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A combination of the main constituents of Fufang Xueshuantong Capsules shows protective effects against streptozotocin-induced retinal lesions in rats
Author’s Accepted Manuscript A Combination of the Main Constituents of Fufang Xueshuantong Capsules Shows Protective Effects against Streptozotocin-induced Retinal Lesions in Rats Weijie Jian, Suyun Yu, Minke Tang, Huihui Duan, Jianmei Huang www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(15)30222-1 http://dx.doi.org/10.1016/j.jep.2015.11.021 JEP9820
To appear in: Journal of Ethnopharmacology Received date: 4 July 2015 Revised date: 8 October 2015 Accepted date: 6 November 2015 Cite this article as: Weijie Jian, Suyun Yu, Minke Tang, Huihui Duan and Jianmei Huang, A Combination of the Main Constituents of Fufang Xueshuantong Capsules Shows Protective Effects against Streptozotocin-induced Retinal Lesions in Rats, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.11.021 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 galley proof before it is published in its final citable 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.
A Combination of the Main Constituents of Fufang Xueshuantong Capsules Shows Protective Effects against Streptozotocin-induced Retinal Lesions in Rats
Weijie Jian1, Suyun Yu1, Minke Tang, HuihuiDuan, Jianmei Huang # School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing, 100102, China.
#
Corresponding author: Dr. Jianmei Huang, School of Chinese Materia Medica, Beijing University of Chinese Medicine, 6#, Wangjing Zhonghuan Nanlu, Chaoyang District, Beijing 100102, China.
Tel: +0086-10-84738619 E-mail: [email protected] 1
These two authors contributed equally to this work.
Abstract Ethnopharmacological Relevance Fufang Xueshuantong Capsule, an herbal formula licensed for clinical use in China, which is composed of Panax notoginseng (Burkill) F.H.Chen, Salvia miltiorrhiza Bunge, Astragalus membranaceus (Fisch.) Bunge, and Scrophularia ningpoensis Hemsl, has proven effective for the treatment of diabetic retinopathy. However, its bioactive constituents are still ambiguous. In this study, the therapeutic effects of a combination of the main constituents of Fufang
Xueshuantong
Capsule
(cFXT)
were
evaluated
in
streptozotocin
(STZ)-induced retinal lesions to identify the bioactive constituents. Methods Sprague-Dawley rats, except for those in the control group (vehicle+ vehicle), were administered a single injection of 60mg/kg STZ. One-week later, STZ-treated rats were randomly divided into three groups—one STZ group (STZ + vehicle) and two cFXT treatment groups (STZ + cFXT). The rats in the latter two groups received cFXT 44.8 mg/kg or cFXT 22.4 mg/kg by intragastric gavage once per day, for 24 consecutive weeks. The rats in the control and STZ groups received the vehicle in the same way. Body weights and fasting blood glucose levels were recorded every four weeks. After treatment, hemorheological tests were performed to record the erythrocyte aggregation indexes, blood viscosity, and plasma viscosity. The trypsin digestion method was used to observe pericyte and acellular capillary counts in the retina. Ultraviolet spectrophotometry was utilized to measure the activity of aldose reductase (AR) by measuring the nicotinamide adenine dinucleotide phosphate (NADPH) consumption at 340 nm. An immunohistochemical assay was used to observe the expressions of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in the retina. The expression levels of intercellular adhesion molecule-1 (ICAM-1), endothelin-1 (RT-1),and occludin in the retina were tested by the western blot assay. Results cFXT is composed of 991.44 mg/g saponins of Panax notoginseng, 1.62 mg/g harpagoside, 0.70 mg/g cryptotanshinone, 0.74 mg/g tanshinone I, and 5.50
mg/g astragaloside A. Although it showed no effects on the increased body weight and blood glucose levels induced by STZ in rats. However, it showed a tendency to attenuate the increase in erythrocyte aggregation, plasma viscosity, and acellular vessel and pericyte loss, paralleled with a reversal of the hyper-activation of AR, the hyper-expression of VEGF, ICAM-1, and ET-1, and the hypo-expression of PEDF and occludin in the retinas of STZ-treated rats. Conclusion The saponins of Panax notoginseng, harpagoside, cryptotanshinone, tanshinone I, and astragaloside A are the main bioactive constituents of Fufang Xueshuantong Capsule and contribute to the attenuation of STZ-induced retinal lesions in rats. These constituents can be used as the base to optimize a new drug for the treatment of diabetic retinopathy, and can be selected for quality control of Fufang Xueshuantong Capsules. Key Words Diabetic
retinopathy;
Fufang
Xueshuantong
Capsule;
Endothelin-1;
Intercellular adhesion molecule-1; Pigment epithelium derived factor; Occludin. Chemical Compounds Notoginsenoside R1 (CAS NO., 80418-24-2), Ginsenoside Rg1 (CAS NO., 22427-39-0), Ginsenoside Rb1 (CAS NO., 41753-43-9), Harpagoside (CAS NO., 19210-12-9), Cryptotanshinone (CAS NO., 35825-57-1), Tanshinone I (CAS NO., 568-73-0), Astragaloside A (CAS NO., 83207-58-3)
1
Introduction Diabetic retinopathy (DR) is a disease induced by diabetes that involves the
retinal capillaries, arterioles, and venules and is accompanied by leakage or occlusion of the small vessels (Nentwich and Ulbig, 2015). Phanerous lesions are observed with the progression of DR, such as microaneurysms, hemorrhages, vessel abnormalities, fibrous proliferation, and angiogenesis (Bandello et al., 2013). Studies have demonstrated that down-regulation of the factors involved in the pathogenesis of DR, such as hyper-activation of aldose reductase (AR) (Hattori et al., 2010), over expression of vascular endothelial growth factor (VEGF) (Matsuda et al., 2014) and intercellular adhesion molecule-1 (ICAM-1), and up-regulation of pigment epithelium-derived factor (PEDF) (Bucolo et al., 2012; Bucolo et al., 2009; Miyamoto et al., 2000; Ogata et al., 2002) are effective strategies for the treatment of DR. Fufang Xueshuantong Capsule (FXT, also called Compound Xueshuantong) is a Chinese herbal formula composed of Panax notoginseng (Burkill) F.H.Chen (Araliaceae), Salvia miltiorrhiza Bunge (Lamiaceae), Astragalus membranaceus (Fisch.)
Bunge
(Leguminosae),
and
Scrophularia
ningpoensis
Hemsl
(Scrophulariaceae)(Sheng et al., 2014). It has been clinically used to treat DR (Cheng, 2013). Our previous research in a model of streptozotocin (STZ)-induced rat diabetes showed that FXT could normalize abnormal whole blood viscosity (WBV), plasma viscosity (PV), and erythrocyte aggregation indexes (HXBJJ). In addition, FXT attenuated the development of microvessel lesions in the retina by decreasing pericyte loss and reducing acellular capillaries. This was in parallel to down-regulation of AR hyper-activity and over expression of VEGF and ICAM-1, and up-regulation of the hypo-expression of PEDF and occludin. FXT thus showed protective effects against STZ-induced retinal lesions in rats (Duan et al., 2013). By reviewing the literature, we found that the characteristic constituents of the four herbs might be related to the pharmacological effects of FXT. Ginsenoside Rb1 from Panax notoginseng showed inhibitory effects on endothelial proliferation with
increased production of superoxide anion (Ohashi et al., 2006). Ginsenoside Rb1, ginsenoside Rg1, and notoginsenoside R1 were effective in treating microcirculatory disturbances induced by lipopolysaccharide (Sun et al., 2007). Cryptotanshinone and tanshinone I from Salvia miltiorrhiza showed inhibitory effects on ICAM-1 and endothelin-1 (ET-1) expression in human umbilical vein endothelial cells (Jin et al., 2009; Onitsuka et al., 1983; Zhou et al., 2006). Harpagoside from Scrophularia ningpoensis showed protective effects against human vascular endothelial cell injury induced by hydrogen peroxide (Kim et al., 2002). Astragaloside A from Astragalus membranaceus reduced retinal ganglion cell apoptosis and down-regulated AR activity to prevent DR (Ding et al., 2014). In this study, the afore mentioned bioactive constituents of FXT were selected to form a constituent combination (cFXT). The quantity of the selected constituents in cFXT was in accordance with that in FXT as had been determined previously (Yu et al., 2014). The therapeutic effects of cFXT on STZ-induced retinal lesions in rats were evaluated in this study, aiming to clarify the roles of the core bioactive components of FXT and provide a foundation to optimize a new medicine with minimum constituents, high efficacy, and easy quality control. 2
Materials and Methods
2.1 Animals and Reagents Male Sprague-Dawley (SD) rats weighing 230 to 250 g (SCXK [Jing] 2007-0001) were purchased from Vital River (Beijing, China). The animal experiment protocol was in accordance with the statement for the Use of Animals in Ophthalmic and Vision Research by the Association for Research in Vision and Ophthalmology and approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. Rats were acclimatized for one week before experiments, housed in a 12-hour light/dark cycle, at constant temperature and humidity, and were provided enough food and water. STZ was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) or Abcam (Cambridge, UK).
