Biomedicine & Pharmacotherapy 96 (2017) 466–470 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha Original article Inhibitory eﬀect of Triperygium wilfordii polyglucoside on dipeptidyl peptidase I in vivo and in vitro Jingjing Wang, Yi Chu, Xiaoying Zhou MARK ⁎ School of Pharmaceutical Engineering and Life Science, Changzhou University, 213164, Jiangsu, China A R T I C L E I N F O A B S T R A C T Keywords: DPPI activity Triperygium Wilfordii Polyglucoside Triptolide CIA rat model Backgroud: Dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease is derived from granule immune cells including mast cell, neutrophils, and toxicity T cells. DPPI can activate serine proteases by removal of dipeptides from N-termini of the pro-proteases, resulting in granule immune cells activation which involved in physiological or pathological responses. Triperygium Wilfordii Polyglucoside (TWP) is one of the traditional Chinese medicines, and commonly used in rheumatoid arthritis (RA) treatment. The present study intended to evaluate the eﬀects of TWP on DPPI activity. Methods: In vivo and in vitro studies were carried out to investigate the functions of TWP or triptolide (TP) on DPPI activities in serum, tissues of CIA rats. Rats were divided into ﬁve groups randomly: normal group, untreated CIA rat group, TWP treatment CIA groups (the low dose 2.5 mg/100 g body-weight and high dose 5 mg/ 100 g body-weight), and TP treatment CIA group (4 μg/100 g body-weight). Arthritis development was monitored visually, and joint pathology was examined radiologically. Total protein concentrations in synovial ﬂuids (SFs) were determined by BCA method. Serums and tissue homogenates from CIA rats were collected and DPPI activities were detected using ﬂuorescence substrate GF-AFC. The in vitro interactions between DPPI in serums or in tissue homogenates and TWP or TP were assessed. Results: TWP-treated CIA rats showed a signiﬁcant improvement in bone erosion. TWP signiﬁcantly suppressed paw swelling and total protein concentration in the SFs of CIA rats compared with untreated CIA rats. The elevated activities of DPPI in serums or tissues of CIA rats were signiﬁcantly inhibited by TWP, but not by TP in vivo. The inhibitory eﬀects of TWP on DPPI activities were also conﬁrm by in vitro study. Conclusion: One of the therapeutic functions of TWP in RA treatment could be inhibiting DPPI activity in serums and synovial tissue produced during RA development, and then reducing inﬂammatory serine proteases activities and further recovering CIA rats from RA symptoms. 1. Introduction Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease derived from granule immune cells . DPPI is constitutively expressed by granule immune cells in a variety of tissues, such as lung, serum, liver and spleen  and plays an important role in the activation of serine proteases, such as mast cell chymase, neutrophil elastase, and T cells granulase A and B by removing dipeptides from N-termini of proserine-proteases [3–5]. Our previous study established a DPPI speciﬁc ﬂuorescent substrate Gly-Phe-AFC (GF-AFC) which can be hydrolyzed by DPPI. The cleaved AFC from GF-AFC displayed strong ﬂuorescence enhancement at Ex400 nm/Em492 nm in a linear relationship with the levels of DPPI activity . Our studies showed a direct correlation between increased DPPI activities in serum samples and arthritis severities in collagen induced arthritis (CIA) rats during the progress of ⁎ disease . The previous study reported that DPPI−/− mice were highly resistant to the development of RA in CIA rats and DPPI regulates the development of collagen-induced arthritis [7,8]. Thus, we hypothesized that the inhibition of elevated DPPI activities would be beneﬁcial for RA treatment. Triperygium Wilfordii Polyglucoside (TWP), a mixed extracts from the eﬀective parts of Tripterygium wilfordii Hook F, is reported as the useful traditional Chinese medicine for RA treatment [9–11]. Triptolide (TP) is a monomer composition included within TWP. A great amount of clinical data have shown that TWP has anti‐inﬂammatory and immuno-suppressive activities in human clinical trials for inﬂammatory and autoimmune diseases, and been historically used for RA treatment [12,13]. Numerous studies indicated that TWP suppressed