Aconitine: a potential novel treatment for systemic lupus erythematosus

Aconitine A potential novel treatment for systemic lupus erythematosus Accepted Manuscript Aconitine A potential novel treatment for systemic lupus erythematosus Xiaodong Li, Liwei Gu, Lan Yang, Dong[.]

Accepted Manuscript Aconitine: A potential novel treatment for systemic lupus erythematosus Xiaodong Li, Liwei Gu, Lan Yang, Dong Zhang, Jianying Shen PII: S1347-8613(17)30026-9 DOI: 10.1016/j.jphs.2017.01.007 Reference: JPHS 327 To appear in: Journal of Pharmacological Science Received Date: September 2016 Revised Date: 30 December 2016 Accepted Date: 21 January 2017 Please cite this article as: Li X, Gu L, Yang L, Zhang D, Shen J, Aconitine: A potential novel treatment for systemic lupus erythematosus, Journal of Pharmacological Science (2017), doi: 10.1016/ j.jphs.2017.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Aconitine: A potential novel treatment for systemic lupus erythematosus Short title: Aconitine for SLE treatment Xiaodong Li1,2, #, Liwei Gu1, #, Lan Yang1, Dong Zhang1 and Jianying Shen1,* RI PT 1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700 2.Gansu Provincial Hospital of Traditional Chinese Medicine, Lanzhou 730050, China #, Equal contribution *, To whom correspondence should be addressed M AN U SC 10 Email: jyshen@icmm.ac.cn, No.16 Nanxiaojie, Dongzhimen Nei Ave Beijing100700, China 11 Tel:86-10-84252805 (Ext: 7200) 12 16 17 18 19 20 21 22 23 24 25 EP 15 AC C 14 TE D 13 ACCEPTED MANUSCRIPT Abstract 27 Background: Aconitum plants have been widely used in China for thousands of years 28 Recent evidences indicate that aconitine, the main active ingredient of Aconitum, has 29 immunomodulatory properties that might be useful for treating autoimmune diseases, 30 such as rheumatoid arthritis In this study, we conducted a pilot study to explore the effect 31 and mechanisms of aconitine on the treatment of systemic lupus erythematosus 32 Methods: A pristine-induced murine model was used The pristine-induced mice were 33 treated with aconitine (25, 75 µg·kg-1·d-1, po) for weeks Every three weeks, proteinuria 34 was detected to monitor the kidney damage and blood was collected to measure serum 35 levels of autoantibodies, besides the kidney pathological examination The major B cell 36 activating factor and major pro-inflammatory mediators, PGE2, IL-17a and IL-6, were 37 also detected, 38 Results: We found that aconitine significantly improved the mouse health, decreased the 39 elevated blood leukocyte counts, reduced the serum level of anti-double-stranded DNA 40 (anti-dsDNA) antibody, greatly ameliorated renal histopathologic damage and reduced 41 IgG deposit in glomerular Furtherly, the levels of PGE2, IL-17a and IL-6, were found to 42 have decreased in aconitine treated mice 43 Conclusion: We have demonstrated that aconitine can inhibit the progression of disease 44 and ameliorate the pathologic lesion of systemic lupus erythematosus SC M AN U TE D EP AC C 45 RI PT 26 46 Key Words: 47 Aconitine; systemic lupus erythematosus (SLE); pristane-induced lupus (PIL); autoantibodies; 48 Traditional Chinese Medicine (TCM) 49 ACCEPTED MANUSCRIPT 50 Introduction Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder affecting 52 multiple organs and characterized by a variety of autoantibodies, immune complex 53 deposition in tissues and subsequent development of glomerulonephritis[1] Several 54 animal models, genetically engineered or chemical-induced, are widely used for SLE 55 studies Pristane (2, 4, 10, 14-tetramethylpentadecane) administration in Balb/c mice, 56 known as the environmentally induced model, mimics human idiopathic lupus syndrome 57 and is characterized by lupus-specific autoantibody production along with arthritis, 58 hemorrhagic pulmonary capillaritis, proteinuria and glomerulonephritis [2, 3] In addition, 59 the autoantibody level of pristine-induced lupus in BALB/c is comparable to that found in 60 MRL/lpr mice, such as antiribonucleoprotein (RNP) antibodies (anti-Su, anti-Sm, and 61 anti-U1RNP), anti-double-stranded (ds) DNA, anti-single-stranded (ss) DNA, and 62 anti-histone[4, 5] This model has been widely used for exploring the pathogenesis of the 63 SLE, which is still poorly understood SC M AN U Standard clinical TE D 64 RI PT 51 therapies for SLE are glucocorticoids combined with immunosuppressive agents, antimalarial drugs and non-steroidal anti-inflammatory drugs 66 They often lead immunotolerance or exhibit various side-effects and disease relapses 67 after therapy discontinuation or tapered doses[6] Belimumab (Benlysta®) is the only 68 drug approved by the US Food and Drug Administration (FDA) for 56 years It’s a fully 69 humanized monoclonal antibody that inhibits B cell activating factor, and used for 70 treatment of autoantibody-positive SLE in adults [7] But the price is unacceptable for 71 most SLE patients To search less costy and more effective novel treatments, we turned to 72 traditional Chinese medicine (TCM) 73 AC C EP 65 Aconitum plants (Ranunculaceae family) have been widely used to treat various ACCEPTED MANUSCRIPT diseases, such as rheumatism, knee pain, wheezing, cough, cyanosis, chronic diarrhea, 75 impotence, dense tinea, herpes zoster, scabies and other disorders in China for thousands 76 of years [8] In TCM, the pharmacological effects of Aconitum plants include reviving 77 Yang for resuscitation, dispelling Wind to eliminate dampness, warming Channels to 78 expel coldness Aconitine (AC, MW: 645.74, molecular formation: C34H47NO11, chemical 79 structure: shown in Fig 1A) is the main active component in Aconitum plants [9] It has 80 been used for treatment of pain and inflammation [10] In this study, using 81 pristane-induced murine model, we have studied the therapeutic effects of aconitine in 82 lupus symptoms and our results for the first time have demonstrated that aconitine may 83 be a potential novel treatment for SLE 84 Materials and methods 85 Materials TE D M AN U SC RI PT 74 Aconitine (C34H47NO11, MW, 645.74) was supplied by the National Institutes for 87 Food and Drug Control (NIFDC), with a purity of 99.5% Prednisone acetate tablets were 88 purchased from Topfond pharmaceutical (Zhumadian, Henan, China) Concanavalin A 89 (Con A) was supplied by Solarbio (Beijing, China) The Prostaglandin E2 (PGE2) levels 90 were measured using an ELISA kit (Fanke industrial co., LTD, Shanghai, China) Other 91 chemicals and reagents were of analytical grade AC C EP 86 92 93 94 Mice Female BALB/c mice (aged 4~6 weeks) were purchased from the Vital River ACCEPTED MANUSCRIPT experimental animal technical co (Beijing, China) The mice were housed under specific 96 pathogen-free conditions and fed standard rodent chow and water ad libitum Mice were 97 injected intraperitoneally (ip) with either 0.5 ml of pristane (Sigma, St Louis, Missouri, 98 USA) or saline (controls) and monitor proteinuria every weeks to track the disease 99 progression Animal experiments were conducted according to the institutional ethical RI PT 95 guidelines for animal experiments and approved by the Institutional Animal Care and Use 101 Committee (IACUC) at Institute of Chinese Materia Medica, China Academy of Chinese 102 Medical Sciences SC 100 104 105 M AN U 103 Preparation of aconitine and treatment of animals When proteinuria appeared obviously in disease model mice, mice (n=12 per group) were treated with aconitine 75 µg/kg or 25 µg/kg body weight by oral gavage daily for 107 nine weeks in experimental groups and the positive control group were administered only 108 with prednisone acetate 6.3 mg/kg The normal control and model control mice (n=14) 109 received the same volume of 0.5% sodium carboxymethylcellulose TE D 106 112 Scoring of proteinuria The assessment was performed every three weeks starting at three weeks after AC C 111 EP 110 113 injection of pristane (or saline in normal control group) Proteinuria test stripes were 114 supplied by Pearl River chemical reagent Co (Guangzhou, China) Urine was collected 115 by catching stress and assessed by using semiquantitative scores (for 0=-，1=±，2=+， 116 3=++，4=+++) The same standard was adopted in each test 117 118 