目的 對(duì)RAW 264.7細(xì)胞炎癥模型進(jìn)行優(yōu)化，并考察異喹啉類生物堿的抗炎活性。方法 采用內(nèi)毒素脂多糖（LPS）誘導(dǎo)RAW264.7細(xì)胞炎癥反應(yīng)，以細(xì)胞上清液中炎癥因子分泌水平為指標(biāo)，優(yōu)化LPS誘導(dǎo)濃度、時(shí)間及陽性藥地塞米松（DEX）孵育時(shí)間，并考察異喹啉類生物堿的抗炎活性。結(jié)果 RAW 264.7細(xì)胞上清液中腫瘤壞死因子-α（TNF-α）分泌水平隨LPS誘導(dǎo)濃度、時(shí)間增加而升高，但當(dāng)LPS質(zhì)量濃度超過100 ng/mL、誘導(dǎo)時(shí)間超過12 h后，TNF-α分泌水平趨于平緩；LPS為20 ng/mL、誘導(dǎo)12 h的TNF-α分泌水平明顯升高。DEX對(duì)TNF-α分泌抑制作用隨孵育時(shí)間延長而增強(qiáng)，但孵育4 h后，TNF-α分泌抑制作用趨于平緩。異喹啉類生物堿呈現(xiàn)不同程度TNF-α分泌抑制作用，其抑制活性與其季胺型母核結(jié)構(gòu)及R基團(tuán)烷氧基取代等密切相關(guān)。結(jié)論 LPS為20 ng/mL、誘導(dǎo)12 h能較好誘導(dǎo)RAW 264.7細(xì)胞炎癥反應(yīng)；異喹啉類生物堿的TNF-α分泌抑制作用與其季胺型母核結(jié)構(gòu)、R基團(tuán)取代等密切相關(guān)。

Objective To optimize the RAW 264.7 cell inflammation model and investigate the anti-inflammatory activity of isoquinoline alkaloids. Methods Endotoxin lipopolysaccharide (LPS) was used to induce an inflammatory response in RAW 264.7 cells, in which the induction concentration and time of LPS, and incubation time of positive drug dexamethasone (DEX) were optimized by determining the secretion level of inflammatory factors in cell supernatant. In addition, the anti-inflammatory activity of isoquinoline alkaloids were investigated. Results The secretion level of TNF-αin supernatant of RAW 264.7 cells increased with the inductive concentration and time of LPS induction, while the level of TNF-α tended to be flat when the induction concentration of LPS exceeded 100 ng/mL, or the induction time exceeded 12 h. In addition, the secretion level of TNF-α was higher when the induction concentration and time of LPS were 20 ng/mL and 12 h, respectively. Besides, the inhibitory effect of DEX on TNF-α increased with its incubation time, while the inhibitory effect of DEX on TNF-α secretion tended to be flat after incubation for 4 h. The anti-inflammatory results showed that the isoquinoline alkaloids exhibited different degrees of inhibition on TNF-α secretion, and the anti-inflammatory activity were closely correlated with their quaternary amine parent nucleus and alkoxy substitution of R group. Conclusion LPS at 20 ng/mL for 12 h incubation could better induce the inflammatory response in RAW 264.7 cells, and the inhibitory effect of isoquinoline alkaloids on the secretion of TNF-α are closely related to their quaternary amine parent nucleus structure and R group substitution.

R285.5

中國博士后科學(xué)基金項(xiàng)目（2019M653087）；廣東省深圳市科技計(jì)劃項(xiàng)目（JCYJ20170413173747440、20180306174903174、ZDSYS201707281432317、CKFW2016082916015476）；山西中醫(yī)藥大學(xué)科技創(chuàng)新能力培育計(jì)劃項(xiàng)目（2019PY-110、2020PY-YC-05）