人参皂苷Rg1的选择性糖皮质激素样作用的研究
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摘要
研究背景
糖皮质激素(Glucocorticoid, GC)是由肾上腺分泌的一类甾体激素,在生物体的生长、发育、生殖、代谢、免疫及维持内环境稳定等过程都有重要的调节作用；同时GC也是临床上广泛应用的抗炎药物,用于治疗哮喘、风湿性关节炎等疾病。然而,由于不可避免的副作用,如骨质疏松、糖尿病、脂肪重新分布、水钠潴留状等,极大地限制了激素的使用。糖皮质激素的生理和药理作用主要是由糖皮质激素受体(Glucocorticoid receptor, GR)介导的。GR作为配基依赖性的转录调节因子调节基因表达,GC与GR结合导致GR的活化,活化的GR以同源二聚体的形式转移到核内,通过转录激活或转录抑制作用调节基因转录。现在普遍认为,转录抑制作用是GC发挥抗炎效应的关键机制；而一些严重副作用则主要与它的转录激活作用有关。因此,如果有一种GC类药物与GR结合后保留转录抑制作用,而没有或较少发挥转录激活作用,从而在发挥治疗作用的同时,没有或较少产生副作用,则对于炎性疾病的临床治疗具有重要价值。
现代药理研究表明,许多中药、中药单体或中药复方都可发挥抗炎作用,并且许多中药提取物中含有与甾体类激素相似的化学结构。人参皂苷(Ginsenosides, GSS)是人参的主要活性成份,具有类似甾体激素样结构,具有抗炎、抗应激、调节免疫等广泛的药理学活性。我科前期的临床研究发现,GSS联合泼尼松能够增强其疗效,减少GC副作用的发生率,比如柯兴征、带状疱疹、细菌感染等。此外,GSS联合地塞米松(Dexamethasone, Dex)还可以有效防治经动脉化疗栓塞(Transcatheter arterial chemoembolization, TACE)后肝肾功能的损伤,防治TACE术后综合征。因此,我们推测：GSS中很可能含有某种成分,具有类似于GC样的抗炎作用,而较少发挥GC样的副作用。通过复习文献,我们发现人参皂苷Rgl (Ginsenosides Rgl, G-Rg1)可以作为GR的功能性配体,激活荧光素酶报告基因的活性。因此,我们推测G-Rg1极有可能能够发挥GC样的抗炎作用,而具有较少的副作用。
研究目的
观察G-Rg1在体内外的抗炎作用及其对糖代谢、骨质代谢的影响,并进一步阐明G-Rg1的作用机制,探讨G-Rg1是否能够作为一种新型的GR调节剂通过抑制NF-κB参与抗炎反应,同时较少引起GC样的副作用。
研究方法
(1)体外抗炎作用及副作用
LPS刺激Raw264.7细胞8h后,收集上清液,酶联免疫吸附法(Enzyme-linked immunosorbent assay; ELISA)检测肿瘤坏死因子a (Tumor necrosis factor-alpha;TNF-α)及白介素6 (Interleukin-6, IL-6)水平。MTT法检测G-Rg1对小鼠原代培养成骨细胞增殖的作用。荧光定量PCR法检测TNF-a mRNA水平。
(2)体内抗炎作用及副作用
采用酵母多糖诱导的小鼠脚掌肿胀急性炎症模型和牛Ⅱ型胶原诱导的小鼠关节炎模型,检验G-Rgl在体内的抗炎作用。观察G-Rg1一次性用药后血糖的变化及长期用药后小鼠体重、胸腺和脾脏重量及骨密度的变化。
(3)G-Rg1作用机制
采用Western blot方法观察G-Rg1对GR核转位和磷酸化,MAPK通路,IκB以及NF-κB的影响,探讨G-Rg1的作用机制。
结果
(1)体外抗炎作用及副作用
G-Rg1和Dex能显著抑制LPS诱导的TNF-α和IL-6分泌,其作用呈一定的浓度依赖性；Dex对TNF-α的抑制作用在10-5-10-7M显著强于G-Rg1,但二者对IL-6的抑制作用在10-5-10-8M时差异无统计学意义。尽管低浓度时Dex对成骨细胞无明显抑制作用,而随着Dex浓度的增加,其抑制小鼠原代培养成骨细胞增殖的作用逐渐增强,呈明显的浓度依赖性；G-Rg1在选取的浓度范围内(10-9-10-5M)对成骨细胞增殖均无显著的抑制作用。GR特异性阻断剂Ru486能够部分阻断G-Rg1的抗炎作用,提示G-Rg1可部分通过GR发挥抗炎作用。
(2)体内抗炎作用及副作用
G-Rgl (12.5mg/kg)和Dex (2.5mg/kg)对酵母多糖诱导的小鼠脚掌肿胀急性炎症模型均具有显著的抗炎作用(与溶剂对照组相比P<0.05或P<0.01),而两种药物之间作用差异无统计学意义(P>0.05)。给牛Ⅱ型胶原诱导的关节炎模型小鼠给药两周后,G-Rg1组(12.5mg/kg-d)和Dex (2.5mg/kg-d)组小鼠关节炎症状评分明显小于溶剂对照组(P<0.01),两组之间差异无统计学意义(P>0.05)。按抗炎剂量给小鼠腹腔注射药物6h后,Dex组小鼠外周血血糖水平较溶剂对照组明显升高(P<0.01),而G-Rg1组小鼠血糖水平无明显升高(P>0.05)。给小鼠腹腔注射药物4周后,Dex组小鼠体重明显轻于溶剂对照组及G-Rg1组(P<0.01)；而G-Rg1组小鼠体重与溶剂对照组差异无统计学意义(P>0.05)。Dex组小鼠脾脏重量明显低于溶剂对照组及G-Rg1组(P<0.01)；而G-Rg1组小鼠脾脏重量与溶剂对照组差异无统计学意义(P>0.05)。