PSP下调miR-345-5p对雨蛙素诱导的AP腺泡细胞氧化应激和炎症因子表达

目的探讨黄精多糖（PSP）对雨蛙素诱导的急性胰腺炎（AP）腺泡细胞氧化应激和炎症因子表达的影响及分子机制。 方法取对数期大鼠胰腺腺泡细胞AR42J，采用100 nmol/L雨蛙素处理细胞6 h，建立AP腺泡细胞损伤模型，并采用不同浓度（1、2、4 mg/mL）PSP处理AP细胞（AP+PSP-L、AP+PSP-M、AP+PSP-H）。试剂盒检测细胞中丙二醛（MDA）含量、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GPx）活性以及培养液中白细胞介素-6 （IL-6）、肿瘤坏死因子-α （TNF-α）和IL-1β水平，实时荧光定量PCR （RT-qPCR）检测miR-345-5p表达水平。将miR-345-5p抑制物转染AR42J细胞，检测下调miR-345-5p表达对AP细胞氧化应激和炎症因子表达的影响。将miR-345-5p模拟物转染AR42J细胞，检测上调miR-345-5p表达和PSP处理对AP细胞氧化应激和炎症因子表达的影响。两组比较采用t检验，多组间比较采用方差分析，进一步两两比较采用LSD-t检验。 结果与对照比较，AP细胞MDA含量、IL-6、TNF-α、IL-1β水平和miR-345-5p表达水平均升高，SOD和GPx活性降低（P < 0.05）。与AP细胞比较，不同浓度（1、2、4 mg/mL）PSP作用的AP细胞中MDA含量（1.08± 0.07）比（0.88±0.06），（0.73±0.06），（0.60±0.05） nmol/mg]降低，SOD （43.01±4.37）比（59.60±5.62），（72.37±6.32），（94.21±8.70） U/mg]和GPx活性（29.03±2.51）比（44.11± 4.71），（58.07±4.20），（72.67± 6.56） U/mg]均升高，培养液中IL-6 （310.72±22.27）比（257.01±20.85），（192.28±17.70），（146.93±11.90） pg/mL]、TNF-α （223.82±21.87）比（175.57±15.85），（137.00±11.31），（89.26±7.05） pg/mL]、IL-1β表达水平（41.66±3.85）比（33.82±3.20），（26.15±2.56），（20.14±1.71） pg/mL]和miR-345-5p表达水平（2.78±0.24比2.38±0.21，1.91±0.12，1.25±0.13）均降低，且呈浓度依赖性，差异有统计学意义（P均< 0.05）。下调miR-345-5p表达后，AP细胞中MDA含量（1.13±0.08）比（0.72±0.06） nmol/mg]降低，SOD活性（41.31±3.98）比（81.73±7.62） U/mg]和GPx （28.82±2.97）比（61.41±5.81） U/mg]升高，培养液中IL-6 （314.65±25.02）比159.76±11.93） pg/mL]、TNF-α （235.18±23.13）比（100.41±8.09） pg/mL]和IL-1β水平（48.67±4.50）比（27.73±2.54） pg/mL]降低（P均< 0.05）。上调miR-345-5p表达可逆转PSP对AP细胞的影响。其中MDA含量（0.58±0.03）比（0.95±0.08） nmol/mg]升高、SOD活性（96.52±9.54）比（54.24±4.15） U/mg]、GPx活性（79.62±6.23）比（39.81±3.84） U/mg]降低、IL-6 （145.38±12.49）比（275.38± 21.55） pg/mL]、TNF-α （84.83±7.81）比（183.73±16.39） pg/mL]和IL-1β （19.38±1.85）比（36.97±3.62） pg/mL]的表达水平升高（P均< 0.05）。 结论PSP以剂量依赖方式减轻雨蛙素诱导AP细胞炎症反应和氧化应激损伤，其机制与下调miR-345-5p表达有关。

Polygonatum sibiricum polysaccharide inhibits caerulein-induced oxidative stress and inflammatory factors expression of acinar cells in acute pancreatitis by down-regulating miR-345-5p

