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Efficacy of Lagopsis supina to promote blood circulation, remove blood stasis, and block inflammation in a rat model of traumatic blood stasis

Abstract

Lagopsis supina (Steph) IK. Gal. was a traditional Chinese medicinal plant for promoting blood circulation and removing blood stasis (PBCRBS), anti-inflammatory and diuresis with little scientific validation. The aims of this study were first to evaluate the PBCRBS and anti-inflammatory effects of L. supina in a rat model of traumatic blood stasis (TBS). We demonstrated that an ethanolic extract of L. supina (LS, 460 mg/kg/d) possessed significant PBCRBS and remarkable inhibitory effect on inflammation cytokines, which were associated with renovated amount of the injured muscle fibers, alleviate the degree of the damaged tissue edema, decreased the number of inflammatory cells, increased the number of the capillary hyperplasia by hematoxylin and eosin (HE), as well as remarkably down-regulated ( p < 0.05 or p < 0.01) the levels of inflammation cytokines, including TNF-α, IL-6, IL-8, and IL-10 by ELISA. This finding provide a pharmacological basis and partial interpretation for the clinical application of L. supina, which has therapeutic properties for blood stasis syndrome (BSS) and inflammation-related diseases.

Keywords:
Lagopsis supina; Promoting blood circulation and removing blood stasis (PBCRBS); Anti-inflammatory; Traumatic blood stasis (TBS)