Dex长期给药(4周)亦可导致小鼠胸腺重量较溶剂对照组明显降低(P<0.01),G-Rg1对小鼠胸腺重量无明显影响(P>0.05)；Dex组小鼠胸腺重量亦显著低于G-Rg1组(P<0.01)。为消除体重变化对小鼠胸腺、脾脏重量的影响,我们计算了其胸腺、脾脏指数。与小鼠胸腺、脾脏重量变化一致,Dex组小鼠脾脏、胸腺指数亦低于溶剂对照组和G-Rg1组(P<0.01)；G-Rg1组小鼠胸腺指数与溶剂对照组无统计学差异(P＞0.05),而脾脏指数大于溶剂对照组(P<0.05)。给药4周后各组小鼠胫骨骨小梁、皮质密度及总骨密度差异均无统计学意义(P>0.05)；然而,Dex组小鼠胫骨皮质厚度明显小于溶剂对照组及G-Rg1组小鼠(P<0.05),其横断面面积亦小于溶剂对照组及G-Rg1组小鼠(P<0.01；P<0.05),而溶剂对照组和G-Rg1组皮质厚度和骨面积差异无统计学意义(P>0.05)；Dex组小鼠骨含量亦较溶剂对照组及G-Rg1组小鼠降低(P<0.01；P<0.05),溶剂对照组和G-Rg1组小鼠骨含量差异无统计学意义(P>0.05)。
(3)G-Rg1作用机制
药物处理1h后,G-Rg1及Dex均可引起GR的核转位,Dex促进GR核转位的作用强于G-Rg1；Dex能引起Ser211位点磷酸化的GR显著增加,而G-Rg1对GRSer211位点的磷酸化无明显影响。LPS刺激后核内p65的水平明显升高,而G-Rg1能够明显的抑制LPS作用后核内p65的水平；LPS作用后可导致IκB的降解,在LPS处理30 min后其基本被完全降解,而在第60 min时,又可以观察到新合成的IκB,G-Rg1预处理2h后可明显增加IKB的水平,抑制LPS对IκB的降解,提示G-Rg1可通过减弱LPS对IκB的降解,降低p65的水平,抑制NF-κB的激活发挥抗炎作用。LPS刺激15min后,JNK、p38和ERK磷酸化水平明显升高：而Rg1预处理则可明显抑制ERK、JNK和p38磷酸化,提示抑制MAPK的磷酸化,进而抑制炎症因子的表达是G-Rg1发挥抗炎作用的机制之一。
结论
(1)G-Rg1在体内外均可发挥较显著的抗炎作用；
(2)G-Rg1在糖代谢、体重、淋巴器官重量及骨质代谢等方面不会引起GC样的副作用；
(3)G-Rg1能够通过抑制MAPK磷酸化、抑制NF-κB的激活发挥抗炎作用；G-Rg1未出现GC样副作用的机制可能与不引起GR Ser211位点磷酸化有关；
(4)G-Rg1是一个潜在的,具有较优治疗指数(抗炎作用/副作用比)的抗炎药物。
Background
Glucocorticoid (GC), a steroid hormone secreted by the adrenal gland, regulates many important physiological processes in the body, including growth, metabolism, and immunological reactivity. GC is also one of the most commonly used medicines in treating chronic asthma, rheumatoid arthritis, etc. However, their use is limited by the unavoidable side effects, such as osteoporosis, diabetes, redistribution of fat and sodium retention, etc. The physiological and pharmacologic effects of GC are mainly mediated by glucocorticoid receptor (GR), a ligand dependent transcription factor. Binding of GC to GR activates GR. Activated GR translocates to nucleus as a dimer, and then regulates gene transcription directly or indirectly. The transrepression is generally considered the key mechanism for the anti-inflammatory activity of GC. In contrast, several side effects are thought to be predominantly mediated via transactivation. Thus, ligands that preferentially induce the transrepression and not transactivation function of GR and could be as effective as standard GC but with fewer undesirable effects would be more valuable for the clinical use of GC.