Detection of auto-antibodies ACCEPTED MANUSCRIPT 119 Serum anti-double-stranded DNA antibody was measured with enzyme-linked 120 immunosorbent assay (ELISA) (Cusabio Biotech Co Ltd, Wuhan, China) Results are 121 presented as ng/ml employing the standard curve provided by the manufacturer 123 RI PT 122 Routine blood tests For laboratory measurements, 100 µl of mouse whole blood was collected into tubes 125 containing EDTA anticoagulant Routine blood tests were immediately performed using a 126 SYSMEX-XS800i automated hematology analyzer (Sysmex Corp., Hyogo, Japan) SC 124 128 129 M AN U 127 Flow cytometry Heparin anticoagulant blood was stained with fluorochrome-conjugated monoclonal antibodies against surface markers: APC-B220, PerCP-CD3e and PE-CD8a (eBioscience, 131 San Diego, California, USA), FITC-CD4 and appropriate isotypic antibodies (BD 132 Pharmingen, NewJersey, USA) were used as controls After remove the red blood cells, 133 the centrifugal pellet was washed with phosphate buffer saline, and then resuspended in 134 0.3 ml of PBS contain 0.02% FBS The cell surface markers were analyzed using a flow 135 cytometer (Accuri C6, BD, NewJersey, USA) 137 138 EP AC C 136 TE D 130 Lymphoproliferation The spleens of the mice were harvested after nine weeks of treatment The spleens 139 were mashed and passed through a 200-mesh sterile sieve to prepare a single cell 140 suspension The cells were suspended in RPMI-1640(Invitrogen, Paisley, Scotland, UK) 141 medium with 10% fetal bovine serum (FBS) and centrifuged The pelleted cells were 142 resuspended in Red Blood Cell Lysis Buffer Lysing Buffer to lyse erythrocytes After ACCEPTED MANUSCRIPT 143 being washed in Phosphate Buffer Solution (PBS), the total number of cells was 144 calculated and 2×106 cells were seeded in 96-well plates, then stimulated with ConA(5 145 µg/ml) for 68 h Lymphoproliferation was determined by MTT assay 147 The culture supernatant were collected after 68 h and kept at -80°C for detecting RI PT 146 PGE2 and cytokines 148 Cytokine detection SC 149 Interleukin-6 (IL-6), IL-4, IL-10, IFN-γ, TNF-α, IL-17a and IL-2 cytokines was 151 detected using a mouse Th1/Th2/Th17 cytometric bead array kit (CBA; BD Biosciences, 152 San Diego, California, USA) and was analyzed on a FACSCalibur flow cytometer 153 Standard curves were determined for each cytokine from a range of 10-5000 pg/ml M AN U 150 154 Histopathology and immunohistochemistry assay TE D 155 Kidney and spleen tissue samples were collected at the time of harvest and fixed in 157 10% buffered formalin, embedded in paraffin Hematoxylin and eosin (H&E), periodic 158 acid-Schiff stain (PAS) and Masson stain were to used check and define the 159 pathologicalstage of fibrosis of the specimen tissue For immunohistochemical staining, the kidney tissues (3 µm thick) were stained with AC C 160 EP 156 161 fluorescein-conjugated anti-mouse IgG and IgM (Abcam, Cambridge, UK) To observe 162 the distribution of collagen under and immunoglobulins deposition used a light 163 microscope 164 165 166 Statistical analysis All group results are expressed as mean± standard deviation (SD), if not stated ACCEPTED MANUSCRIPT otherwise ANOVA, Student’s t test or Fisher’s exact test (two tailed) were used for the 168 comparison of group values For comparing group values that did not follow Gaussian 169 distribution, Mann-Whitney’s test (two tailed) was used 171 Results 172 Gross observation and body weight measurement SC 170 RI PT 167 In the positive control group, prednisone induced deleterious effects in this 174 experiment, and two mice died at the end of treatment During the treatment, mice 175 manifested emaciation, listlessness and dry and dull hair in model and prednisone groups 176 However, aconitine-treated mice demonstrated glossy hair (data not shown) and less body 177 weight loss (Fig.1B) 178 179 Time course of proteinuria TE D M AN U 173 Proteinuria levels were measured for detecting renal injury degree The proteinuria 181 levels in aconitine groups were decreased more significantly than control group and 182 prednisone 183 administration (p