INTRODUCTION

Traditional Chinese medicines (TCMs), one of the oldest medical systems of health care in China and other Asian countries, have been used to treat a variety of human diseases for many centuries due to their therapeutic efficacy, little side effects and broad applications (He et al., 2016He JW, Yang L, Zhu JX, Wang XM, Zhou ZR, He WW, et al. Comparison of anti-inflammatory effects and HPLC detection on different extracts from the flowers of Hosta plantaginea in mice. J Jiangxi Norm Univ. (Nat Sci.) 2016;40(2):183-185.; Pang et al., 2016Pang HQ, Wu L, Tang YP, Zhou GS, Qu C, Duan JA. Chemical analysis of the herbal medicine Salviae miltiorrhizae radix et rhizoma (Danshen). Molecules. 2016;21:51.; Li et al., 2017Li PL, Su WW, Yun S, Liao YQ, Liu H, Li PB, et al. Toward a scientific understanding of the effectiveness, material basis and prescription compatibility of a Chinese herbal formula Dan-hong injection. Sci Rep. 2017;7:46266.; Qiu et al., 2017Qiu HH, Zhang L, Zhu MM, Zhang MH, Chen J, Feng L, et al. Capture of anti-coagulant active ingredients from Moutan cortex by platelet immobilized chromatography and evaluation of anticoagulant activity in rats. Biomed Pharmacother. 2017;95:235-244.; Zhou et al., 2018Zhou JM, Wang HM, Lv YZ, Wang ZZ, Xiao W. Anti- atherosclerotic effect of Longxuetongluo Capsule in high cholesterol diet induced atherosclerosis model rats. Biomed Pharmacother . 2018;97:793-801.). In recent years, TCMs have been recognized as ideal examples of complementary and alternative medicines and are typically composed of multiple components that have multiple disease targets. TCMs increasingly attract attention from researchers worldwide. Blood stasis syndrome (BSS) is a serious pathological syndrome that can be treated with TCMs and Korean medicine (Dang et al., 2015Dang X, Miao JJ, Chen AQ, Li P, Chen L, Liang JR. The antithrombotic effect of RSNK in blood-stasis model rats. J Ethnopharmacol. 2015;173:266-272.; He et al., 2019He JW, Zeng LB, Wei RR, Zhong GY, Zhu YY, Xu TT, et al. Lagopsis supina exerts its diuretic effect via inhibition of aquaporin-1, 2 and 3 expression in a rat model of traumatic blood stasis. J Ethnopharmacol . 2019;231:446-452.; Jung et al., 2018Jung J, Ko MM, Lee JA, Lee MS. Recognition of association between blood stasis syndrome and traumatic injury among doctors of Korean medicine: a cross-sectional observation study. Chin J Integr Med. 2018;24(4):254-259.; Liu et al., 2012Liu Y, Yin HJ, Shi DZ, Chen KJ. Chinese herb and formulas for promoting blood circulation and removing blood stasis and antiplatelet therapies. Evid-Base Compl Alt. 2012;2012:184503.). BSS is defined as a state in which blood circulation is turbulent or stagnant occurring as a result of traumatic injury, cardiovascular disease, and other diseases (Dang et al., 2015Dang X, Miao JJ, Chen AQ, Li P, Chen L, Liang JR. The antithrombotic effect of RSNK in blood-stasis model rats. J Ethnopharmacol. 2015;173:266-272.; Jung et al., 2018Jung J, Ko MM, Lee JA, Lee MS. Recognition of association between blood stasis syndrome and traumatic injury among doctors of Korean medicine: a cross-sectional observation study. Chin J Integr Med. 2018;24(4):254-259.; Liu et al., 2012Liu Y, Yin HJ, Shi DZ, Chen KJ. Chinese herb and formulas for promoting blood circulation and removing blood stasis and antiplatelet therapies. Evid-Base Compl Alt. 2012;2012:184503.). Many TCM formulas or extracts have been used to treat BSS and have achieved good clinical outcomes via promoting blood circulation and removing blood stasis (PBCRBS). Such outcomes include an inhibition of platelet aggregation, the promotion of the hemostasis release reaction, and other salutary outcomes (Dang et al., 2015Dang X, Miao JJ, Chen AQ, Li P, Chen L, Liang JR. The antithrombotic effect of RSNK in blood-stasis model rats. J Ethnopharmacol. 2015;173:266-272.; Li et al., 2009Li HX, Han SY, Wang XW, Ma X, Zhang K, Wang L, et al. Effect of the carthamins yellow from Carthamus tinctorius L. on hemorheological disorders of blood stasis in rats. Food Chem Toxicol. 2009;47(8):1797-1802.; Liu et al., 2012Liu Y, Yin HJ, Shi DZ, Chen KJ. Chinese herb and formulas for promoting blood circulation and removing blood stasis and antiplatelet therapies. Evid-Base Compl Alt. 2012;2012:184503.; Zhang et al., 2010Zhang H, Wang WR, Lin R, Zhang JY, Ji QL, Lin QQ, et al. Buyang Huanwu decoction ameliorates coronary heart disease with Qi deficiency and blood stasis syndrome by reducing CRP and CD40 in rats. J Ethnopharmacol . 2010;130:98-102.). However, the lack of scientific evidence regarding the ability of TCMs to treat disease may lead to an underestimation of their value in modern medicine. Furthermore, the clinical application and quality control of TCMs still faces enormous challenges. To the best of our knowledge, a pattern or syndrome that reflects multi-system changes is an ideal candidate for TCM treatment. Therefore, the establishment of a whole animal model that reflects a certain functional state or a certain syndrome is of great importance in the pharmacological evaluation of TCMs. According to TCM theory, the rat of traumatic blood stasis (TBS) model was a simulation of the BSS in human patients (Tian et al., 2006Tian JZ, Wang YY, Xu Y, Shi J. Type, evaluation and research of animal model of blood stasis syndrome. J Beijing Univ Tradit Chin Med. 2006;29(6):396-400.; Yan et al., 2004Yan SS, Dou WH, Dong SH, Liang JF. Making animal model of the blood stasis syndrome and its problems. Chin J Basic Med Tradit Chin Med. 2004;10(2):35-37.). Moreover, inflammatory cytokines, such as IL-6, IL-8, TNF, IL-1β, and NOS2, are involved in the processes by which TCMs act on PBCRBS according to experimental researches and network pharmacology analysis (Dang et al., 2015Dang X, Miao JJ, Chen AQ, Li P, Chen L, Liang JR. The antithrombotic effect of RSNK in blood-stasis model rats. J Ethnopharmacol. 2015;173:266-272.; Lv et al., 2015Lv M, Wang TY, Tian XX, Shi XH, Fan GW, Zhang Y, et al. Interaction of anti-thrombotic and anti-inflammatory activities of commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis revealed by network pharmacology analysis. Acta Pharmaceut. 2015;50(9):1135-1141.; Song et al., 2015Song GH, Zhang QM, Pang BZ, He LJ, Saimaiti J, Tulahong A, et al. Effects of different Chinese herbal prescriptions on cytokines in autoimmune prostatitis rats. J Tradit Chin Med. 2015;35(2):211-217.). Accordingly, TBS rat model has been used to evaluate efficacy for PBCRBS and anti-inflammatory activity of TCMs or prescriptions, such as Salvia miltiorrhiza Bunge (Dong et al., 2013Dong XJ, Yang Y, Ren TY, Xu JQ, Li F, Chen XL. Verifying the part nature of “promoting blood circulation and diuresis” by adjusting the AQP1’s characteristic expression with Salvia. J Emerg Tradit Chin. Med. 2013;22(5):732-735., 2014Dong XJ, Guo LF, Yao R, Xue SY, Li F. Relationship between regulation effect of Salvia miltiorrhiza on AQP2 in kidney and promoting blood circulation and diuresis. China J Chin Mater Med. 2014;39(16):3162-3165.), arisaema plants (Wang et al., 2017Wang FJ, Yang YH, Wang T, Miao MS, Guo XF. Effects of different sources of external arisaema rat traumatic blood stasis model. Acta Chin Med. 2017;32(12):2408-2414.), Asarum sieboldii Miq. (Bai et al., 2014Bai M, Liu DD, Yan X, Miao ML. External curative effect of wine paste of different varieties of asari radix et rhizoma by external use on traumatic impact blood stasis model in rats. Chin J Mod Appl Pharm. 2014;31(5):517-522.), Pinellia ternata (Lv et al., 2013Lv JD, Miu JX, Miao MS. External curative effect of Pinellia ternata wine paste on traumatic blood stasis rat model. China J Tradit Chin Med Pharm. 2013;28(3):616-619.), and Huo-xue-cu-yu capsules (Xu et al., 2013Xu LB, Chen LP, Xiao MY. Effects of Huoxue cuyu capsule on rats with acute blood stasis. Chin J Exp Tradit Med Form. 2013;19(4):270-273.).