It has been demonstrated that many Chinese herbs, herbs'monomers and herbs compounds may exert anti-inflammatory effects. Many herbs' extraction also has a similar structure to steroid hormones. Ginsenosides (GSS) which have a steroid-like structure and possess anti-inflammatory, anti-stress and modulating immunology effects are the main activity components of ginseng. In our previous clinical studies, GSS may enhance the effectiveness of prednisolone and attenuate its side effects, such as Cushing's syndrome, Shingles and Bacterial infection, etc. Furthermore, GSS combined with dexamethasone (Dex) could prevent and cure the injury of liver and kidney after transcatheter arterial chemoembolization (TACE) effectively. Previous studies have reported that Ginsenosides Rgl (G-Rgl) could transactivate the activity of GRE-luciferase reporter gene as a GR functional ligand. Therefore, we supposed that G-Rgl may exert GC-like anti-inflammatory effect but with little side effects.
Objective
To observe the anti-inflammatory effects in vitro and in vivo and the effects on glucose and bone metabolism of G-Rgl; to investigate the molecular mechanisms of G-Rg1 effects; to study whether G-Rgl may repress NF-κB through GR then participate anti-inflammation and induce little GC-like side effects meanwhile.
Methods
(1) Anti-inflammatory effect and side effects of G-Rgl in vitro. Eight hours after stimulation of Raw264.7 cells with lipopolysaccharide (LPS), supernatant was collected. Tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) were determined with enzyme-linked immunosorbent assay (ELISA). The effect of G-Rgl on mouse primary osteoblasts proliferation was examined by MTT method.
(2) Anti-inflammatory effect and side effects of G-Rg1 in vivo. The anti-inflammatory effect of G-Rg1 in vivo was examined in two mouse models, zymosan-induced inflamed paw model and bovine type II collagen-induced arthritis (CIA) mouse model. Glucose level was determined 6 h after administration of G-Rgl. Body, spleen and thymus weight and bone metabolism were determined following a four-week mouse administration.
(3) The impacts of G-Rgl on the nuclear translocation and phosphorylation of GR, NF-κB and MAPK pathway were investigated by western blot to study the mechanisms of G-Rg1 actions.