2.2 Drug preparation The cFXT was composed of 991.44 mg/g saponins of Panax notoginseng (NO. 20110831, containing 9.55% notoginsenoside R1, 35.52% ginsenoside Rg1, 36.64% ginsenoside Rb1, and 18.29% other saponins), 1.62 mg/g harpagoside (NO. 20110923, ≥99%) 0.70 mg/g cryptotanshinone (NO. 20110520, ≥98%), 0.74 mg/g tanshinone I (NO. 20111125, ≥95%), and 5.50 mg/g astragaloside A (NO. 20111216, ≥99%). Saponins, harpagoside, cryptotanshinone, and tanshinone I were purchased from Zelang Pharmaceutical Technology Co., Ltd. (Nanjing, China) and astragaloside A was purchased from Kang Bang Technology Co., Ltd. (Chengdu, China). The proportions of these constituents in cFXT were in accordance with those in FXT as determined previously (Yu et al., 2014). cFXT solution was prepared by mixing 10 ml solution A with 100 ml solution B and diluting to 500 ml with distilled water. Solution A was prepared by suspending cryptotanshinone and tanshinone I with 5% carboxymethylcellulose sodium solution (CMC-Na, NO. 20031019, ≥ 6.5%, purchased from Guangfu institute of fine chemicals, Tianjin, China) and diluting with 5% CMC-Na to a final contents of 0.156 mg·ml-1 cryptotanshinone and 0.164 mg·ml-1 tanshinone I respectively. Solution B was prepared by dissolving saponins of Panax notoginseng, harpagoside and astragaloside A with distilled water and diluting with distilled water to a final contents of 22.2 mg·ml-1 saponins of Panax notoginseng, 0.036 mg·ml-1 harpagoside and 0.121 mg·ml-1 astragaloside A respectively. All afore mentioned solutions were stored at 4 ℃ and used at room temperature within 3 days. 2.3 Animal modeling, grouping, and drug treatment All rats (n=180), except those in the control group (vehicle + vehicle, n=35), were treated with a single intraperitoneal injection of 60 mg/kg STZ. One week after STZ injection, the rats were randomly divided into three groups: one STZ group (STZ + vehicle) and two cFXT treatment groups (STZ + cFXT) that received either 44.8 mg/kg or 22.4 mg/kg cFXT. cFXT was administered by intragastric gavage daily for 24 consecutive weeks at 1 ml/100g body weight. The rats in the control and STZ
group received the vehicle in the same way. 2.4 Recording of body weight and blood glucose levels of rats During cFXT treatments, the fasting body weight and fasting blood glucose levels of all rats was measured every four weeks. 2.5 Hemorheological analysis After cFXT treatments, fresh arteriopuncture blood samples were collected in heparin (10 mg/ml) pre-coated tubes. Plasma samples were collected by centrifugation at 3000 rpm for 15 min. WBV and PV at a shear rate of 60 s -1 were assayed with a blood viscometer (STEELLEX, China). HXBJJ was determined using an RBC deformation and aggregation test instrument (STEELLEX, China). 2.6 Trypsin digestion method After cFXT treatments, the trypsin digestion method was used to determine the counts of pericytes and acellular capillaries as described in a previous study (Duan et al., 2013). Briefly, the isolated retina was incubated with 3% crude trypsin (Difco Laboratories, Detroit, MI, USA) within Tris buffer (0.1 M, pH=7.8) in an incubator shaker (37°C). The retina was transferred to phosphate-buffered saline (pH=7.4) when the inner limiting membrane began separating from the retina after about 2–3 h of incubation. While being observed under the microscope, the separated vascular tree was washed with distilled water until free of neural tissue, set onto glass slides, air-dried, and stained with hematoxylin and periodic acid-Schiff stain. The number of pericytes and acellular occluded vessel segments were counted using an image system (ECLIPSE 80i, Nikon, Japan). The results were presented as the relative capillary density (numbers of cells per millimeter square of capillary area). 2.7 Measurement of aldose reductase activity After cFXT treatment, the separated retina of the rats was homogenized with 10
volumes (v/w) of potassium phosphate buffer (100 mM, pH=6.2). The suspension solution was centrifuged at 15000 rpm for 30 min. The supernatant (crude AR solution) was stored at -20°C. Protein concentrations of the supernatant were determined with a Bradford Protein Assay Kit (Biomiga, San Diego, CA, USA). AR activity was assayed in accordance with the method reported previously (Akileshwari et al., 2012; Duan et al., 2013). Briefly, the reactant was pre-incubated at 37 °C. Nicotinamide adenine dinucleotide phosphate (NADPH) was added to the reactant to initiate the reaction for 5 min. Subsequently, 2 ml of NaOH solution (0.3 mM) was added to stop the reaction at 60 °C for 15 min. The consumption of NADPH was tested according to the decline in absorbance at 340 nm using a spectrophotometer. An absorbance decrease of 0.001 per min induced by 1 mg of protein was defined as one unit (U) of AR activity. 2.8 Immunohistochemical assay After cFXT treatments, the expression levels of VEGF and PEDF were assessed using an immunohistochemical assay as previously described (De Juan et al., 2000; Duan et al., 2013). Indirect immunoperoxidase staining was performed on a paraffin section of the paraformaldehyde-stabilized bulbus oculi using immunostaining reagents (Beijing Zhong Shan-Golden Bridge Biological Technology Co., LTD, China). The primary antibodies, including rabbit anti-VEGF (ab46154, 1:50 dilution, Abcam, Cambridge, UK) and goat anti-PEDF antibodies (sc16956, 1:50 dilution, Santa Cruz, Dallas, TX, USA), were omitted for negative controls. Immunostaining slides of VEGF or PEDF were analyzed with the Image Pro Plus Analysis Software (Media Cybernetics, Rockville, MD, USA). 2.9 Western blot analysis After cFXT treatments, the expression of ICAM-1, ET-1, and occludin in the retinas was assayed by western blot analysis according to the previously reported method (Park et al., 2009). Protein extracts of ICAM-1, ET-1, and occludin were
prepared from the retinas of rats. The target proteins, mouse anti-ICAM-1 antibody (ab2213, 1:2000 dilution, Abcam, Cambridge, UK), rabbit anti-ET-1 antibody (ab117757,1:2000 dilution, Abcam, Cambridge,UK), rabbit anti-occludin antibody (ab31721,1:500 dilution, Abcam, Cambridge, UK), goat anti-rabbit lgG (1:500), and goat anti-mouse lgG (1:2000) were visualized with an enhanced chemiluminescence kit (Sc-2048, Santa Cruz, Dallas, TX, USA). The gray intensities of the target bands of each sample were analyzed using Image Pro Plus Analysis Software (Media Cybernetics, Rockville, MD, USA). 2.10 Data analysis Data for all observed parameters were analyzed by one-way ANOVA followed by a Fischer’s least significant difference post hoc test for multiple comparisons. Values were presented as means±SEM. P<0.05 indicated a statistically significant difference. 3
Results
3.1 cFXT treatment had no effects on body weight and blood glucose level in STZ rats As shown in Fig. 1, rats in the STZ groups were found to gain less weight during the 24-week period than the control rats, and this phenomenon was not reversed by 44.8 mg/kg or 22.4 mg/kg cFXT treatments (F
(3,211)=37.961,
P<0.01, Fig. 1A).
Meanwhile, sustained high blood glucose levels were induced by STZ treatments. The increased blood glucose levels were not affected by 44.8 mg/kg or 22.4 mg/kg cFXT treatments (F (3,211)=78.109, P<0.01, Fig. 1B).
Fig. 1 Effects of cFXT on fasting body weight and blood glucose level. One week after streptozotocin (STZ) injection, the fasting body weight (A) and blood glucose level (B) of rats was measured every four weeks for 25 weeks. N=60 for each STZ group and n=35 for control group. ▲ or ▼, P<0.01 for all STZ groups vs. Control. 3.2 cFXT treatment reduced HXBJJ and WBV, but not PV, in STZ rats As shown in Fig. 2, STZ treatment led to enhanced HXBJJ (F (3, 28) =5.064, P<0.01, Fig. 2A) and elevated WBV (F (3, 28) =7.861, P<0.01, Fig. 2B) and PV (F (3, 28) =3.320, P=0.03 Fig. 2C) in rats. Daily treatment of 44.8 mg/kg or 22.4 mg/kg cFXT for 24 weeks significantly alleviated HXBJJ and decreased WBV as compared with STZ injection only. Regarding the elevated PV in STZ rats, neither 44.8 mg/kg nor 22.4 mg/kg cFXT treatment showed a significant down-regulating effect.