Lagopsis supina (Steph) IK. Gal. is a perennial herbaceous plant species of the Labiatae family and widely distributed in northeast Asia. It was first described in the authoritative medical book of ancient China “Shennong’s Herbal Classics (Shen Nong Ben Cao Jing)” and is known to induce PBCRBS, block inflammation, and promote diuresis. The whole plants of L. supina, known as “Xiazhicao (夏至草)” in Chinese, has been used in TCM, specifically in Han, Tibetan, and Mongolian medicines (State Administration of Traditional Chinese Medicine 1999State Administration of Traditional Chinese Medicine. Chinese Materia Medica. Shanghai Science and Technology Press: Shanghai, China, Volume VII, 1999;pp:55-56.; Jia et al., 2016Jia MR, Zhang Y. Dictionary of Chinese Ethnic Medicine. China Medical Science Press: Beijing, China, 2016;468.). Previous phytochemical studies on L. supina have identified the presence of diterpenoids (Li et al., 2014Li H, Li MM, Su SQ, Sun J, Gu YF, Zeng KW, et al. Anti- inflammatory labdane diterpenoids from Lagopsis supina. J Nat Prod. 2014;77(4):1047-1053.), flavonoid glycosides (Zhang et al., 2015aZhang J, Pang DR, Huang Z, Huo HX, Li YT, Zheng J, et al. Flavonoids from the whole plants of Lagopsis supina. Chin J Chin Mater Med. 2015a;40(16):3224-3228.), phenylethanoid glycosides (Zhang et al., 2015bZhang J, Huang Z, Huo HX, Li YT, Pang DR, Zheng J, et al. Chemical constituents from Lagopsis supina (Steph.) IK.- Gal. ex Knorr. Biochem Syst Ecol. 2015b;61:424-428.), and monoterpenes (Zhang et al., 2015bZhang J, Huang Z, Huo HX, Li YT, Pang DR, Zheng J, et al. Chemical constituents from Lagopsis supina (Steph.) IK.- Gal. ex Knorr. Biochem Syst Ecol. 2015b;61:424-428.). It has been suggested that L. supina improves blood and lymph microcirculation (Zhang et al., 2004Zhang LM, Jiang H, Liu YK, Zhang XF, Niu CY, Zhang WM. The effects of extracts from herba Lagopsis on microcirculation of acute blood stasis rats. Chin Med Mater. 2004;27(7):509-511.), myocardioprotective (Liang et al., 2008Liang HF, Wang WP, Zahng YP, Du ST, Jiang H. Effect of ethanol extract from Mrrubium incisum against the myocardium injury in experimental DIC rats. Lishizhen Med Mater Med Res. 2008;19(7):1650-1651.), antioxidative (Zhang et al., 2008Zhang YC, Han R, Hou YL, LI BL, Niu FL, Zhao ZG, et al. Effects of ethanol extract from Mrrubium incisum on free radical injury in shock rats. Lishizhen Med Mater Med Res. 2008;19(8):1909-1910.), and antiviral (Wang et al., 2014Wang X, Yao MY, Kong J, Pang R, Cui YX, Zhou CZ. Study of Lagopsis supina in vitro anti-herpes simples virus. Shangdong Tradit Chin Med. 2014;33(11):928-931.) effects. However, the efficacy of L. supina for PBCRBS and for the inhibition of inflammation has not yet been studied in detail. Therefore, the purpose of the present study is to evaluate the efficacy for PBCRBS of L. supina in the TBS rat model and to evaluate the capacity of L. supina to inhibit inflammatory cytokines.