Results
(1) Anti-inflammatory effect and side effects of G-Rg1 in vitro
G-Rgl may significantly inhibit the secretion of TNF-a and IL-6 in a concentration-dependent manner. Its anti-inflammatory effect was less than Dex at the same concentration. Dex exhibited a proliferation inhibition effect on mouse primary osteoblasts cells in a concentration-dependent manner, but G-Rg1 had little inhibition effect on mouse primary osteoblasts cells at indicated concentrations (10-9-10-5 M).
(2) Anti-inflammatory effect and side effects of G-Rg1 in vivo
Both G-Rg1 and Dex could exert anti-inflammatory effects in zymosan-induced inflamed paw model (P<0.05 or P<0.01, vs vehicle group), but no significant difference was found between the two groups (P>0.05). In CIA mouse model, after 2-week treatment with G-Rgl or Dex, the symptom score was significantly less than that of vehicle group (P<0.01) and there was no significant difference between G-Rg1 and Dex groups (P>0.05). Six hours after intraperitoneal injection of Dex, mouse blood glucose level significantly increased compared with vehicle; however, there was no significant difference between G-Rgl and vehicle groups (P>0.05). after 4 weeks treatment of Dex, mouse body weight notablely decreased (P<0.01), but G-Rg1 treatment did not decrease the body weight compared with vehicle (P>0.05). The spleen weight in Dex treatment group was light than that of G-Rg1 and vehicle groups; no notable difference was detected between the G-Rgl and vehicle groups(P>0.05). The thymus weight of Dex treated mice was also much light than that treated with G-Rgl or vehicle (P<0.01); G-Rg1 may not affect the thymus weight (P>0.05). To eliminate the affect of body weight changes on spleen and thymus weight, we also calculated the spleen and thymus index, consistent with spleen and thymus weight changes, the spleen and thymus index of Dex treated mice were also lower than of G-Rgl and vehicle treated mice (P<0.01); there was no significant difference of the thymus index between G-Rgl and vehicle groups (P>0.05), but significant difference was found of spleen index between the two groups (P<0.05). There was no significant difference of mouse bone trabeculae, cortex and total density between the three groups (P>0.05); however, the cortex thickness of Dex group was thinner than that of G-Rgl and vehicle groups (P<0.05), and the bone area was also less that another two groups (P<0.01; P<0.05). The bone mineral content was decreased by four-week injection of Dex (P<0.01), but G-Rg1 didn't decrease that compared with vehicle. There was also a significant difference between Dex and G-Rgl groups (P<0.05).
(3) Mechanisms of G-Rgl effects
One hour after the treatment, both G-Rgl and Dex could induce nuclear translocation of GR. G-Rgl could not induce the phosphorylation of GR Ser-211 site, but Dex may induce the phosphorylation of GR Ser-211 site significantly. LPS stimulation could increase the level of p65 protein in nucleus, but G-Rgl pretreatment may attenuate it. IκB was degraded by LPS and was completely degraded after 30 min of LPS treatment. After 60 min of LPS treatment, new synthetical IκB can be detected again. Two hours pretreatment of G-Rgl could markedly increase the stability of IκB, attenuate its degradation. Fifteen minutes after LPS stimulation, phosphorylation of JNK, p38, ERK was increased significantly; pretreatment with G-Rgl may inhibit LPS induced-phosphorylation of ERK, JNK and p38, indicating that inhibiting the phosphorylation of MAPK pathway, then inhibiting inflammatory cytokines secretion is one of the mechanisms of anti-inflammatory effects of G-Rgl.
Conclusion
(1) G-Rgl may exert anti-inflammatory effect in vitro and in vivo;
(2) G-Rgl may not induce GC-like side effects on the glucose metabolism, body and lymphoid organs weight and bone metabolism;
(3) G-Rgl may exert anti-inflammatory effect through inhibiting MAPK phosphorylation and NF-κB activation; the mechanism that G-Rgl may not induce GC-like side effects maybe related with non-phosphorylation of GR at Ser 211 site.
(4) G-Rgl is a potential anti-inflammatory medicine with better therapy index (anti-inflammatory effect/side effects).