production of cytokines, including TNF-α, interleukin-2 (IL-2), interferon (IFN)-γ, IL-6 and IL-8 in serum, and inhibited endothelial growth factor (VEGF) Corresponding author. E-mail address: firstname.lastname@example.org (X. Zhou). http://dx.doi.org/10.1016/j.biopha.2017.09.139 Received 21 May 2017; Received in revised form 19 September 2017; Accepted 26 September 2017 0753-3322/ © 2017 Published by Elsevier Masson SAS. Biomedicine & Pharmacotherapy 96 (2017) 466–470 J. Wang et al. 2.3. Clinical assessment of arthritis mRNA expression in CIA rats, also TWP can induce apoptosis of synoviocytes in RA [14–16]. TP was also reported as an immune-suppressor in the treatment of some inﬂammatory and autoimmune diseases [17–19]. But, the mechanisms of TWP or TP in RA treatment were still not fully understood. Our study has ﬁrstly explored the function of TWP or TP in inhibition of serum DPPI activities and investigated the association of the reductions of DPPI activities with the recoveries of CIA rat from RA symptoms in vivo using CIA rat model, a widely used animal model that shares many features with human RA especially [20,21]. Also, our study employed the molecular models to evaluate the enzymatic interactions between TWP or TP and DPPI activities (in serum or tissue) in vitro. The human recombinant DPPI (hrDPPI) and speciﬁc DPPI inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) were used as the control compounds [22,23], aiming to explore the potential mechanisms of TWP in the treatment of RA. The thickness of hind paws of each rats in diﬀerent groups were measured every 2 days using a vernier caliper from the 7th day after intragastrically administered with TWP, the body weight of each individual animals was also recorded. 2.4. SFs and serums samples preparation After two weeks of administration, all rats were sacriﬁced; the canthus blood samples and synovial membranes were collected. The canthus blood samples were stood at room temperature for 2 h, then centrifuged at 4000 rpm for 20 min, the serums were isolated and stored at −80 °C for later analysis. Synovial membranes were cut into pieces, and mixed (1: 9) with PBS buﬀer containing protease inhibitors (0.1 mM), and then homogenized for 2–3 min. The homogenates of SFs were obtained after centrifugation in 3000 rpm for 20 min at 4 °C. 2. Materials and methods Six-week wistar rats (male, 250–300 g) were purchased from Changzhou Cavens Lab Animal Company (China). Bovine type II collagen (CII) (Chondrex, USA) and Freund's incomplete adjuvant (IFA) (Sigma, USA) were used for rat immunization. TWP pills (containing 10 mg of TWP per pill) were obtained from DND Pharmaceuticals (Zhejiang Province, China). TP, purity (HPLC) > 99%, were from ZiyiReagent of China. Vernier caliper was obtained from Harbin Measuring & Cutting Tool Group Co., Ltd. Fluorescence spectra were recorded on LS55 (Perkin Elmer, USA) and the slits for excitation and emission were both set to 10 nm. Fluorescent substrate GF-AFC was made in our laboratory, The School of Pharmaceutical Engineering and Life Science, Changzhou University, Jiangsu, China . The hrDPPI used as a positive control was from R & D Systems (USA) and DPPI speciﬁc inhibitor Gly-Phe-CHN2 was purchased from Sigma (USA). Total protein concentrations in serums or SFs were determined using BCA assay kit (Pierce Chemical Co., Rockland, IL). 2.5. Determination of total protein concentration in SFs by BCA method The protein concentrations in SFs of all rats were determined using BCA Protein Concentration Assay Kit. A standard curve was generated by a series of dilutions of bovine serum albumin (BSA). The total protein concentrations in SFs were calculated based on the standard curve according to the OD value obtained from each SF samples. 2.6. DPPI activity assay As previous described , brieﬂy, 90 μl of assay buﬀer (25 mM citric acid, 10 mM NaCl, pH6.0, and 0.5 mM GF-AFC) was mixed with 10 μl of serums or SFs collected from diﬀerent groups. The ﬂuorescence intensity was monitored at Ex400/Em492 nm by ﬂuorescence spectrometer. Maximum speed of kinetic reaction (Vmax) was calculated as the changes in ﬂorescent intensity per min over 10 min in each reaction. Michaelis-Menten equation: v = Vmax[S]/(Km + [S]) where v is the initial velocity of the enzyme reaction, Vmax is the maximal velocity, [S] is the substrate concentration, and Km is the Michaelis-Menten constant (concentration of substrate at 0.5 of Vmax). 