MATERIAL AND METHODS

Materials and reagents

L. supina whole plants were collected in Keerqin District, Tongliao City, Inner Mongolia, China in June 2016 having been identified by one of the authors (Guoyue Zhong). A voucher specimen (no. XZC201606) was deposited at the Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Chinese Medicine, Nanchang, China. The preparation of the crude extract was conducted according to our previously method (He et al., 2019He JW, Zeng LB, Wei RR, Zhong GY, Zhu YY, Xu TT, et al. Lagopsis supina exerts its diuretic effect via inhibition of aquaporin-1, 2 and 3 expression in a rat model of traumatic blood stasis. J Ethnopharmacol . 2019;231:446-452.): briefly, the air-dried and powdered whole plants (38 kg) were exhaustively extracted using 95% EtOH (300 L × 3) and subsequently 50% EtOH (300 L × 3) by maceration at room temperature for seven days. After filtration, combination, and solvent evaporation, an ethanol crude extract of L. supina (LS, 8.7 kg, the yield was 23 %) was obtained. The crude ethanol extract was resuspended in 0.3% sodium carboxymethyl cellulose (CMC-Na) for subsequent intragastric administration and kept at -20°C until use.

Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β, IL-4, IL-6, IL-8, and IL-10 were purchased from Nanjing SenBeiJia Biological Technology Co., Ltd. (Nanjing, China). All of the other chemicals used in this study were of analytical reagent grade.