糖皮质激素(Glucocorticoid, GC)是由肾上腺分泌的一类甾体激素,在生物体的生长、发育、生殖、代谢、免疫及维持内环境稳定等过程都有重要的调节作用；同时GC也是临床上广泛应用的抗炎药物,用于治疗哮喘、风湿性关节炎等疾病。然而,由于不可避免的副作用,如骨质疏松、糖尿病、脂肪重新分布、水钠潴留状等,极大地限制了激素的使用。糖皮质激素的生理和药理作用主要是由糖皮质激素受体(Glucocorticoid receptor, GR)介导的。GR作为配基依赖性的转录调节因子调节基因表达,GC与GR结合导致GR的活化,活化的GR以同源二聚体的形式转移到核内,通过转录激活或转录抑制作用调节基因转录。现在普遍认为,转录抑制作用是GC发挥抗炎效应的关键机制；而一些严重副作用则主要与它的转录激活作用有关。因此,如果有一种GC类药物与GR结合后保留转录抑制作用,而没有或较少发挥转录激活作用,从而在发挥治疗作用的同时,没有或较少产生副作用,则对于炎性疾病的临床治疗具有重要价值。
现代药理研究表明,许多中药、中药单体或中药复方都可发挥抗炎作用,并且许多中药提取物中含有与甾体类激素相似的化学结构。人参皂苷(Ginsenosides, GSS)是人参的主要活性成份,具有类似甾体激素样结构,具有抗炎、抗应激、调节免疫等广泛的药理学活性。我科前期的临床研究发现,GSS联合泼尼松能够增强其疗效,减少GC副作用的发生率,比如柯兴征、带状疱疹、细菌感染等。此外,GSS联合地塞米松(Dexamethasone, Dex)还可以有效防治经动脉化疗栓塞(Transcatheter arterial chemoembolization, TACE)后肝肾功能的损伤,防治TACE术后综合征。因此,我们推测：GSS中很可能含有某种成分,具有类似于GC样的抗炎作用,而较少发挥GC样的副作用。通过复习文献,我们发现人参皂苷Rgl (Ginsenosides Rgl, G-Rg1)可以作为GR的功能性配体,激活荧光素酶报告基因的活性。因此,我们推测G-Rg1极有可能能够发挥GC样的抗炎作用,而具有较少的副作用。
研究目的
观察G-Rg1在体内外的抗炎作用及其对糖代谢、骨质代谢的影响,并进一步阐明G-Rg1的作用机制,探讨G-Rg1是否能够作为一种新型的GR调节剂通过抑制NF-κB参与抗炎反应,同时较少引起GC样的副作用。
研究方法
(1)体外抗炎作用及副作用
LPS刺激Raw264.7细胞8h后,收集上清液,酶联免疫吸附法(Enzyme-linked immunosorbent assay; ELISA)检测肿瘤坏死因子a (Tumor necrosis factor-alpha;TNF-α)及白介素6 (Interleukin-6, IL-6)水平。MTT法检测G-Rg1对小鼠原代培养成骨细胞增殖的作用。荧光定量PCR法检测TNF-a mRNA水平。
(2)体内抗炎作用及副作用
采用酵母多糖诱导的小鼠脚掌肿胀急性炎症模型和牛Ⅱ型胶原诱导的小鼠关节炎模型,检验G-Rgl在体内的抗炎作用。观察G-Rg1一次性用药后血糖的变化及长期用药后小鼠体重、胸腺和脾脏重量及骨密度的变化。
(3)G-Rg1作用机制
采用Western blot方法观察G-Rg1对GR核转位和磷酸化,MAPK通路,IκB以及NF-κB的影响,探讨G-Rg1的作用机制。
结果
(1)体外抗炎作用及副作用
G-Rg1和Dex能显著抑制LPS诱导的TNF-α和IL-6分泌,其作用呈一定的浓度依赖性；Dex对TNF-α的抑制作用在10-5-10-7M显著强于G-Rg1,但二者对IL-6的抑制作用在10-5-10-8M时差异无统计学意义。尽管低浓度时Dex对成骨细胞无明显抑制作用,而随着Dex浓度的增加,其抑制小鼠原代培养成骨细胞增殖的作用逐渐增强,呈明显的浓度依赖性；G-Rg1在选取的浓度范围内(10-9-10-5M)对成骨细胞增殖均无显著的抑制作用。GR特异性阻断剂Ru486能够部分阻断G-Rg1的抗炎作用,提示G-Rg1可部分通过GR发挥抗炎作用。
(2)体内抗炎作用及副作用
G-Rgl (12.5mg/kg)和Dex (2.5mg/kg)对酵母多糖诱导的小鼠脚掌肿胀急性炎症模型均具有显著的抗炎作用(与溶剂对照组相比P<0.05或P<0.01),而两种药物之间作用差异无统计学意义(P>0.05)。给牛Ⅱ型胶原诱导的关节炎模型小鼠给药两周后,G-Rg1组(12.5mg/kg-d)和Dex (2.5mg/kg-d)组小鼠关节炎症状评分明显小于溶剂对照组(P<0.01),两组之间差异无统计学意义(P>0.05)。按抗炎剂量给小鼠腹腔注射药物6h后,Dex组小鼠外周血血糖水平较溶剂对照组明显升高(P<0.01),而G-Rg1组小鼠血糖水平无明显升高(P>0.05)。给小鼠腹腔注射药物4周后,Dex组小鼠体重明显轻于溶剂对照组及G-Rg1组(P<0.01)；而G-Rg1组小鼠体重与溶剂对照组差异无统计学意义(P>0.05)。Dex组小鼠脾脏重量明显低于溶剂对照组及G-Rg1组(P<0.01)；而G-Rg1组小鼠脾脏重量与溶剂对照组差异无统计学意义(P>0.05)。Dex长期给药(4周)亦可导致小鼠胸腺重量较溶剂对照组明显降低(P<0.01),G-Rg1对小鼠胸腺重量无明显影响(P>0.05)；Dex组小鼠胸腺重量亦显著低于G-Rg1组(P<0.01)。