2.1. Induction of CIA rats and treatments using TWP or TP in-vivo All animal works were taken with the approval of the Changzhou University Ethics Committee. The animals were housed in controlled environment under the Guidelines of Animal Ethics and Welfare. CIA rat models were set up twice (30 wistar rats each time) using the modiﬁed method described previously [6,24,25]. Rats were divided into ﬁve groups randomly (six rats in each group). Control groups: normal group and untreated CIA rat group; three CIA treatment group: TWP treatment groups including low-dose group (2.5 mg/100 g bodyweight) and high-dose group (5 mg/100 g body-weight), TP treatment group (4 μg/100 g body-weight). For CIA rat groups, including untreated CIA control group and TWP or TP treatment groups, 300 μl of collagen II-IFA emulsiﬁed liquid (1 mg/ml) was intra-dermal injected at the base of each rat tail on day 0. The boosting injections were at day 3 and 7 in same method. After two weeks post-injections, the CIA rats of treatment groups were intragastrically administered with TWP or TP every day for 14 days (TWP and TP dissolved in 0.5% sodium carboxymethyl cellulose (CMC) in required concentrations). The rats in untreated CIA rat group were administered with equal volume of 0.5% CMC only for same period. 2.7. The interactions between DPPI and TWP or TP in-vivo TWP or TP were dissolved in dimethysulphoxide (DMSO) to give stock solution of 4000 μg/ml, and diﬀerent concentrations (0, 250, 500, 1000, 2000 μg/ml) were prepared with PBS. 1 μl of diﬀerent concentrations of TWP, TP or DPPI (20 mM)  were separately added into hrDPPI (0.5 mU), or into serums or SFs of CIA rats with high DPPI levels. The interactions of the mixtures were left in shaker at 35 °C for 2 h. After incubation, the DPPI activities in each interation-condition were determined using GF-AFC by ﬂuorescence spectrometer at Ex400/ Em492 nm. The inhibition rate relative to untreated sample in percentage was calculated as: Inhibition rate (%) = [Vmax (TWPtreatedsample) – Vmax (untreatedsample)]/Vmax (untreatedsample) × 100%. The concentration of TWP resulting in 50% inhibition rate of enzyme activity (IC50) was determined by SPSS software. 2.8. Statistical analysis 2.2. Radiography Statistical analyses and graphs were performed using GrapPad Prism 7 package. All the results were expressed as mean ± SEM. The MannWhitney non-parametric test for unpaired variables was used to compare diﬀerences between groups. *P < 0.05 was taken as statistically signiﬁcant, **P < 0.01, statistically very signiﬁcant. Radiography analysis of the hind limbs of rats in diﬀerent groups was carried out using a HP Faxitron (Faxitron X-Ray LLC, Wheeling, IL, USA). Images were visualized using a standard light box and photographed. 467 Biomedicine & Pharmacotherapy 96 (2017) 466–470 J. Wang et al. Fig. 1. Inhibition of bone degradation in the joint by TWP treatment. A. normal rat; B. untreated CIA rat; C. TWP (5 mg/100 g body weight) treated CIA rat. 3. Results Table 2 Total protein concentration in SFs of rats from diﬀerent groups. 3.1. Radiological observation Bone erosion is one of the major features of joint damage caused by RA. Joint narrowing was observed in most of the arthritis-bearing rats . The X-ray results (Fig. 1) showed that the toes joints could barely be seen in untreated CIA rats (Fig. 1B). With TWP treatment, TWP signiﬁcantly improved on joint destruction of CIA rat (Fig. 1C). 3.4. TWP reduced DPPI activities in serum or SFs elevated over RA development of CIA Rats in vivo The results from ﬂuorescent kinetic activity assay (Fig. 2) demonstrated the activities of DPPI in SFs or serums of CIA rats after 2wks administration of TWP or TP. The value of DPPI activity in SFs of CIA Table 1 The clinical assessment variables of rat paw swollen thickness (cm) in untreated CIA group and TWP treatment CIA rats group. Day 33 untreated rats swollen thickness TWP(5 mg/ml) swollen thickness 0.86 ± 0.02 0.86 ± 0.02 0.84 ± 0.02 0.83 ± 0.01 0.51 ± 0.05 0.51 ± 0.047 0.44 ± 0.03 0.39 ± 0.01 Total protein concentration 526 ± 145 1652 ± 32.8 932 ± 105 Serums