| Forsythia suspensa is the dried fruit of Forsythia suspense （Thunb.）Vahl, which commonly used as traditional Chinese medicine by traditional Chinese medicine. More than 40 Chinese medicinal preparations containing Forsythia suspense （FST） are listed in Chinese Pharmacopoeia, such as Shuanghuanglian oral solution, Yinqiao Jiedu tablet and Qinlian tablet. The crude drug is widely used as an antipyretic, antidotal and detumescent agent for the treatment of ulcers and infections, such as respiratory tract infection and erysipelas. Modern pharmacological experiments showed that the Chinese herb forsythia had the effect of antioxidant, antibacterial, anti-inflammatory, anti-viral, anti-liver injury and so on. As the effective component of Fructus Forsythiae, forsythoside A （FTA） has a variety of pharmacological actions, including antibacterial activity, anti-viral, antipyretic analgesic, antioxidant and immunity regulation effects. According to the experience of traditional Chinese medicine, Fructus Forsythiae is good to "upper-jiao" pulmonary disease, and the airway epithelium is the primary line of defense pathogens, which plays an important role in the innate immune system. When inflammation occurs, airway epithelial cells can secrete cytokines, chemokines, antimicrobial peptides and other immune factors which are involved in the inflammatory response. Therefore, in this paper, human airway epithelial cells （BEAS-2B） were used as models to research the anti-inflammatory activity and mechanism of action with forsythia extract （FSE）, which had different percentages of forsythoside A. Besides, this paper also compared anti-oxidative capacity and resistance to cell damage from different regions of forsythia.1. Study on the anti-oxidative activity of FSE from different regions in vivoBy scavenging DPPH-, we studied on the anti-oxidative activity of FST’s 40% ethanol extract from 21 regions in vivo, and the results showed that all the FSE had a good activity of anti-oxidative in vivo. The detection of the IC5o of scavenging DPPH-showed the difference of their places of origin. The activity of anti-oxidative of the FSE from 21 regions had been distinguished by discriminant analysis.2.The protection of FSE from different regions to BEAS-2B cellular damageCultured cells were divided into three groups, Control group, without any treatment; Model group,400 μg/mL（wet weight） Staphylococcus aureus cell lysate were used on cells for 24 h; Medicine group:after the pretreatment by FSE（50 μg/mL） on BEAS-2B cell for 2 h,400 μg/mL（wet weight） Staphylococcus aureus cell lysate were used on cells for 24 h. Observing the morphological through microscope, we found the cells of control group grew well, some cells of the model group had blurred edges, furthermore, the cell antennae of which reduced and had a tendency to become round, whereas, the medicine group could improve this state to some extent. Cell viability was assayed by MTT analysis, there was significant difference between model group and control group, and medicine group could take precautions against the injury by Staphylococcus aureus cell lysate to the BEAS-2B cell.3. Anti-inflammatory mechanism research of FSE to BEAS-2B cellular damageBEAS-2B cell inflammation was induced by LPS, and different concentrations of FSE were set to give cell pretreatment. The anti-inflammatory effect of FSE was detected by secretion of cytokines and inflammatory medium. Through phosphorylation of IKK-a and p65, it can display whether the NF-kB signaling pathways are activated, which demonstrates the anti-inflammatory mechanism of FSE.Experience groups:The experiment was carried out in thirteen groups, Control group, Model group （LPS,1 μg/mL）, positive medicine group （indomethacin, dexamethasone,1 μM）,90%FTA group （25,50,100 μg/mL）, 60%FTA group （25,50,100 μg/mL）, FSEE group （25,50,100 μg/mL）. All treatment agents in positive medicine group and medicine group were taken 2 hours before modeling, the cells and supernatant were collected to detect every index 24h after modeling.Experimental results:（a） morphological changes were observed with microscope, there was no morphological difference between model group and control group. （b） NO in supernatant and ROS, SOD in cells were detected with kits:after being induced by LPS （1 μg/mL） for 24 h, the release of NO and ROS were significantly increased comparing with model group （P＜0.01）. All the FSE group showed strong effect on reducing NO and ROS, and the higher concentration in 90%FTA,60% FTA and FSEE were, the more obviously they could reduce the NO and ROS content, which was dose-dependent. SOD in Model group reduced significantly （P<0.01）. 90% FTA group and 60% FTA group had a preventive effect when the concentration was 25 and 50 μg/mL, and FSEE group had the same effect when the concentration was 50 μg/mL.90% FTA had the best effect on prevention the injury of BEAS-2B. （c） IL-6 in supernatant was measured by ELISA, after LPS was added 24 h, IL-6 in supernatant increased palpable （P＜0.01）. All groups of FSEE,60% FTA and 90% FTA could inhibit the production of IL-6, and 90% FTA did the best work. （d） Expression of IL-6、 IL-8、TNF-α mRNA were detected by Q-RT-PCR, after LPS（1 μg/mL） was added to BEAS-2B cell for 24h, the expression of IL-6, IL-8 and TNF-α in model group showed a clearly enhancement（P<0.01）, however the expression of IL-6, IL-8, TNF-α in positive medicine group （dexamethasone） showed a clearly reduction （P＜0.01）. All the concentrations of 90%FTA group could inhibit the expression of IL-6, IL-8 and TNF-α, furthermore, 60%FTA group and FSEE group could inhibit the expression of IL-6, IL-8 but the expression of TNF-α couldn’t be inhibited by them. （e） Protein IKK-α, P65 and their phosphorylation level were measured by Western Blot analysis, phosphorylation of KK-a and p65 were very low in Control group, but they could be up-regulated by the addition of LPS（1 μg/mL）, which indicated that NF-kB signaling pathway was highly activated. Comparing the level of p-IKK/IKK a and p-p65/p65,90% FTA group,60% FTA group and FSEE group could inhibit the activation of the NF-kB signaling pathway in different levels. Among them 90% FTA group had the most significant effect, whereas Dex and FSEE group had no effect. This study indicated that 90%FTA can do better on anti-endotoxin, its underlying mechanism may due to interference in LPS-TLR4-MyD88-NF-kB signaling pathway.In a word, FSE can improve the cell viability of BEAS-2B which was injured by Staphylococcus aureus cell lysate. Besides it can inhibit BEAS-2B cells of secreting inflammatory cytokines IL-6, IL-8 and TNF-a, and increase the antioxidant capacity of cells. This paper confirmed the anti-inflammatory activity of FSE at the molecular and cellular level, and its anti-inflammatory ingredients might be FTA. |
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