4. Scoring of proteinuria2.5. Detection of auto-antibodies2.6. Routine blood tests2.7. Flow cytometry2.8. Lymphoproliferation2.9. Cytokine detection2.10. Histopathology and immunohistochemistry assay2.11. Statistical analysis

3. Results3.1. Gross observation and body weight measurement3.2. Time course of proteinuria3.3. Time course of autoantibodies in BALB/c PIL3.4. Effects of aconitine on blood cell subsets3.5. Effect of aconitine on PGE2 and cytokines production in the cultured supernatant of splenocyte3.6. Effects of aconitine treatment on histopathology of spleen and kidney3.7. Aconitine reduced the production of lupus-associated IgG but not IgM autoantibodies

4. DiscussionAuthor contributionConflict of interestAcknowledgmentsAppendix A. Supplementary dataReferences

aconitine: a potential novel treatment for systemic lupus...

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