and tissue homogenates were prepared as described above and the eﬀects of TWP in vitro on DPPI activities were determined. The data showed the increased inhibitory rates of TWP on DPPI activities in diﬀerent tissues (serums, SFs, liver, spleen, and lung) and all in dosedependent manners（Fig. 3). However, After incubation with diﬀerent TWP doses of 25, 50, 100 and 200 μg/ml, the activities of hrDPPI were inhibited by 15.1, 36.1, 43.6 and 55.2%. The serums containing high DPPI activity collected from CIA rats were incubated with TWP (25, 50, 100 and 200 μg/ml), serums DPPI activities were inhibited by 22.3, 34.2, 45.1% and 75.6% respectively. After incubation of TWP (25, 50, 100 and 200 μg/ml) with SFs of CIA rats, DPPI activities were inhibited by 8.84, 11.5, 22.1%, 34.0%. In vitro study demonstrated that TWP has a direct inhibitory eﬀects on DPPI activity in serums or in SFs. The IC50 of TWP against hrDPPI activity, serums DPPI activity and SFs DPPI activity were 158 μg/ml; 112 μg/ml and 266 μg/ml. The lower the IC50 values for TWP toward DPPI, the stronger the inhibition of DPPI. Gly-Phe-CHN2 is a DPPI speciﬁc inhibitor and used in our study to assess the strength of inhibitory eﬀects between Gly-Phe-CHN2, TWP, TP and solvent (0.01% DMSO) by adding them separately into hrDPPI (Fig. 4A) or SFs (Fig. 4B) and serums (Fig. 4C) from CIA rats. As shown in Fig. 4, compared with untreated controls, the eﬀects of Gly-PheCHN2 signiﬁcantly decreased DPPI activities of hrDPPI (P = 0.001), SFs (P = 0.004) and serums (P = 0.006) of CIA rats; TWP also signiﬁcantly reduced DPPI activities of hrDPPI (P = 0.021), SFs (P = 0.016) and serums (P = 0.02) of CIA rats. However, there was no signiﬁcant difference between TP and untreated controls in all of hrDPPI, SFs or serums in vitro. Total protein concentrations in SFs of rats were shown in Table 2. In untreated CIA rat group, the total protein concentration in SFs were 68.1% (P = 0.01) higher than that in the normal group. In contrast, CIA rats treated with TWP (5 mg/100 g body weight) exhibited a marked decrease of total protein concentration by 43.5% compared with untreated CIA rat group. There was no signiﬁcant diﬀerence between TWP treatment group and normal group. Day 31 TWP 3.5. TWP inhibited DPPI activity in vitro 3.3. TWP recovered total protein concentration that elevated in SFs over RA development of CIA rats Day 29 Untreated rat group was 16.4 folds higher (P = 0.03) than that in the normal group. The activity of DPPI was decreased 1.3 times in TWP treatment (5 mg/100 g body-weight) group (P = 0.18) compared with the untreated CIA rat group (Fig. 2A). In the serums of untreated CIA rat group the DPPI activity was increased by 63% compared to the normal rat group. Compared with the untreated CIA rat group, the DPPI activity of TWP treatment (5 mg/100 g body-weight) CIA rat group was signiﬁcantly dropped by 58.9% (P = 0.002) (Fig. 2B). There were no signiﬁcant diﬀerence in serums DPPI activities between untreated CIA rat group and TP treatment CIA rat group (Fig. 2C). Rat paws swollen and the changes of the thickness are typical symptom over RA development. The data in Table 1 from pathological assessments have shown the paw swollen thickness of CIA rats with or without TWP treatment. The paw swollen thickness was kept a high level in untreated CIA rats. TWP treatment decreased the severity of paw swelling of CIA rats in comparison with the untreated CIA rats. TWP (5 mg/100 g body weight) exhibited recovery eﬀects from the development of arthritis. At the same time, TWP treatment could effectively suppress the loss of body weight of CIA rats. Day 27 Normal Data are expressed as Mean ± SEM (n = 6). 3.2. Pathological changes in CIA rat after treatment with TWP CIA rat group Group Data are expressed as Mean ± SEM (n = 6). 