Animals and sample collection

All animal experimental protocols were reviewed and approved by the Animal Care and Research Committee of Jiangxi University of Traditional Chinese Medicine. Male Sprague-Dawley rats (weighing 180-220 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). Rats were housed in polyethylene boxes with free access to autoclaved water and food under a controlled room temperature (23 ± 2)°C with 12 h light and dark cycles. All of the experiments were carried out in adherence with the guidelines of the Institutional Animal Care and Use Committee of China and were approved by the Animal Care and Research Committee of Jiangxi University of Traditional Chinese Medicine.

After a week of adaptive feeding, 30 rats were randomly assigned to the sham operated group (control group), traumatic injury group (TBS model group) and LS-treated group (TBS + LS group, referred to as LS group), with 10 rats in each group. The TBS model group and LS group animals were established according to the method of TBS model published previously (Dong et al., 2013Dong XJ, Yang Y, Ren TY, Xu JQ, Li F, Chen XL. Verifying the part nature of “promoting blood circulation and diuresis” by adjusting the AQP1’s characteristic expression with Salvia. J Emerg Tradit Chin. Med. 2013;22(5):732-735., 2014Dong XJ, Guo LF, Yao R, Xue SY, Li F. Relationship between regulation effect of Salvia miltiorrhiza on AQP2 in kidney and promoting blood circulation and diuresis. China J Chin Mater Med. 2014;39(16):3162-3165.). Briefly, rats were fixed on a wooden board, and a 500 g weight was dropped from a height of 58 cm, hitting the midsection of the right hind limb (potential energy was approximately 2.86 Joule (J) and the striking area was 19.2 cm2). Unilateral claudication without obvious skin damage, bleeding, or obvious fracture confirmed the successful procedure.

According to clinical TCM practice the dosage of LS for adults (60 kg/person) is 9-30 g/kg/day (State Administration of Traditional Chinese Medicine, 1999State Administration of Traditional Chinese Medicine. Chinese Materia Medica. Shanghai Science and Technology Press: Shanghai, China, Volume VII, 1999;pp:55-56.). For rats, this dosage is 2 g raw herb/kg/day (equivalent 460 mg crude extract/kg/day) after adjusting for the difference in body surface area. Rats of LS group were orally administered crude drug (LS, 460 mg/kg) once daily for 7 sucessive days. The control group and TBS group received an equivalent amount of 0.3% CMC-Na. Each rat was housed individually in a metabolic cage, and the cumulative urine output was determined at daily intervals for 7 sucessive days and stored at -20°C for the examination of TNF-α, IL-1β, IL-4, IL-6, IL-8, and IL-10 levels. At the end of the experimental period, 4 mL of blood was collected in vacuum tubes from the abdominal aorta, allowed to clot on ice, and subsequently subjected to centrifugation at 3000 rpm at 4°C for 10 min. All experiments were completed within 3 h after blood collection. Then, all of the rats were killed by cervical dislocation. Injured muscle tissue and kidney tissue were dissected for histopathological evaluation within 3 h.

Biochemical analysis

The serum and urine levels of TNF-α, IL-1β, IL-4, IL-6, IL-8, and IL-10 were detected using ELISA kits, according to the manufacturer’s instructions.

Histopathological analysis

Injured muscle and kidney tissue were fixed in 4% (w/v) paraformaldehyde over 24 h for histopathological examination (Liu et al., 2018Liu ZK, Ng CF, Shiu HT, Wong HL, Chin WC, Zhang JF, et al. Neuroprotective effect of Da Chuanxiong Formula against cognitive and motor deficits in a rat controlled cortical impact model of traumatic brain injury. J Ethnopharmacol . 2018;217:11-22.). Following graded ethanol dehydration, xylene and liquid paraffin series, specimens were embedded in paraffin wax. 4 µm sections were obtained from paraffin embedded specimens. Subsequently, sections were deparaffinized, rehydrated and stained with hematoxylin and eosin (HE). Mounted sections were examined using a BX53 microscope (Olympus Corporation, Japan).