Fig. 2 Effects of cFXT on HXBJJ, WBV, and PV of rats. At the end of cFXT treatment, the erythro-agglutination index (HXBJJ) (A), whole blood viscosity at a shear rate of 60 per second (WBV) (B), and plasma viscosity at a shear rate of 60 per second (PV) (C) were assayed. N=8 for each group. or * P<0.05 vs. STZ group.
▲
P<0.01 vs. Control;
**
P<0.01
3.3 cFXT treatment relieved retinal lesions in STZ rats STZ treatment induced significant pericyte loss and acellular capillary proliferation in rat retinas as shown in Fig. 3. Although both 44.8 mg/kg and 22.4 mg/kg cFXT treatments did not significantly reduce pericyte loss in STZ rats (F (3, 34) =7.035, P=0.103, Fig. 3A), cFXT at 44.8 mg/kg significantly attenuated the proliferation of acellular capillaries (F (3, 34) =27.285, P<0.01, Fig. 3B).
Fig. 3 Effects of cFXT on pericyte loss and acellular capillary proliferation. The pericyte counts (A) and acellular capillary counts (B) in the retina were assayed after 24weeks of cFXT treatment. N=10 for each STZ group and n=8 for Control.
▲
or
▼
P<0.01 vs. Control; * P<0.01 vs.STZ group. 3.4 cFXT treatment inhibited the activated AR in STZ rats As shown in Fig. 4, STZ treatment increased AR activity in the retinas of rats.
The activity of AR was remarkably inhibited by treatment with 44.8 mg/kg or 22.4 mg/kg cFXT (F (3, 28) =120.943, P<0.01).
Fig. 4 Effect of cFXT on increased AR activity. At the end of cFXT treatment, the activity of aldose reductase (AR) was assayed. N=8 for each group.
▲
P<0.01 vs.
Control; * P<0.01 vs. STZ group. 3.5 cFXT treatment reversed the abnormal expression of VEGF and PEDF in STZ rats The expression of VEGF in rat retinas was notably increased by STZ treatment, and the elevated expression of VEGF was markedly down-regulated by 44.8 mg/kg or 22.4mg/kg cFXT treatment (F
(3, 16)=19.308,
P<0.01, Fig. 5A). The expression of
PEDF in the retina was decreased by STZ treatment, and the depressed expression of PEDF was up regulated distinctly by 44.8 mg/kg or 22.4mg/kg cFXT (F (3, 16) =11.963, P<0.01, Fig. 5B).
Fig. 5 Effects of cFXT on the expression of VEGF and PEDF. At the end of cFXT treatment, the expression of vascular endothelial growth factor (VEGF) (A) and pigment epithelium derived factor (PEDF) (B) in the retina was assayed with an immunohistochemical assay. N=5 for each group.
▲
or
▼
P<0.01 vs. Control;
**
P<0.01 or * P<0.05 vs. STZ group. 3.6 cFXT treatment regulated the abnormal expressions of ICAM-1, ET-1, and occludin in STZ rats As shown in Fig. 6, the expression of ICAM-1, ET-1, and occludin in the retinas of rats in the STZ group was remarkably dysregulated in comparison with that in the control group. The over expression of ICAM-1 (F (3, 16) =12.948,Fig. 6A) and ET-1 (F (3, 16)
=7.443, P<0.01, Fig. 6B) were well down regulated by treatment with 44.8
mg/kg or 22.4 mg/kg cFXT. The hypo-expression of occludin in the retinas of rats
showed a trend towards recovery after treatment with 44.8 mg/kg or 22.4 mg/kg cFXT, but had no significant difference compared to that in STZ rats (F(3, 16)=24.885, P<0.01, Fig. 6C).
Fig. 6 Effects of cFXT on the expression of ICAM-1, ET-1, and occludin. At the end of cFXT treatment, the expression of intercellular adhesion molecule-1 (ICAM-1) (A), endothelin-1 (ET-1) (B),and occludin (C) in the retina was assayed with western
blot assays. N=5 for each group.
▲
or
▼
P<0.01 vs. the Control;
**
P<0.01, * P<0.05
vs. STZ group. 4
Conclusion and Discussion In this study, a combination of the main constituents of Fufang Xueshuantong
Capsule (cFXT) proved to have the majority of pharmacological effects possessed by the herbal formula FXT (Duan et al., 2013). That is, cFXT reduced STZ-induced retinal lesions in rats by decreasing HXBJJ and WBV levels, and attenuating the proliferation of acellular capillaries. Furthermore, cFXT inhibited AR activity, and rectified the over expression of VEGF, ICAM-1, and ET-1,and the hypo-expression of PEDF in the retina. These results indicate that cFXT contains the bioactive constituents of FXT important for protection against retinal lesions, and suggests that the potential optimization of FXT with easily controlled formulas and minimum constituents would be possible by further research. However, it should be noted that there were some differences found between the therapeutic effects of cFXT compared to FXT. These include a lack of significant attenuation of PV, reduction in pericyte loss, or enhanced expression of occludin (Duan et al., 2013). Obviously, there are constituents in FXT other than those in cFXT that contribute to the retinal protective effects of FXT. One way of further improving cFXT is to identify the other bioactive chemicals in FXT and incorporate them into the combination. During diabetes, the polyol pathway is stimulated by sustained hyperglycemia (Safi et al., 2014). In most cases, the activated polyol pathway leads to activation of AR within cells, accompanied with proliferation of reactive oxygen species (ROS) and advanced glycation end products (AGEs), subsequently leading to a cascade of events, such as the augmentation of oxidative stress, apoptosis, inflammatory response, and promotion of angiogenesis (Yang et al., 2010). DR develops as a result of these pathological alterations within the diabetics retina (Abu El-Asrar et al., 2013). Inhibition of AR activity has been demonstrated to be effective for treatment of DR (Hattori et al., 2010). However, inhibitors have seldom been used in clinical practice
(Hotta et al., 2012), mostly because of pharmacokinetic drawbacks, adverse side effects, or low in vivo efficacy. Our research demonstrated that cFXT inhibits AR activity in the retina, suggesting that the constituents of cFXT may be candidates as AR inhibitors. As shown in the preparation of cFXT, saponins from Panax notoginseng account for a major proportion of the combination. These results should encourage the investigation to determine whether the ginsenosides are AR inhibitors. Molecules other than AR may also be involved in the effects of cFXT. It has been reported that AGEs increase the expression of ICAM-1 and VEGF in microvascular endothelial cells (Ibrahim et al., 2011; Stitt et al., 2000; Yamagishi and Matsui, 2011; Yamagishi et al., 2008a; Yamagishi et al., 2008b). ICAM-1 is a crucial mediator of vascular permeability (Bucolo et al., 2009; Miyamoto et al., 2000). VEGF damages the blood-retinal barrier by inducing the rapid phosphorylation of occludin (Bucolo et al., 2009). PEDF can inhibit the angiogenesis induced by VEGF; however, the balance of VEGF and PEDF is disrupted in diabetes (Ogata et al., 2002). Our research showed that cFXT reduced retinal lesions by the depressing expression of ICAM-1 and VEGF and increasing the expression of PEDF which may due to the decrease of AGEs. This indicated that cFXT may regulate multiple factors involved in the DR pathological pathway. Careful identification of causal relationships between the activity of AR and molecular alterations of ICAM-1, PEDF, and VEGF by further experiments is another way to optimize the combination of cFXT at the pharmacological mechanism level. In conclusion, our study has furthered our understanding of the bioactive constituents of a Chinese herbal medicine, the FXT Capsule. Saponins of Panax notoginseng, harpagoside, cryptotanshinone, tanshinone I, and astragaloside A contribute the protective effects of FXT on STZ-induced retinal lesions. The combination of the above chemicals can be used as the basis to optimize a new drug for the treatment of diabetic retinopathy, and it also demonstrates a promising method to optimize the formulation of FXT for better, easily controlled efficacy with only the
minimum of constituents. 5
Acknowledgements This study was supported by grants from Ministry of Science and Technology of
China (Grant No., 2011ZX09201-201-22). 6
Conflict of interest The authors declare that there are no conflicts of interest.
7
Abbreviations The abbreviations utilized in this paper are presented in Table 1.