为消除体重变化对小鼠胸腺、脾脏重量的影响,我们计算了其胸腺、脾脏指数。与小鼠胸腺、脾脏重量变化一致,Dex组小鼠脾脏、胸腺指数亦低于溶剂对照组和G-Rg1组(P<0.01)；G-Rg1组小鼠胸腺指数与溶剂对照组无统计学差异(P＞0.05),而脾脏指数大于溶剂对照组(P<0.05)。给药4周后各组小鼠胫骨骨小梁、皮质密度及总骨密度差异均无统计学意义(P>0.05)；然而,Dex组小鼠胫骨皮质厚度明显小于溶剂对照组及G-Rg1组小鼠(P<0.05),其横断面面积亦小于溶剂对照组及G-Rg1组小鼠(P<0.01；P<0.05),而溶剂对照组和G-Rg1组皮质厚度和骨面积差异无统计学意义(P>0.05)；Dex组小鼠骨含量亦较溶剂对照组及G-Rg1组小鼠降低(P<0.01；P<0.05),溶剂对照组和G-Rg1组小鼠骨含量差异无统计学意义(P>0.05)。
(3)G-Rg1作用机制
药物处理1h后,G-Rg1及Dex均可引起GR的核转位,Dex促进GR核转位的作用强于G-Rg1；Dex能引起Ser211位点磷酸化的GR显著增加,而G-Rg1对GRSer211位点的磷酸化无明显影响。LPS刺激后核内p65的水平明显升高,而G-Rg1能够明显的抑制LPS作用后核内p65的水平；LPS作用后可导致IκB的降解,在LPS处理30 min后其基本被完全降解,而在第60 min时,又可以观察到新合成的IκB,G-Rg1预处理2h后可明显增加IKB的水平,抑制LPS对IκB的降解,提示G-Rg1可通过减弱LPS对IκB的降解,降低p65的水平,抑制NF-κB的激活发挥抗炎作用。LPS刺激15min后,JNK、p38和ERK磷酸化水平明显升高：而Rg1预处理则可明显抑制ERK、JNK和p38磷酸化,提示抑制MAPK的磷酸化,进而抑制炎症因子的表达是G-Rg1发挥抗炎作用的机制之一。
结论
(1)G-Rg1在体内外均可发挥较显著的抗炎作用；
(2)G-Rg1在糖代谢、体重、淋巴器官重量及骨质代谢等方面不会引起GC样的副作用；
(3)G-Rg1能够通过抑制MAPK磷酸化、抑制NF-κB的激活发挥抗炎作用；G-Rg1未出现GC样副作用的机制可能与不引起GR Ser211位点磷酸化有关；
(4)G-Rg1是一个潜在的,具有较优治疗指数(抗炎作用/副作用比)的抗炎药物。
Background
Glucocorticoid (GC), a steroid hormone secreted by the adrenal gland, regulates many important physiological processes in the body, including growth, metabolism, and immunological reactivity. GC is also one of the most commonly used medicines in treating chronic asthma, rheumatoid arthritis, etc. However, their use is limited by the unavoidable side effects, such as osteoporosis, diabetes, redistribution of fat and sodium retention, etc. The physiological and pharmacologic effects of GC are mainly mediated by glucocorticoid receptor (GR), a ligand dependent transcription factor. Binding of GC to GR activates GR. Activated GR translocates to nucleus as a dimer, and then regulates gene transcription directly or indirectly. The transrepression is generally considered the key mechanism for the anti-inflammatory activity of GC. In contrast, several side effects are thought to be predominantly mediated via transactivation. Thus, ligands that preferentially induce the transrepression and not transactivation function of GR and could be as effective as standard GC but with fewer undesirable effects would be more valuable for the clinical use of GC.