Statistical analysis

Data was analyzed using SPSS Statistics V17.0 software and represented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for multi-group comparisons following Tukey’s test. p < 0.05 was considered to be statistically significant.

RESULTS

LS improves the behavior of TBS rats

The behavior of the TBS rats was slightly depressed as compared to control rats, and claudication was present. Additionally, the TBS rats exhibited reduced food intake and symptoms in the injured muscles, such as swelling, purple skin, and hypokinesia. In the LS group, the swelling in the injured muscles was significantly reduced. Furthermore, food intake and activity in LS group rats were similar to that of the control rats.

Biochemical results

The levels of TNF-α, IL-6, and IL-8 in serum were significantly increased in TBS group rats compared to control group rats (p < 0.01, Figure 1). However, the levels of IL-1β, IL-4, and IL-10 in serum were no significant changes in TBS group rats compared to control group rats (p < 0.01, Figure 1). Following LS treatment, these high serum levels of TNF-α, IL-6, and IL-8 were decreased remarkably (p < 0.01).

FIGURE 1
The levels of TNF-α, IL-1β, IL-4, IL-6, IL-8 and IL-10 from rat serum (pg/mL). Data shown are mean ± SD (n = 10 per group). ## p < 0.01 vs control group, ** p < 0.01 vs TBS group.

As shown in Figure 2, the ELISA results revealed that the levels of TNF-α, IL-6, IL-8, and IL-10 in urine from TBS group rats were significantly upregulated compared to control group rats (p < 0.01). Moreover, the levels of IL- 1β and IL-4 in serum were no significant changes in TBS group rats compared to control group rats (p < 0.01, Figure 2). Conversely, the LS treatment group rats had significantly lower levels of TNF-α, IL-6, IL-8, and IL-10 in urine than the TBS group rats (p < 0.01 or p < 0.05).

FIGURE 2
The levels of TNF-α, IL-1β, IL-4, IL-6, IL-8, and IL-10 from rat urine (pg/mL). Data shown are mean ± SD (n = 10 per group). ## p < 0.01 vs control group, ** p < 0.01 and * p < 0.05 vs TBS group.

Histopathological results

Muscle tissue

In the control group, muscle tissue exhibited a normal appearance and morphology by light microscopy (Figure 3A). However, muscles of TBS group rats showed significant tissue damage, including muscle fibers rearrangement (black arrow), inflammatory cell infiltration (yellow arrow), blood stasis (green arrow), and edema (blue circle, Figure 3B). Treatment with LS improved these histopathological measures of tissue injury, including restoration of the injured muscle fibers, the alleviation of tissue edema, a decrease in inflammatory cell infiltration, and by the promotion of capillary hyperplasia (Figure 3C).

FIGURE 3
Effect of LS on injured muscle in the TBS rats by HE staining (400 × magnification, black arrow-muscle fiber, yellow arrow-inflammatory cell, green arrow-blood stasis, blue circle-edema). (A) Control group; (B) TBS group; and (C) LS group.

Kidney tissue

Control group rats exhibited normal kidney appearance by light microscopy (Figure 4A). Glomerular atrophy (blue circle), renal tubular epithelial cell swelling and deformation (blue arrow), capillary congestion (green arrow), and inflammatory cell infiltration (yellow arrow) were observed in TBS group rats (by arrows, Figure 4B). In contrast, treatment with LS improved the histopathological measures of renal injury, including recovery of the damaged glomerulus and renal tubules, the alleviation of capillary congestion, and by a decrease in inflammatory cell infiltration (Figure 4C).

FIGURE 4
Effect of LS on renal damage in the TBS rats by HE staining (400 × magnification, blue circle-glomerular atrophy, blue arrow-renal tubular epithelial cell, green arrow-capillary, yellow arrow-inflammatory cell). (A) control group; (B) TBS group; and (C) LS group.