Table 1 Abbreviations Utilized Abbreviation
Full Name
AGEs
advanced glycation end products
AR
aldose reductase
cFXT
combination of main constituents of Fufang Xueshuantong Capsule
Contr.
control group
DR
diabetic retinopathy
HXBJJ
erythrocyte aggregation indexes
FXT
Fufang Xueshuantong Capsule
ICAM-1
intercellular adhesion molecule-1
NADPH
nicotinamide adenine dinucleotide phosphate
PEDF
pigment epithelium-derived factor
PV
plasma viscosity
ROS
reactive oxygen species
STZ
streptozotocin
VEGF
vascular endothelial growth factor
WBV
whole blood viscosity
8
References
Abu El-Asrar, A.M., Midena, E., Al-Shabrawey, M., Mohammad, G., 2013. New developments in the pathophysiology and management of diabetic retinopathy. J Diabetes Res 2013, 424258. Akileshwari, C., Muthenna, P., Nastasijevic, B., Joksic, G., Petrash, J.M., Reddy, G.B., 2012. Inhibition of aldose reductase by Gentiana lutea extracts. Exp Diabetes Res 2012, 147965. Bandello, F., Lattanzio, R., Zucchiatti, I., Del Turco, C., 2013. Pathophysiology and treatment of diabetic retinopathy. Acta Diabetol 50, 1-20. Bucolo, C., Leggio, G.M., Drago, F., Salomone, S., 2012. Eriodictyol prevents early retinal and plasma abnormalities in streptozotocin-induced diabetic rats. Biochem Pharmacol 84, 88-92. Bucolo, C., Ward, K.W., Mazzon, E., Cuzzocrea, S., Drago, F., 2009. Protective effects of a coumarin derivative in diabetic rats. Invest Ophthalmol Vis Sci 50, 3846-3852. Cheng, Y.X., 2013. Clinical Effect Observation of Compound Xueshuantong Capsule in the Treatment of Diabetic Retinopathy. Guide China Med 11, 215-216. De Juan, J.A., Moya, F.J., Ripodas, A., Bernal, R., Fernandez-Cruz, A., Fernandez-Durango, R., 2000. Changes in the density and localisation of endothelin receptors in the early stages of rat diabetic retinopathy and the effect of insulin treatment. Diabetologia 43, 773-785. Ding, Y., Yuan, S., Liu, X., Mao, P., Zhao, C., Huang, Q., Zhang, R., Fang, Y., Song, Q., Yuan, D., Xie, P., Liu, Y., Liu, Q., 2014. Protective effects of astragaloside IV on db/db mice with diabetic retinopathy. PLoS One 9, e112207. Duan, H., Huang, J., Li, W., Tang, M., 2013. Protective effects of fufang xueshuantong on diabetic retinopathy in rats. Evid Based Complement Alternat Med 2013, 408268. Hattori, T., Matsubara, A., Taniguchi, K., Ogura, Y., 2010. Aldose reductase inhibitor fidarestat attenuates leukocyte-endothelial interactions in experimental diabetic rat retina in vivo. Curr Eye Res 35, 146-154. Hotta, N., Kawamori, R., Fukuda, M., Shigeta, Y., 2012. Long-term clinical effects of epalrestat, an aldose reductase inhibitor, on progression of diabetic neuropathy and other microvascular complications: multivariate epidemiological analysis based on patient background factors and severity of diabetic neuropathy. Diabet Med 29, 1529-1533. Ibrahim, A.S., El-Remessy, A.B., Matragoon, S., Zhang, W., Patel, Y., Khan, S., Al-Gayyar, M.M., El-Shishtawy, M.M., Liou, G.I., 2011. Retinal microglial activation and inflammation induced by amadori-glycated albumin in a rat model of diabetes. Diabetes 60, 1122-1133. Jin, Y.C., Kim, C.W., Kim, Y.M., Nizamutdinova, I.T., Ha, Y.M., Kim, H.J., Seo, H.G., Son, K.H., Jeon, S.J., Kang, S.S., Kim, Y.S., Kam, S.C., Lee, J.H., Chang, K.C., 2009. Cryptotanshinone, a lipophilic compound of Salvia miltiorrriza root, inhibits TNF-alpha-induced expression of adhesion molecules in HUVEC and attenuates rat myocardial ischemia/reperfusion injury in vivo. Eur J Pharmacol 614, 91-97. Kim, S.R., Lee, K.Y., Koo, K.A., Sung, S.H., Lee, N.G., Kim, J., Kim, Y.C., 2002. Four new neuroprotective iridoid glycosides from Scrophularia buergeriana roots. J Nat Prod 65, 1696-1699. Matsuda, S., Tam, T., Singh, R.P., Kaiser, P.K., Petkovsek, D., Carneiro, G., Zanella, M.T., Ehlers, J.P., 2014. The impact of metabolic parameters on clinical response to VEGF inhibitors for diabetic macular edema. J Diabetes Complications 28, 166-170. Miyamoto, K., Khosrof, S., Bursell, S.E., Moromizato, Y., Aiello, L.P., Ogura, Y., Adamis, A.P., 2000.
Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability is mediated by intercellular adhesion molecule-1 (ICAM-1). Am J Pathol 156, 1733-1739. Nentwich, M.M., Ulbig, M.W., 2015. Diabetic retinopathy - ocular complications of diabetes mellitus. World J Diabetes 6, 489-499. Ogata, N., Wada, M., Otsuji, T., Jo, N., Tombran-Tink, J., Matsumura, M., 2002. Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization. Invest Ophthalmol Vis Sci 43, 1168-1175. Ohashi, R., Yan, S., Mu, H., Chai, H., Yao, Q., Lin, P.H., Chen, C., 2006. Effects of homocysteine and ginsenoside Rb1 on endothelial proliferation and superoxide anion production. J Surg Res 133, 89-94. Onitsuka, M., Fujiu, M., Shinma, N., Maruyama, H.B., 1983. New platelet aggregation inhibitors from Tan-Shen; radix of Salvia miltiorrhiza Bunge. Chem Pharm Bull (Tokyo) 31, 1670-1675. Park, K., Chen, Y., Hu, Y., Mayo, A.S., Kompella, U.B., Longeras, R., Ma, J.X., 2009. Nanoparticle-mediated expression of an angiogenic inhibitor ameliorates ischemia-induced retinal neovascularization and diabetes-induced retinal vascular leakage. Diabetes 58, 1902-1913. Safi, S.Z., Qvist, R., Kumar, S., Batumalaie, K., Ismail, I.S., 2014. Molecular mechanisms of diabetic retinopathy, general preventive strategies, and novel therapeutic targets. Biomed Res Int 2014, 801269. Sheng, S., Wang, Y., Long, C., Su, W., Rong, X., 2014. Chinese medicinal formula Fufang Xueshuantong capsule could inhibit the activity of angiotensin converting enzyme. Biotechnol Biotechnol Equip 28, 322-326. Stitt, A.W., Bhaduri, T., McMullen, C.B., Gardiner, T.A., Archer, D.B., 2000. Advanced glycation end products induce blood-retinal barrier dysfunction in normoglycemic rats. Mol Cell Biol Res Commun 3, 380-388. Sun, K., Wang, C.S., Guo, J., Horie, Y., Fang, S.P., Wang, F., Liu, Y.Y., Liu, L.Y., Yang, J.Y., Fan, J.Y., Han, J.Y., 2007. Protective effects of ginsenoside Rb1, ginsenoside Rg1, and notoginsenoside R1 on lipopolysaccharide-induced microcirculatory disturbance in rat mesentery. Life Sci 81, 509-518. Yamagishi, S., Matsui, T., 2011. Advanced glycation end products (AGEs), oxidative stress and diabetic retinopathy. Curr Pharm Biotechnol 12, 362-368. Yamagishi, S., Matsui, T., Nakamura, K., Inoue, H., Takeuchi, M., Ueda, S., Okuda, S., Imaizumi, T., 2008a. Olmesartan blocks inflammatory reactions in endothelial cells evoked by advanced glycation end products by suppressing generation of reactive oxygen species. Ophthalmic Res 40, 10-15. Yamagishi, S., Matsui, T., Nakamura, K., Ueda, S., Noda, Y., Imaizumi, T., 2008b. Pigment epithelium-derived factor (PEDF): its potential therapeutic implication in diabetic vascular complications. Curr Drug Targets 9, 1025-1029. Yang, Y., Hayden, M.R., Sowers, S., Bagree, S.V., Sowers, J.R., 2010. Retinal redox stress and remodeling in cardiometabolic syndrome and diabetes. Oxid Med Cell Longev 3, 392-403. Yu, S., Yang, L., Wang, Y., Fu, Q., Huang, J., Xie, C., Chen, X., 2014. Application of Quantitative Analysis of Multi-components by Single-marker in Detection of Eight Components of Fufang Xueshuantong Capsule. J Int Pharm Res 42, 231-237.