Objective To study the effects of polygonatum sibiricum polysaccharide(PSP)on the oxidative stress and inflammatory factors expression in caerulein-induced acute pancreatitis(AP)acinar cells and relevant mechanisms.Methods Rat pancreatic acinar cells(AR42J)were treated with 100 nmol/L caerulein for 6 h to establish a model of acinar cell injury in AP.Caerulein-induced AP cells were incubated with different concentrations(1,2,4 mg/mL)of PSP.Malonaldehyde(MDA)content,superoxide dismutase(SOD)and glutathion peroxidase(GPx)activities in the cell,and the levels of intedeukin-6(IL-6),tumor necrosis factorα(TNF-α)and IL-1βin the culture medium were evaluated by commercial kits.The expression level of miR-345-5p was measured by real-time quantitative PCR(RT-qPCR).AR42J cells were transfected with miR-345-5p inhibitors.The effect of down-regulating miR-345-5p on caerulein-induced oxidative stress and inflammatory factor expression in AP cells was examined by the above methods.The miR-345-5p mimics was transfected into AP cells,and the effects of upregulation of miR-345-5p and PSP treatment on oxidative stress and inflammatory cytokine expression was examined by the above methods.For data with homogeneity of variance,t test was used for comparison between two groups,analysis of variance for comparison between multiple groups,and LSD-t test for further pair comparison.Results Compared with control,MDA content,the levels of IL-6,TNF-α,IL-1βand miR-345-5p were increased in AP cells,whereas SOD and GPx activities were decreased.The MDA content(1.08±0.07)v s(0.88±0.06),(0.73±0.06),(0.60±0.05)nmol/mg]in AP cells was significantly reduced after PSP treatment,while the activities of SOD(43.01±4.37)vs(59.60±5.62),(72.37±6.32),(94.21±8.70)U/mg]and GPx(29.03±2.51)vs(44.11±4.71),(58.07±4.20),(72.67±6.56)U/mg]were significantly increased.In addition,the expression levels of IL-6(310.72±22.27)vs(257.01±20.85),(192.28±17.70),(146.93±11.90)pg/mL],TNF-α(310.72±22.27)vs(257.01±20.85),(192.28±17.70),(146.93±11.90)pg/mL]and IL-1β(41.66±3.85)vs(33.82±3.20),(26.15±2.56),(20.14±1.71)pg/mL]in the culture medium and the expression level of miR-345-5p(2.78±0.24 vs 2.38±0.21,1.91±0.12,1.25±0.13)were significantly reduced after PSP treatment in a dose-dependent manner(all P<0.05).After down-regulating the expression of miR-345-5p,the MDA content(1.13±0.08)vs(0.72±0.06)nmol/mg]in AP cells was significantly reduced,the activities of SOD(41.31±3.98)vs(81.73±7.62)U/mg]and GPx(28.82±2.97)vs(61.41±5.81)U/mg]were significantly increased,and the levels of IL-6(314.65±25.02)vs(159.76±11.93)pg/mL],TNF-α(235.18±23.13)vs(100.41±8.09)pg/mL]and IL-1β(48.67±4.50)vs(27.73±2.54)pg/mL]in the culture medium were significantly reduced,with statistically significant differences(all P<0.05).Up-regulating miR-345-5p expression reduced the above impacts of PSP on AP cells,with an increase of expressions of MDA content(0.58±0.03)vs(0.95±0.08)nmol/mg],decreased SOD(96.52±9.54)vs(54.24±4.15)U/mg]and GPx(79.62±6.23)vs(39.81±3.84)U/mg]activity,as well as increased expression levels of IL-6(145.38±12.49]vs(275.38±21.55)pg/mL],TNF-α(84.83±7.81)vs(183.73±16.39)pg/mL]and IL-1β(19.38±1.85)vs(36.97±3.62)pg/mL]in culture medium(P<0.05).Conclusion PSP can alleviate caerulein-induced acinar cells'inflammatory response and oxidative stress injury in acute pancreatitis in a dose-dependent manner,which is related to the down-regulation of miR-345-5p expression.