It has been demonstrated that many Chinese herbs, herbs'monomers and herbs compounds may exert anti-inflammatory effects. Many herbs' extraction also has a similar structure to steroid hormones. Ginsenosides (GSS) which have a steroid-like structure and possess anti-inflammatory, anti-stress and modulating immunology effects are the main activity components of ginseng. In our previous clinical studies, GSS may enhance the effectiveness of prednisolone and attenuate its side effects, such as Cushing's syndrome, Shingles and Bacterial infection, etc. Furthermore, GSS combined with dexamethasone (Dex) could prevent and cure the injury of liver and kidney after transcatheter arterial chemoembolization (TACE) effectively. Previous studies have reported that Ginsenosides Rgl (G-Rgl) could transactivate the activity of GRE-luciferase reporter gene as a GR functional ligand. Therefore, we supposed that G-Rgl may exert GC-like anti-inflammatory effect but with little side effects.
Objective
To observe the anti-inflammatory effects in vitro and in vivo and the effects on glucose and bone metabolism of G-Rgl; to investigate the molecular mechanisms of G-Rg1 effects; to study whether G-Rgl may repress NF-κB through GR then participate anti-inflammation and induce little GC-like side effects meanwhile.
Methods
(1) Anti-inflammatory effect and side effects of G-Rgl in vitro. Eight hours after stimulation of Raw264.7 cells with lipopolysaccharide (LPS), supernatant was collected. Tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) were determined with enzyme-linked immunosorbent assay (ELISA). The effect of G-Rgl on mouse primary osteoblasts proliferation was examined by MTT method.
(2) Anti-inflammatory effect and side effects of G-Rg1 in vivo. The anti-inflammatory effect of G-Rg1 in vivo was examined in two mouse models, zymosan-induced inflamed paw model and bovine type II collagen-induced arthritis (CIA) mouse model. Glucose level was determined 6 h after administration of G-Rgl. Body, spleen and thymus weight and bone metabolism were determined following a four-week mouse administration.
(3) The impacts of G-Rgl on the nuclear translocation and phosphorylation of GR, NF-κB and MAPK pathway were investigated by western blot to study the mechanisms of G-Rg1 actions.