Zhou, Z., Wang, S.Q., Liu, Y., Miao, A.D., 2006. Cryptotanshinone inhibits endothelin-1 expression and stimulates nitric oxide production in human vascular endothelial cells. Biochim Biophys Acta 1760, 1-9.
PII: DOI: Reference:
S0378-8741(15)30222-1 http://dx.doi.org/10.1016/j.jep.2015.11.021 JEP9820
To appear in: Journal of Ethnopharmacology Received date: 4 July 2015 Revised date: 8 October 2015 Accepted date: 6 November 2015 Cite this article as: Weijie Jian, Suyun Yu, Minke Tang, Huihui Duan and Jianmei Huang, A Combination of the Main Constituents of Fufang Xueshuantong Capsules Shows Protective Effects against Streptozotocin-induced Retinal Lesions in Rats, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.11.021 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 galley proof before it is published in its final citable 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.
A Combination of the Main Constituents of Fufang Xueshuantong Capsules Shows Protective Effects against Streptozotocin-induced Retinal Lesions in Rats
Weijie Jian1, Suyun Yu1, Minke Tang, HuihuiDuan, Jianmei Huang # School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing, 100102, China.
#
Corresponding author: Dr. Jianmei Huang, School of Chinese Materia Medica, Beijing University of Chinese Medicine, 6#, Wangjing Zhonghuan Nanlu, Chaoyang District, Beijing 100102, China.
Tel: +0086-10-84738619 E-mail: [email protected] 1
These two authors contributed equally to this work.
Abstract Ethnopharmacological Relevance Fufang Xueshuantong Capsule, an herbal formula licensed for clinical use in China, which is composed of Panax notoginseng (Burkill) F.H.Chen, Salvia miltiorrhiza Bunge, Astragalus membranaceus (Fisch.) Bunge, and Scrophularia ningpoensis Hemsl, has proven effective for the treatment of diabetic retinopathy. However, its bioactive constituents are still ambiguous. In this study, the therapeutic effects of a combination of the main constituents of Fufang
Xueshuantong
Capsule
(cFXT)
were
evaluated
in
streptozotocin
(STZ)-induced retinal lesions to identify the bioactive constituents. Methods Sprague-Dawley rats, except for those in the control group (vehicle+ vehicle), were administered a single injection of 60mg/kg STZ. One-week later, STZ-treated rats were randomly divided into three groups—one STZ group (STZ + vehicle) and two cFXT treatment groups (STZ + cFXT). The rats in the latter two groups received cFXT 44.8 mg/kg or cFXT 22.4 mg/kg by intragastric gavage once per day, for 24 consecutive weeks. The rats in the control and STZ groups received the vehicle in the same way. Body weights and fasting blood glucose levels were recorded every four weeks. After treatment, hemorheological tests were performed to record the erythrocyte aggregation indexes, blood viscosity, and plasma viscosity. The trypsin digestion method was used to observe pericyte and acellular capillary counts in the retina. Ultraviolet spectrophotometry was utilized to measure the activity of aldose reductase (AR) by measuring the nicotinamide adenine dinucleotide phosphate (NADPH) consumption at 340 nm. An immunohistochemical assay was used to observe the expressions of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in the retina. The expression levels of intercellular adhesion molecule-1 (ICAM-1), endothelin-1 (RT-1),and occludin in the retina were tested by the western blot assay. Results cFXT is composed of 991.44 mg/g saponins of Panax notoginseng, 1.62 mg/g harpagoside, 0.70 mg/g cryptotanshinone, 0.74 mg/g tanshinone I, and 5.50
mg/g astragaloside A. Although it showed no effects on the increased body weight and blood glucose levels induced by STZ in rats. However, it showed a tendency to attenuate the increase in erythrocyte aggregation, plasma viscosity, and acellular vessel and pericyte loss, paralleled with a reversal of the hyper-activation of AR, the hyper-expression of VEGF, ICAM-1, and ET-1, and the hypo-expression of PEDF and occludin in the retinas of STZ-treated rats. Conclusion The saponins of Panax notoginseng, harpagoside, cryptotanshinone, tanshinone I, and astragaloside A are the main bioactive constituents of Fufang Xueshuantong Capsule and contribute to the attenuation of STZ-induced retinal lesions in rats. These constituents can be used as the base to optimize a new drug for the treatment of diabetic retinopathy, and can be selected for quality control of Fufang Xueshuantong Capsules. Key Words Diabetic
retinopathy;
Fufang
Xueshuantong
Capsule;
Endothelin-1;
Intercellular adhesion molecule-1; Pigment epithelium derived factor; Occludin. Chemical Compounds Notoginsenoside R1 (CAS NO., 80418-24-2), Ginsenoside Rg1 (CAS NO., 22427-39-0), Ginsenoside Rb1 (CAS NO., 41753-43-9), Harpagoside (CAS NO., 19210-12-9), Cryptotanshinone (CAS NO., 35825-57-1), Tanshinone I (CAS NO., 568-73-0), Astragaloside A (CAS NO., 83207-58-3)
1
Introduction Diabetic retinopathy (DR) is a disease induced by diabetes that involves the
retinal capillaries, arterioles, and venules and is accompanied by leakage or occlusion of the small vessels (Nentwich and Ulbig, 2015). Phanerous lesions are observed with the progression of DR, such as microaneurysms, hemorrhages, vessel abnormalities, fibrous proliferation, and angiogenesis (Bandello et al., 2013). Studies have demonstrated that down-regulation of the factors involved in the pathogenesis of DR, such as hyper-activation of aldose reductase (AR) (Hattori et al., 2010), over expression of vascular endothelial growth factor (VEGF) (Matsuda et al., 2014) and intercellular adhesion molecule-1 (ICAM-1), and up-regulation of pigment epithelium-derived factor (PEDF) (Bucolo et al., 2012; Bucolo et al., 2009; Miyamoto et al., 2000; Ogata et al., 2002) are effective strategies for the treatment of DR. Fufang Xueshuantong Capsule (FXT, also called Compound Xueshuantong) is a Chinese herbal formula composed of Panax notoginseng (Burkill) F.H.Chen (Araliaceae), Salvia miltiorrhiza Bunge (Lamiaceae), Astragalus membranaceus (Fisch.)
Bunge
(Leguminosae),
and
Scrophularia
ningpoensis
Hemsl
(Scrophulariaceae)(Sheng et al., 2014). It has been clinically used to treat DR (Cheng, 2013). Our previous research in a model of streptozotocin (STZ)-induced rat diabetes showed that FXT could normalize abnormal whole blood viscosity (WBV), plasma viscosity (PV), and erythrocyte aggregation indexes (HXBJJ). In addition, FXT attenuated the development of microvessel lesions in the retina by decreasing pericyte loss and reducing acellular capillaries. This was in parallel to down-regulation of AR hyper-activity and over expression of VEGF and ICAM-1, and up-regulation of the hypo-expression of PEDF and occludin. FXT thus showed protective effects against STZ-induced retinal lesions in rats (Duan et al., 2013). By reviewing the literature, we found that the characteristic constituents of the four herbs might be related to the pharmacological effects of FXT. Ginsenoside Rb1 from Panax notoginseng showed inhibitory effects on endothelial proliferation with
increased production of superoxide anion (Ohashi et al., 2006). Ginsenoside Rb1, ginsenoside Rg1, and notoginsenoside R1 were effective in treating microcirculatory disturbances induced by lipopolysaccharide (Sun et al., 2007). Cryptotanshinone and tanshinone I from Salvia miltiorrhiza showed inhibitory effects on ICAM-1 and endothelin-1 (ET-1) expression in human umbilical vein endothelial cells (Jin et al., 2009; Onitsuka et al., 1983; Zhou et al., 2006). Harpagoside from Scrophularia ningpoensis showed protective effects against human vascular endothelial cell injury induced by hydrogen peroxide (Kim et al., 2002). Astragaloside A from Astragalus membranaceus reduced retinal ganglion cell apoptosis and down-regulated AR activity to prevent DR (Ding et al., 2014). In this study, the afore mentioned bioactive constituents of FXT were selected to form a constituent combination (cFXT). The quantity of the selected constituents in cFXT was in accordance with that in FXT as had been determined previously (Yu et al., 2014). The therapeutic effects of cFXT on STZ-induced retinal lesions in rats were evaluated in this study, aiming to clarify the roles of the core bioactive components of FXT and provide a foundation to optimize a new medicine with minimum constituents, high efficacy, and easy quality control. 2
Materials and Methods
2.1 Animals and Reagents Male Sprague-Dawley (SD) rats weighing 230 to 250 g (SCXK [Jing] 2007-0001) were purchased from Vital River (Beijing, China). The animal experiment protocol was in accordance with the statement for the Use of Animals in Ophthalmic and Vision Research by the Association for Research in Vision and Ophthalmology and approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. Rats were acclimatized for one week before experiments, housed in a 12-hour light/dark cycle, at constant temperature and humidity, and were provided enough food and water. STZ was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) or Abcam (Cambridge, UK).