DISCUSSION

Although some studies have indicated that the crude extract of L. supina might improve blood and lymph microcirculation in a rat model of acute blood stasis induced by chemical drugs, such as Dextran 500 (Zhang et al., 2004Zhang LM, Jiang H, Liu YK, Zhang XF, Niu CY, Zhang WM. The effects of extracts from herba Lagopsis on microcirculation of acute blood stasis rats. Chin Med Mater. 2004;27(7):509-511.), it cannot fully reflect the efficacy of PBCRBS. BSS is a result of a series of pathological changes caused by blood stasis and can lead to edema, tissue degeneration, and inflammation (Dong et al., 2013Dong XJ, Yang Y, Ren TY, Xu JQ, Li F, Chen XL. Verifying the part nature of “promoting blood circulation and diuresis” by adjusting the AQP1’s characteristic expression with Salvia. J Emerg Tradit Chin. Med. 2013;22(5):732-735., 2014Dong XJ, Guo LF, Yao R, Xue SY, Li F. Relationship between regulation effect of Salvia miltiorrhiza on AQP2 in kidney and promoting blood circulation and diuresis. China J Chin Mater Med. 2014;39(16):3162-3165.; Kim et al., 2018Kim JH, Lee EY, Cho MR, Lee CK, Cho JH. Effects of Dangguixu-san on acute lateral ankle sprain: study protocol for a randomized controlled trial. Trials. 2018;19(202).). In this experiment, we observed that muscles of BSS model group (TBS group) rats showed significant tissue damage, including muscle fibers rearrangement, inflammatory cell infiltration, blood stasis, and edema compared with those of the model group. Moreover, glomerular atrophy, renal tubular epithelial cell swelling and deformation, capillary congestion, and inflammatory cell infiltration were observed in TBS group rats compared with the model group. Treatment with LS improved these histopathological measures of tissue injury, edema, and inflammation. These results indicate that LS could remarkably improve the pathological changes of BSS in a rat model of TBS.

Inflammation is a very important and common regulated process of the host defence system. Moreover, many factors including physical stress, chemical stress, and microbiological toxins can cause chronic inflammatory diseases, such as inflammatory bowel disease, blood stasis disease, arthritis and hyperlipidemia (Song et al., 2015Song GH, Zhang QM, Pang BZ, He LJ, Saimaiti J, Tulahong A, et al. Effects of different Chinese herbal prescriptions on cytokines in autoimmune prostatitis rats. J Tradit Chin Med. 2015;35(2):211-217.; Zhai et al., 2016Zhai XT, Chen JQ, Jiang CH, Song J, Li DY, Zhang H, et al. Corydalis bungeana Turcz. Attenuates LPS-induced inflammatory responses via the suppression of NF-kB signaling pathway in vitro and in vivo. J Ethnopharmacol . 2016;194:153-161.). Inflammatory cytokines, such as IL-6, IL-8, TNF, IL-1β, and NOS2, are involved in the processes by which TCMs act on PBCRBS according to experimental researches and network pharmacology analysis (Dang et al., 2015Dang X, Miao JJ, Chen AQ, Li P, Chen L, Liang JR. The antithrombotic effect of RSNK in blood-stasis model rats. J Ethnopharmacol. 2015;173:266-272.; Lv et al., 2015Lv M, Wang TY, Tian XX, Shi XH, Fan GW, Zhang Y, et al. Interaction of anti-thrombotic and anti-inflammatory activities of commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis revealed by network pharmacology analysis. Acta Pharmaceut. 2015;50(9):1135-1141.; Song et al., 2015Song GH, Zhang QM, Pang BZ, He LJ, Saimaiti J, Tulahong A, et al. Effects of different Chinese herbal prescriptions on cytokines in autoimmune prostatitis rats. J Tradit Chin Med. 2015;35(2):211-217.). Therefore, it is very important to assess the efficacy of L. supina for anti-inflammatory activity in the TBS rat model. However, the anti-inflammatory activity of L. supina has only been reported by one study (Li et al., 2014Li H, Li MM, Su SQ, Sun J, Gu YF, Zeng KW, et al. Anti- inflammatory labdane diterpenoids from Lagopsis supina. J Nat Prod. 2014;77(4):1047-1053.), which showed that four diterpenoids isolated from L. supina moderately inhibited lipopolysaccharide (LPS) induced nitric oxide production by BV-2 microglial cells. In the present study, this is the first reported notable anti- inflammatory effect by an ethanolic extract of L. supina, which probably was through downregulated the levels of the inflammatory cytokines TNF-α, IL-6, IL-8 and IL-10 in a rat model of TBS.