Results
(1) Anti-inflammatory effect and side effects of G-Rg1 in vitro
G-Rgl may significantly inhibit the secretion of TNF-a and IL-6 in a concentration-dependent manner. Its anti-inflammatory effect was less than Dex at the same concentration. Dex exhibited a proliferation inhibition effect on mouse primary osteoblasts cells in a concentration-dependent manner, but G-Rg1 had little inhibition effect on mouse primary osteoblasts cells at indicated concentrations (10-9-10-5 M).
(2) Anti-inflammatory effect and side effects of G-Rg1 in vivo
Both G-Rg1 and Dex could exert anti-inflammatory effects in zymosan-induced inflamed paw model (P<0.05 or P<0.01, vs vehicle group), but no significant difference was found between the two groups (P>0.05). In CIA mouse model, after 2-week treatment with G-Rgl or Dex, the symptom score was significantly less than that of vehicle group (P<0.01) and there was no significant difference between G-Rg1 and Dex groups (P>0.05). Six hours after intraperitoneal injection of Dex, mouse blood glucose level significantly increased compared with vehicle; however, there was no significant difference between G-Rgl and vehicle groups (P>0.05). after 4 weeks treatment of Dex, mouse body weight notablely decreased (P<0.01), but G-Rg1 treatment did not decrease the body weight compared with vehicle (P>0.05). The spleen weight in Dex treatment group was light than that of G-Rg1 and vehicle groups; no notable difference was detected between the G-Rgl and vehicle groups(P>0.05). The thymus weight of Dex treated mice was also much light than that treated with G-Rgl or vehicle (P<0.01); G-Rg1 may not affect the thymus weight (P>0.05). To eliminate the affect of body weight changes on spleen and thymus weight, we also calculated the spleen and thymus index, consistent with spleen and thymus weight changes, the spleen and thymus index of Dex treated mice were also lower than of G-Rgl and vehicle treated mice (P<0.01); there was no significant difference of the thymus index between G-Rgl and vehicle groups (P>0.05), but significant difference was found of spleen index between the two groups (P<0.05). There was no significant difference of mouse bone trabeculae, cortex and total density between the three groups (P>0.05); however, the cortex thickness of Dex group was thinner than that of G-Rgl and vehicle groups (P<0.05), and the bone area was also less that another two groups (P<0.01; P<0.05). The bone mineral content was decreased by four-week injection of Dex (P<0.01), but G-Rg1 didn't decrease that compared with vehicle. There was also a significant difference between Dex and G-Rgl groups (P<0.05).
(3) Mechanisms of G-Rgl effects
One hour after the treatment, both G-Rgl and Dex could induce nuclear translocation of GR. G-Rgl could not induce the phosphorylation of GR Ser-211 site, but Dex may induce the phosphorylation of GR Ser-211 site significantly. LPS stimulation could increase the level of p65 protein in nucleus, but G-Rgl pretreatment may attenuate it. IκB was degraded by LPS and was completely degraded after 30 min of LPS treatment. After 60 min of LPS treatment, new synthetical IκB can be detected again. Two hours pretreatment of G-Rgl could markedly increase the stability of IκB, attenuate its degradation. Fifteen minutes after LPS stimulation, phosphorylation of JNK, p38, ERK was increased significantly; pretreatment with G-Rgl may inhibit LPS induced-phosphorylation of ERK, JNK and p38, indicating that inhibiting the phosphorylation of MAPK pathway, then inhibiting inflammatory cytokines secretion is one of the mechanisms of anti-inflammatory effects of G-Rgl.
Conclusion
(1) G-Rgl may exert anti-inflammatory effect in vitro and in vivo;
(2) G-Rgl may not induce GC-like side effects on the glucose metabolism, body and lymphoid organs weight and bone metabolism;
(3) G-Rgl may exert anti-inflammatory effect through inhibiting MAPK phosphorylation and NF-κB activation; the mechanism that G-Rgl may not induce GC-like side effects maybe related with non-phosphorylation of GR at Ser 211 site.
(4) G-Rgl is a potential anti-inflammatory medicine with better therapy index (anti-inflammatory effect/side effects).
引文
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