2.2 Drug preparation The cFXT was composed of 991.44 mg/g saponins of Panax notoginseng (NO. 20110831, containing 9.55% notoginsenoside R1, 35.52% ginsenoside Rg1, 36.64% ginsenoside Rb1, and 18.29% other saponins), 1.62 mg/g harpagoside (NO. 20110923, ≥99%) 0.70 mg/g cryptotanshinone (NO. 20110520, ≥98%), 0.74 mg/g tanshinone I (NO. 20111125, ≥95%), and 5.50 mg/g astragaloside A (NO. 20111216, ≥99%). Saponins, harpagoside, cryptotanshinone, and tanshinone I were purchased from Zelang Pharmaceutical Technology Co., Ltd. (Nanjing, China) and astragaloside A was purchased from Kang Bang Technology Co., Ltd. (Chengdu, China). The proportions of these constituents in cFXT were in accordance with those in FXT as determined previously (Yu et al., 2014). cFXT solution was prepared by mixing 10 ml solution A with 100 ml solution B and diluting to 500 ml with distilled water. Solution A was prepared by suspending cryptotanshinone and tanshinone I with 5% carboxymethylcellulose sodium solution (CMC-Na, NO. 20031019, ≥ 6.5%, purchased from Guangfu institute of fine chemicals, Tianjin, China) and diluting with 5% CMC-Na to a final contents of 0.156 mg·ml-1 cryptotanshinone and 0.164 mg·ml-1 tanshinone I respectively. Solution B was prepared by dissolving saponins of Panax notoginseng, harpagoside and astragaloside A with distilled water and diluting with distilled water to a final contents of 22.2 mg·ml-1 saponins of Panax notoginseng, 0.036 mg·ml-1 harpagoside and 0.121 mg·ml-1 astragaloside A respectively. All afore mentioned solutions were stored at 4 ℃ and used at room temperature within 3 days. 2.3 Animal modeling, grouping, and drug treatment All rats (n=180), except those in the control group (vehicle + vehicle, n=35), were treated with a single intraperitoneal injection of 60 mg/kg STZ. One week after STZ injection, the rats were randomly divided into three groups: one STZ group (STZ + vehicle) and two cFXT treatment groups (STZ + cFXT) that received either 44.8 mg/kg or 22.4 mg/kg cFXT. cFXT was administered by intragastric gavage daily for 24 consecutive weeks at 1 ml/100g body weight. The rats in the control and STZ
group received the vehicle in the same way. 2.4 Recording of body weight and blood glucose levels of rats During cFXT treatments, the fasting body weight and fasting blood glucose levels of all rats was measured every four weeks. 2.5 Hemorheological analysis After cFXT treatments, fresh arteriopuncture blood samples were collected in heparin (10 mg/ml) pre-coated tubes. Plasma samples were collected by centrifugation at 3000 rpm for 15 min. WBV and PV at a shear rate of 60 s -1 were assayed with a blood viscometer (STEELLEX, China). HXBJJ was determined using an RBC deformation and aggregation test instrument (STEELLEX, China). 2.6 Trypsin digestion method After cFXT treatments, the trypsin digestion method was used to determine the counts of pericytes and acellular capillaries as described in a previous study (Duan et al., 2013). Briefly, the isolated retina was incubated with 3% crude trypsin (Difco Laboratories, Detroit, MI, USA) within Tris buffer (0.1 M, pH=7.8) in an incubator shaker (37°C). The retina was transferred to phosphate-buffered saline (pH=7.4) when the inner limiting membrane began separating from the retina after about 2–3 h of incubation. While being observed under the microscope, the separated vascular tree was washed with distilled water until free of neural tissue, set onto glass slides, air-dried, and stained with hematoxylin and periodic acid-Schiff stain. The number of pericytes and acellular occluded vessel segments were counted using an image system (ECLIPSE 80i, Nikon, Japan). The results were presented as the relative capillary density (numbers of cells per millimeter square of capillary area). 2.7 Measurement of aldose reductase activity After cFXT treatment, the separated retina of the rats was homogenized with 10
volumes (v/w) of potassium phosphate buffer (100 mM, pH=6.2). The suspension solution was centrifuged at 15000 rpm for 30 min. The supernatant (crude AR solution) was stored at -20°C. Protein concentrations of the supernatant were determined with a Bradford Protein Assay Kit (Biomiga, San Diego, CA, USA). AR activity was assayed in accordance with the method reported previously (Akileshwari et al., 2012; Duan et al., 2013). Briefly, the reactant was pre-incubated at 37 °C. Nicotinamide adenine dinucleotide phosphate (NADPH) was added to the reactant to initiate the reaction for 5 min. Subsequently, 2 ml of NaOH solution (0.3 mM) was added to stop the reaction at 60 °C for 15 min. The consumption of NADPH was tested according to the decline in absorbance at 340 nm using a spectrophotometer. An absorbance decrease of 0.001 per min induced by 1 mg of protein was defined as one unit (U) of AR activity. 2.8 Immunohistochemical assay After cFXT treatments, the expression levels of VEGF and PEDF were assessed using an immunohistochemical assay as previously described (De Juan et al., 2000; Duan et al., 2013). Indirect immunoperoxidase staining was performed on a paraffin section of the paraformaldehyde-stabilized bulbus oculi using immunostaining reagents (Beijing Zhong Shan-Golden Bridge Biological Technology Co., LTD, China). The primary antibodies, including rabbit anti-VEGF (ab46154, 1:50 dilution, Abcam, Cambridge, UK) and goat anti-PEDF antibodies (sc16956, 1:50 dilution, Santa Cruz, Dallas, TX, USA), were omitted for negative controls. Immunostaining slides of VEGF or PEDF were analyzed with the Image Pro Plus Analysis Software (Media Cybernetics, Rockville, MD, USA). 2.9 Western blot analysis After cFXT treatments, the expression of ICAM-1, ET-1, and occludin in the retinas was assayed by western blot analysis according to the previously reported method (Park et al., 2009). Protein extracts of ICAM-1, ET-1, and occludin were
prepared from the retinas of rats. The target proteins, mouse anti-ICAM-1 antibody (ab2213, 1:2000 dilution, Abcam, Cambridge, UK), rabbit anti-ET-1 antibody (ab117757,1:2000 dilution, Abcam, Cambridge,UK), rabbit anti-occludin antibody (ab31721,1:500 dilution, Abcam, Cambridge, UK), goat anti-rabbit lgG (1:500), and goat anti-mouse lgG (1:2000) were visualized with an enhanced chemiluminescence kit (Sc-2048, Santa Cruz, Dallas, TX, USA). The gray intensities of the target bands of each sample were analyzed using Image Pro Plus Analysis Software (Media Cybernetics, Rockville, MD, USA). 2.10 Data analysis Data for all observed parameters were analyzed by one-way ANOVA followed by a Fischer’s least significant difference post hoc test for multiple comparisons. Values were presented as means±SEM. P<0.05 indicated a statistically significant difference. 3
Results
3.1 cFXT treatment had no effects on body weight and blood glucose level in STZ rats As shown in Fig. 1, rats in the STZ groups were found to gain less weight during the 24-week period than the control rats, and this phenomenon was not reversed by 44.8 mg/kg or 22.4 mg/kg cFXT treatments (F
(3,211)=37.961,
P<0.01, Fig. 1A).
Meanwhile, sustained high blood glucose levels were induced by STZ treatments. The increased blood glucose levels were not affected by 44.8 mg/kg or 22.4 mg/kg cFXT treatments (F (3,211)=78.109, P<0.01, Fig. 1B).
Fig. 1 Effects of cFXT on fasting body weight and blood glucose level. One week after streptozotocin (STZ) injection, the fasting body weight (A) and blood glucose level (B) of rats was measured every four weeks for 25 weeks. N=60 for each STZ group and n=35 for control group. ▲ or ▼, P<0.01 for all STZ groups vs. Control. 3.2 cFXT treatment reduced HXBJJ and WBV, but not PV, in STZ rats As shown in Fig. 2, STZ treatment led to enhanced HXBJJ (F (3, 28) =5.064, P<0.01, Fig. 2A) and elevated WBV (F (3, 28) =7.861, P<0.01, Fig. 2B) and PV (F (3, 28) =3.320, P=0.03 Fig. 2C) in rats. Daily treatment of 44.8 mg/kg or 22.4 mg/kg cFXT for 24 weeks significantly alleviated HXBJJ and decreased WBV as compared with STZ injection only. Regarding the elevated PV in STZ rats, neither 44.8 mg/kg nor 22.4 mg/kg cFXT treatment showed a significant down-regulating effect.