468 Biomedicine & Pharmacotherapy 96 (2017) 466–470 J. Wang et al. Fig. 2. CIA rats model in vivo study after TWP or TP intragastric administrations for 14 days, the levels of DPPI activities in (A) SFs and (B) serums of normal rats, untreated CIA rats and TWP treated CIA rats; (C) the levels of DPPI activities in serums of normal rats, untreated CIA rats and TP treated CIA rats, (n = 6). The values are expressed as mean ± SEM. 4. Discussion Our both in vivo and in vitro studies highlighted that TWP is able to inhibit the increasment of DPPI activity in serum, SFs and tissues of CIA rat and suppress RA development, giving a new insight that the possible pharmacological mechanism of TWP in treating RA may be related to the decrease of elevated DPPI activity to keep synovial homeostasis. In present study, we demonstrated that TWP can ameliorate the inﬂammatory condition of arthritis and inhibit the progression of RA in CIA rats. The major pathological changes of RA are chronic inﬂammatory responses of synovial joints. Our radiological observation indicated that TWP treatment lead to reduced cartilage destruction and bone erosion, contributing to a protective eﬀect against RA. Our results showed that the total protein concentrations in SFs of untreated CIA rat group were signiﬁcantly higher than that in the normal group, while the total protein concentration in the SFs of TWP treatment group were recovered to the level of normal group. It was also observed that the paw’s swollen thickness of CIA rats in TWP treatment group were also recovered to normal level and well decreased compared with the untreated CIA rats. DPPI activities in SFs and serum samples of CIA rats in vivo were much more increased compared with that in normal rats, while TWP signiﬁcantly reduced DPPI activity in SFs and serum samples of CIA rats in dose-dependent manner after treatment, and signiﬁcantly recovered to normal levels. In our in vitro study, TWP showed the inhibitory eﬀect on hr-DPPI activity, and was very eﬀective in inhibiting DPPI activity in serum and in SFs from CIA rats in vitro. However TP had no inhibitory eﬀect on DPPI activity in vivo and in vitro respectively. 5. Conclusion In summary, our studies discovered that the elevated DPPI activities in SFs and serums of CIA rats during RA progress can be decreased by TWP treatment in vivo; the increased DPPI activities or decreased DPPI activities were associated with the development of RA progress or recovery from RA development. The inhibitory eﬀects of TWP on DPPI activities were also conﬁrm by in vitro study. We suggest that a possible therapeutic function of TWP in treatment of RA might be relating to inhibition of increments of DPPI activities and then further reduction of serine proteases activations, as well as indirectly inhibiting and weakening the roles of activated serine proteases in inﬂammatory symptoms. However, TP is a very monomer in the components of TWP and had fewer eﬀects on TWP in this case ; also TP did not show the inhibitory function on DPPI activity in vivo and in vitro. Fig. 3. In vitro study DPPI activities (A) of hrDPPI (0.5mU), (B) in serums, (C) in SFs, (D) in spleen, (E) in liver and (F) in lung of CIA rats were inhibited by addition of TWP (0, 25, 50, 100, 200 μg/ml). The inhibition rate was calculated: Inhibition rate (%) = [Vmax (TWPtreatedsample) – Vmax (untreatedsample)]/Vmax (untreatedsample) × 100%, and expressed as mean ± SEM (n = 3–6, in triplicates). 469 Biomedicine & Pharmacotherapy 96 (2017) 466–470 J. Wang et al. Fig. 4. Eﬀects of Gly-Phe-CHN2 (20 μM), TWP (100 μg/ml) or TP (75 μg/ml) on DPPI activities of (A) hrDPPI(0.5mU), (B) SFs and (C) serums of CIA rats in-vitro. Values are expressed as mean ± SEM (n = 3-6, in triplicates), *P < 0.05, **P < 0.01. Acknowledgements We thank Changzhou University Talent Introdution Researh Fund (ZMF14020066); Start-up Research Laboratory for Over-sea Talent Fund (Z391405); Changzhou Science and Technology Bureau International Cooperation Research Fund, China (CZ20150014) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17-2082) for ﬁnancial supports.  References   W. Yang, W. Xia, J. Mao, D. Xu, J. Chen, S. Feng, J. Wang, H. Li, C.F. Theisen, J.M. Petersen, High level expression, puriﬁcation and activation of human dipeptidyl peptidase I from mammalian cells, Protein Expr. Purif. 76 (2011) 59–64.  C.T. Pham, R.J. Armstrong, D.B. Zimonjic, N.C. Popescu, D.G. Payan, T.J. Ley, Molecular cloning chromosomal localization, and expression of murine dipeptidyl peptidase I, J. Biol. Chem. 272 (1997) 10695–10703.  B. Ruﬀell, N.I. Aﬀara, L. Cottone, S. Junankar, M. Johansson, D.G. Denardo, L. Korets, T. Reinheckel, B.F. Sloane, M. 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