CONCLUSIONS

To summarize our fingdings, this is the first study to investigate the effects of L. supina on PBCRBS and anti-inflammatory properties in a TBS rat model. Based on our results, we conclude that L. supina carries out its PBCRBS and anti-inflammatory effect mainly by improve the pathological changes of BSS and downregulated the levels of the inflammatory cytokines (TNF-α, IL-6, IL-8, and IL-10) in a rat model of TBS, respectively. These finding suggest that LS may warrant further evaluation as a possible agent for the treatment of BSS and inflammatory diseases. Further work is needed to interpret the precise active components of LS and the underlying molecular mechanisms by which they exert their effects.

ACKNOWLEDGMENT

This work was financially supported by grants from the Subject of Jiangxi Education Department (Nos. GJJ170754 and GJJ180272), and the Jiangxi University of Chinese Medicine (Nos. JXSYLXK-ZHYA0031, 2016RC001 and JXXT2017008).

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  • Dang X, Miao JJ, Chen AQ, Li P, Chen L, Liang JR. The antithrombotic effect of RSNK in blood-stasis model rats. J Ethnopharmacol. 2015;173:266-272.
  • Dong XJ, Yang Y, Ren TY, Xu JQ, Li F, Chen XL. Verifying the part nature of “promoting blood circulation and diuresis” by adjusting the AQP1’s characteristic expression with Salvia. J Emerg Tradit Chin. Med. 2013;22(5):732-735.
  • Dong XJ, Guo LF, Yao R, Xue SY, Li F. Relationship between regulation effect of Salvia miltiorrhiza on AQP2 in kidney and promoting blood circulation and diuresis. China J Chin Mater Med. 2014;39(16):3162-3165.
  • He JW, Yang L, Zhu JX, Wang XM, Zhou ZR, He WW, et al. Comparison of anti-inflammatory effects and HPLC detection on different extracts from the flowers of Hosta plantaginea in mice. J Jiangxi Norm Univ. (Nat Sci.) 2016;40(2):183-185.
  • He JW, Zeng LB, Wei RR, Zhong GY, Zhu YY, Xu TT, et al. Lagopsis supina exerts its diuretic effect via inhibition of aquaporin-1, 2 and 3 expression in a rat model of traumatic blood stasis. J Ethnopharmacol . 2019;231:446-452.
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  • Li PL, Su WW, Yun S, Liao YQ, Liu H, Li PB, et al. Toward a scientific understanding of the effectiveness, material basis and prescription compatibility of a Chinese herbal formula Dan-hong injection. Sci Rep. 2017;7:46266.
  • Liang HF, Wang WP, Zahng YP, Du ST, Jiang H. Effect of ethanol extract from Mrrubium incisum against the myocardium injury in experimental DIC rats. Lishizhen Med Mater Med Res. 2008;19(7):1650-1651.
  • Liu Y, Yin HJ, Shi DZ, Chen KJ. Chinese herb and formulas for promoting blood circulation and removing blood stasis and antiplatelet therapies. Evid-Base Compl Alt. 2012;2012:184503.
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Publication Dates

  • Publication in this collection
    06 June 2022
  • Date of issue
    2022

History

  • Received
    16 May 2019
  • Accepted
    15 Sept 2020
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br