Fig. 2 Effects of cFXT on HXBJJ, WBV, and PV of rats. At the end of cFXT treatment, the erythro-agglutination index (HXBJJ) (A), whole blood viscosity at a shear rate of 60 per second (WBV) (B), and plasma viscosity at a shear rate of 60 per second (PV) (C) were assayed. N=8 for each group. or * P<0.05 vs. STZ group.
▲
P<0.01 vs. Control;
**
P<0.01
3.3 cFXT treatment relieved retinal lesions in STZ rats STZ treatment induced significant pericyte loss and acellular capillary proliferation in rat retinas as shown in Fig. 3. Although both 44.8 mg/kg and 22.4 mg/kg cFXT treatments did not significantly reduce pericyte loss in STZ rats (F (3, 34) =7.035, P=0.103, Fig. 3A), cFXT at 44.8 mg/kg significantly attenuated the proliferation of acellular capillaries (F (3, 34) =27.285, P<0.01, Fig. 3B).
Fig. 3 Effects of cFXT on pericyte loss and acellular capillary proliferation. The pericyte counts (A) and acellular capillary counts (B) in the retina were assayed after 24weeks of cFXT treatment. N=10 for each STZ group and n=8 for Control.
▲
or
▼
P<0.01 vs. Control; * P<0.01 vs.STZ group. 3.4 cFXT treatment inhibited the activated AR in STZ rats As shown in Fig. 4, STZ treatment increased AR activity in the retinas of rats.
The activity of AR was remarkably inhibited by treatment with 44.8 mg/kg or 22.4 mg/kg cFXT (F (3, 28) =120.943, P<0.01).
Fig. 4 Effect of cFXT on increased AR activity. At the end of cFXT treatment, the activity of aldose reductase (AR) was assayed. N=8 for each group.
▲
P<0.01 vs.
Control; * P<0.01 vs. STZ group. 3.5 cFXT treatment reversed the abnormal expression of VEGF and PEDF in STZ rats The expression of VEGF in rat retinas was notably increased by STZ treatment, and the elevated expression of VEGF was markedly down-regulated by 44.8 mg/kg or 22.4mg/kg cFXT treatment (F
(3, 16)=19.308,
P<0.01, Fig. 5A). The expression of
PEDF in the retina was decreased by STZ treatment, and the depressed expression of PEDF was up regulated distinctly by 44.8 mg/kg or 22.4mg/kg cFXT (F (3, 16) =11.963, P<0.01, Fig. 5B).
Fig. 5 Effects of cFXT on the expression of VEGF and PEDF. At the end of cFXT treatment, the expression of vascular endothelial growth factor (VEGF) (A) and pigment epithelium derived factor (PEDF) (B) in the retina was assayed with an immunohistochemical assay. N=5 for each group.
▲
or
▼
P<0.01 vs. Control;
**
P<0.01 or * P<0.05 vs. STZ group. 3.6 cFXT treatment regulated the abnormal expressions of ICAM-1, ET-1, and occludin in STZ rats As shown in Fig. 6, the expression of ICAM-1, ET-1, and occludin in the retinas of rats in the STZ group was remarkably dysregulated in comparison with that in the control group. The over expression of ICAM-1 (F (3, 16) =12.948,Fig. 6A) and ET-1 (F (3, 16)
=7.443, P<0.01, Fig. 6B) were well down regulated by treatment with 44.8
mg/kg or 22.4 mg/kg cFXT. The hypo-expression of occludin in the retinas of rats
showed a trend towards recovery after treatment with 44.8 mg/kg or 22.4 mg/kg cFXT, but had no significant difference compared to that in STZ rats (F(3, 16)=24.885, P<0.01, Fig. 6C).
Fig. 6 Effects of cFXT on the expression of ICAM-1, ET-1, and occludin. At the end of cFXT treatment, the expression of intercellular adhesion molecule-1 (ICAM-1) (A), endothelin-1 (ET-1) (B),and occludin (C) in the retina was assayed with western
blot assays. N=5 for each group.
▲
or
▼
P<0.01 vs. the Control;
**
P<0.01, * P<0.05
vs. STZ group. 4
Conclusion and Discussion In this study, a combination of the main constituents of Fufang Xueshuantong
Capsule (cFXT) proved to have the majority of pharmacological effects possessed by the herbal formula FXT (Duan et al., 2013). That is, cFXT reduced STZ-induced retinal lesions in rats by decreasing HXBJJ and WBV levels, and attenuating the proliferation of acellular capillaries. Furthermore, cFXT inhibited AR activity, and rectified the over expression of VEGF, ICAM-1, and ET-1,and the hypo-expression of PEDF in the retina. These results indicate that cFXT contains the bioactive constituents of FXT important for protection against retinal lesions, and suggests that the potential optimization of FXT with easily controlled formulas and minimum constituents would be possible by further research. However, it should be noted that there were some differences found between the therapeutic effects of cFXT compared to FXT. These include a lack of significant attenuation of PV, reduction in pericyte loss, or enhanced expression of occludin (Duan et al., 2013). Obviously, there are constituents in FXT other than those in cFXT that contribute to the retinal protective effects of FXT. One way of further improving cFXT is to identify the other bioactive chemicals in FXT and incorporate them into the combination. During diabetes, the polyol pathway is stimulated by sustained hyperglycemia (Safi et al., 2014). In most cases, the activated polyol pathway leads to activation of AR within cells, accompanied with proliferation of reactive oxygen species (ROS) and advanced glycation end products (AGEs), subsequently leading to a cascade of events, such as the augmentation of oxidative stress, apoptosis, inflammatory response, and promotion of angiogenesis (Yang et al., 2010). DR develops as a result of these pathological alterations within the diabetics retina (Abu El-Asrar et al., 2013). Inhibition of AR activity has been demonstrated to be effective for treatment of DR (Hattori et al., 2010). However, inhibitors have seldom been used in clinical practice
(Hotta et al., 2012), mostly because of pharmacokinetic drawbacks, adverse side effects, or low in vivo efficacy. Our research demonstrated that cFXT inhibits AR activity in the retina, suggesting that the constituents of cFXT may be candidates as AR inhibitors. As shown in the preparation of cFXT, saponins from Panax notoginseng account for a major proportion of the combination. These results should encourage the investigation to determine whether the ginsenosides are AR inhibitors. Molecules other than AR may also be involved in the effects of cFXT. It has been reported that AGEs increase the expression of ICAM-1 and VEGF in microvascular endothelial cells (Ibrahim et al., 2011; Stitt et al., 2000; Yamagishi and Matsui, 2011; Yamagishi et al., 2008a; Yamagishi et al., 2008b). ICAM-1 is a crucial mediator of vascular permeability (Bucolo et al., 2009; Miyamoto et al., 2000). VEGF damages the blood-retinal barrier by inducing the rapid phosphorylation of occludin (Bucolo et al., 2009). PEDF can inhibit the angiogenesis induced by VEGF; however, the balance of VEGF and PEDF is disrupted in diabetes (Ogata et al., 2002). Our research showed that cFXT reduced retinal lesions by the depressing expression of ICAM-1 and VEGF and increasing the expression of PEDF which may due to the decrease of AGEs. This indicated that cFXT may regulate multiple factors involved in the DR pathological pathway. Careful identification of causal relationships between the activity of AR and molecular alterations of ICAM-1, PEDF, and VEGF by further experiments is another way to optimize the combination of cFXT at the pharmacological mechanism level. In conclusion, our study has furthered our understanding of the bioactive constituents of a Chinese herbal medicine, the FXT Capsule. Saponins of Panax notoginseng, harpagoside, cryptotanshinone, tanshinone I, and astragaloside A contribute the protective effects of FXT on STZ-induced retinal lesions. The combination of the above chemicals can be used as the basis to optimize a new drug for the treatment of diabetic retinopathy, and it also demonstrates a promising method to optimize the formulation of FXT for better, easily controlled efficacy with only the
minimum of constituents. 5
Acknowledgements This study was supported by grants from Ministry of Science and Technology of
China (Grant No., 2011ZX09201-201-22). 6
Conflict of interest The authors declare that there are no conflicts of interest.
7
Abbreviations The abbreviations utilized in this paper are presented in Table 1.
Table 1 Abbreviations Utilized Abbreviation
Full Name
AGEs
advanced glycation end products
AR
aldose reductase
cFXT
combination of main constituents of Fufang Xueshuantong Capsule
Contr.
control group
DR
diabetic retinopathy
HXBJJ
erythrocyte aggregation indexes
FXT
Fufang Xueshuantong Capsule
ICAM-1
intercellular adhesion molecule-1
NADPH
nicotinamide adenine dinucleotide phosphate
PEDF
pigment epithelium-derived factor
PV
plasma viscosity
ROS
reactive oxygen species
STZ
streptozotocin
VEGF
vascular endothelial growth factor
WBV
whole blood viscosity
8
References
Abu El-Asrar, A.M., Midena, E., Al-Shabrawey, M., Mohammad, G., 2013. New developments in the pathophysiology and management of diabetic retinopathy. J Diabetes Res 2013, 424258. Akileshwari, C., Muthenna, P., Nastasijevic, B., Joksic, G., Petrash, J.M., Reddy, G.B., 2012. Inhibition of aldose reductase by Gentiana lutea extracts. Exp Diabetes Res 2012, 147965. Bandello, F., Lattanzio, R., Zucchiatti, I., Del Turco, C., 2013. Pathophysiology and treatment of diabetic retinopathy. Acta Diabetol 50, 1-20. Bucolo, C., Leggio, G.M., Drago, F., Salomone, S., 2012. Eriodictyol prevents early retinal and plasma abnormalities in streptozotocin-induced diabetic rats. Biochem Pharmacol 84, 88-92. Bucolo, C., Ward, K.W., Mazzon, E., Cuzzocrea, S., Drago, F., 2009. Protective effects of a coumarin derivative in diabetic rats. Invest Ophthalmol Vis Sci 50, 3846-3852. Cheng, Y.X., 2013. Clinical Effect Observation of Compound Xueshuantong Capsule in the Treatment of Diabetic Retinopathy. Guide China Med 11, 215-216. De Juan, J.A., Moya, F.J., Ripodas, A., Bernal, R., Fernandez-Cruz, A., Fernandez-Durango, R., 2000. Changes in the density and localisation of endothelin receptors in the early stages of rat diabetic retinopathy and the effect of insulin treatment. Diabetologia 43, 773-785. Ding, Y., Yuan, S., Liu, X., Mao, P., Zhao, C., Huang, Q., Zhang, R., Fang, Y., Song, Q., Yuan, D., Xie, P., Liu, Y., Liu, Q., 2014. Protective effects of astragaloside IV on db/db mice with diabetic retinopathy. PLoS One 9, e112207. Duan, H., Huang, J., Li, W., Tang, M., 2013. Protective effects of fufang xueshuantong on diabetic retinopathy in rats. Evid Based Complement Alternat Med 2013, 408268. Hattori, T., Matsubara, A., Taniguchi, K., Ogura, Y., 2010. Aldose reductase inhibitor fidarestat attenuates leukocyte-endothelial interactions in experimental diabetic rat retina in vivo. Curr Eye Res 35, 146-154. Hotta, N., Kawamori, R., Fukuda, M., Shigeta, Y., 2012. Long-term clinical effects of epalrestat, an aldose reductase inhibitor, on progression of diabetic neuropathy and other microvascular complications: multivariate epidemiological analysis based on patient background factors and severity of diabetic neuropathy. Diabet Med 29, 1529-1533. Ibrahim, A.S., El-Remessy, A.B., Matragoon, S., Zhang, W., Patel, Y., Khan, S., Al-Gayyar, M.M., El-Shishtawy, M.M., Liou, G.I., 2011. Retinal microglial activation and inflammation induced by amadori-glycated albumin in a rat model of diabetes. Diabetes 60, 1122-1133. Jin, Y.C., Kim, C.W., Kim, Y.M., Nizamutdinova, I.T., Ha, Y.M., Kim, H.J., Seo, H.G., Son, K.H., Jeon, S.J., Kang, S.S., Kim, Y.S., Kam, S.C., Lee, J.H., Chang, K.C., 2009. Cryptotanshinone, a lipophilic compound of Salvia miltiorrriza root, inhibits TNF-alpha-induced expression of adhesion molecules in HUVEC and attenuates rat myocardial ischemia/reperfusion injury in vivo. Eur J Pharmacol 614, 91-97. Kim, S.R., Lee, K.Y., Koo, K.A., Sung, S.H., Lee, N.G., Kim, J., Kim, Y.C., 2002. Four new neuroprotective iridoid glycosides from Scrophularia buergeriana roots. J Nat Prod 65, 1696-1699. Matsuda, S., Tam, T., Singh, R.P., Kaiser, P.K., Petkovsek, D., Carneiro, G., Zanella, M.T., Ehlers, J.P., 2014. The impact of metabolic parameters on clinical response to VEGF inhibitors for diabetic macular edema. J Diabetes Complications 28, 166-170. Miyamoto, K., Khosrof, S., Bursell, S.E., Moromizato, Y., Aiello, L.P., Ogura, Y., Adamis, A.P., 2000.
Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability is mediated by intercellular adhesion molecule-1 (ICAM-1). Am J Pathol 156, 1733-1739. Nentwich, M.M., Ulbig, M.W., 2015. Diabetic retinopathy - ocular complications of diabetes mellitus. World J Diabetes 6, 489-499. Ogata, N., Wada, M., Otsuji, T., Jo, N., Tombran-Tink, J., Matsumura, M., 2002. Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization. Invest Ophthalmol Vis Sci 43, 1168-1175. Ohashi, R., Yan, S., Mu, H., Chai, H., Yao, Q., Lin, P.H., Chen, C., 2006. Effects of homocysteine and ginsenoside Rb1 on endothelial proliferation and superoxide anion production. J Surg Res 133, 89-94. Onitsuka, M., Fujiu, M., Shinma, N., Maruyama, H.B., 1983. New platelet aggregation inhibitors from Tan-Shen; radix of Salvia miltiorrhiza Bunge. Chem Pharm Bull (Tokyo) 31, 1670-1675. Park, K., Chen, Y., Hu, Y., Mayo, A.S., Kompella, U.B., Longeras, R., Ma, J.X., 2009. Nanoparticle-mediated expression of an angiogenic inhibitor ameliorates ischemia-induced retinal neovascularization and diabetes-induced retinal vascular leakage. Diabetes 58, 1902-1913. Safi, S.Z., Qvist, R., Kumar, S., Batumalaie, K., Ismail, I.S., 2014. Molecular mechanisms of diabetic retinopathy, general preventive strategies, and novel therapeutic targets. Biomed Res Int 2014, 801269. Sheng, S., Wang, Y., Long, C., Su, W., Rong, X., 2014. Chinese medicinal formula Fufang Xueshuantong capsule could inhibit the activity of angiotensin converting enzyme. Biotechnol Biotechnol Equip 28, 322-326. Stitt, A.W., Bhaduri, T., McMullen, C.B., Gardiner, T.A., Archer, D.B., 2000. Advanced glycation end products induce blood-retinal barrier dysfunction in normoglycemic rats. Mol Cell Biol Res Commun 3, 380-388. Sun, K., Wang, C.S., Guo, J., Horie, Y., Fang, S.P., Wang, F., Liu, Y.Y., Liu, L.Y., Yang, J.Y., Fan, J.Y., Han, J.Y., 2007. Protective effects of ginsenoside Rb1, ginsenoside Rg1, and notoginsenoside R1 on lipopolysaccharide-induced microcirculatory disturbance in rat mesentery. Life Sci 81, 509-518. Yamagishi, S., Matsui, T., 2011. Advanced glycation end products (AGEs), oxidative stress and diabetic retinopathy. Curr Pharm Biotechnol 12, 362-368. Yamagishi, S., Matsui, T., Nakamura, K., Inoue, H., Takeuchi, M., Ueda, S., Okuda, S., Imaizumi, T., 2008a. Olmesartan blocks inflammatory reactions in endothelial cells evoked by advanced glycation end products by suppressing generation of reactive oxygen species. Ophthalmic Res 40, 10-15. Yamagishi, S., Matsui, T., Nakamura, K., Ueda, S., Noda, Y., Imaizumi, T., 2008b. Pigment epithelium-derived factor (PEDF): its potential therapeutic implication in diabetic vascular complications. Curr Drug Targets 9, 1025-1029. Yang, Y., Hayden, M.R., Sowers, S., Bagree, S.V., Sowers, J.R., 2010. Retinal redox stress and remodeling in cardiometabolic syndrome and diabetes. Oxid Med Cell Longev 3, 392-403. Yu, S., Yang, L., Wang, Y., Fu, Q., Huang, J., Xie, C., Chen, X., 2014. Application of Quantitative Analysis of Multi-components by Single-marker in Detection of Eight Components of Fufang Xueshuantong Capsule. J Int Pharm Res 42, 231-237.
Zhou, Z., Wang, S.Q., Liu, Y., Miao, A.D., 2006. Cryptotanshinone inhibits endothelin-1 expression and stimulates nitric oxide production in human vascular endothelial cells. Biochim Biophys Acta 1760, 1-9.