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OBJECTIVE To identify an d analyze chemical cons tituents of Pleione yunnanensis with origin of Pleione yunnanensis. METHODS UPLC-Q-Exactive-Plus-Orbitrap-MS was adopted. The determination was performed on Hyperdil GOLD column with mobile phase consisted of 0.1％ formic acid solution-0.1％ formic acid acetonitrile solution （gradient elution ）at the flow rate of 0.3 mL/min. The column temperature was set at 40 ℃，and sample size was 2 µL. The electrospray ion source was adopted，and the scanning range was m/z 100-1 500，and the scanning mode was positive and negative ion exchange mode of full scan+ddMS2. The structure of chemical constituents were determined by using Compound Discoverer 3.1 software，comparing with mzCloud，PubChem network database and OTCML ，on the basis of reference substance and published literatures. RESULTS & CONCLUSIONS A total of 42 chemical constituents were identified （positive ion mode has 24，negative ion mode has 27）， including 13 benzyl succinate glycosides （such as dactylorhin C ，coelovirin A ，militarine），4 phenol glycosides （such as adenosine ， guanosine，gastrodin），3 alkaloids（choline，betaine，berberine），and one flavonoid （nobiletin），7 aromatics（such as DL-lysine ， DL-arginine，DL-glutamine），one sugar （sucrose），3 benzenes（shancigusin H ，shancigusin H isomer ，batatasin Ⅲ）and 10 others （such as p-methoxybenzoic acid ，monomethyl dodecanedioate ，diphenylamine）. Glucose oxybenzyl and some small mole cules are easy to be lost in the cleavage of benzyl succinate glycosides；glycosyl is easy to be lost in the cleavage of phenol glycosides；the cleavage of alkaloids mainly manifest as the cleavage and loss of small molecular substituents ；demethyla- tion reaction is occurred in most flavonoids.
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Objective:To investigate the effects of miR-497 on cytobiology behaviors of papillary thyroid carcinoma (PTC) by targetingly regulating the expression of Yes-associated protein 1 (YAP1) .Methods:Human TPC-1 cells were divided into control group, miR-497 group, si-YAP1 group and miR-497+si-YAP1 group. The liposome transfection was conducted by LipofectamineTM3000. The targeted relationship between miR-497 and YAP1 was validated by Luciferase Reporter Assay. The cell proliferation activity in each group was detected by MTT method. The apoptosis rates were analyzed by flow cytometry. The number of invasion cells was detected by Transwell. The cell migration rates were detected by scratch assay. The expression of Cyclin D1, matrix metalloproteinase 2 (MMP-2) , MMP-9, matrix metalloproteinase inhibitor-1 (TIMP-1) and activated caspase 3 (cleaved Caspase-3) was detected by Western blot. SPSS 22.0 was used to analyze data, and normally distributed measurement data were expressed as ( ± s) . One-way ANOVA was analyzed for the difference between multiple groups, and SNK-q were analyzed for the difference between two groups. Results:Compared with the control group, the expression of YAP1 mRNA and protein was decreased in miR-497 group and si-YAP1 group ( q=14.682, 14.597; 23.743, 23.571; P<0.05) , cell proliferation activity, number of invasion cells and migration rate were decreased ( q=4.724, 4.568, 3.841; 4.216, 3.952, 3.274; P<0.05) , apoptosis rate was increased ( q=3.783; 4.336; P<0.05) , expression of CyclinD1, MMP-2, MMP-9 and cleared Caspase-3 proteins was decreased ( q=5.823, 5.981, 6.036, 6.485; 5.934, 6.110, 6.573, 6.614; P<0.05) , and expression of TIMP-1 protein was increased ( q=6.071; 6.148; P<0.05) . Compared with si-YAP1 group, miR-497 level was increased in miR-497+si-YAP1 group ( q=14.726, P<0.05) , the expression of YAP1 mRNA and protein was decreased ( q=3.089, 3.126; P<0.05) , cell proliferation activity, number of invasion cells and migration rate were decreased ( q=2.654, 2.537, 2.246; P<0.05) , apoptosis rate was increased ( q=2.875, P<0.05) , expression of CyclinD1, MMP-2, MMP-9 and cleared Caspase-3 proteins was decreased ( q=4.371, 4.365, 4.383, 4.368; P<0.05) , and expression of TIMP-1 protein was increased ( q=4.275, P<0.05) . Conclusion:MiR-497 can negatively targetingly regulate the expression of YAP1, inhibit proliferation, invasion and migration of TPC-1 cells.
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Objective To investigate any protective effect of low-intensity pulsed ultrasound (LIPUS) and pioglitazone on chondrocytes in osteoarthritic patients using the pathway from peroxisome proliferator-activated γreceptor (PPARγ) to nuclear factor kappa B (NF-κB) to inducible nitric oxide synthase (iNOS).Methods Normal chondrocytes of 24 healthy adult New Zealand white rabbits were extracted and divided into a normal group,a lipopolysaccharide (LPS) group,a LIPUS group (LPS+LIPUS) and a pioglitazone group (LPS+pioglitazone),each of 6 using a random number table.Each group was given the intervention their names implies.The levels of tumor necrosis factor-α (TNF-α),leptin (LEP) and nitric oxide (NO) in the chondrocytes were detected using enzyme-linked immune sorbent assays.The expression of type Ⅱ collagen (COL2) in the chondrocytes of each groups was detected using immunocytochemistry and fluorescent staining.The mRNA and protein expressions of PPARγ,NF-κB and iNOS were detected using reverse transcription polymerase chain reactions and western blotting respectively.Results Compared with the LPS group,the average level of TNF-α,LEP and NO in the LIPUS and pioglitazone groups was significantly lower,with the levels in the pioglitazone group significantly lower than in the LIPUS group.Compared with the LPS group,COL2 expression in the LIPUS group was significantly greater.The mRNA and protein expressions of PPARγ in the chondrocytes in the LIPUS and pioglitazone groups were significantly higher than those in the LPS group.Compared with the LPS group,the mRNA and protein expressions of NF-κB and iNOS in the pioglitazone and LIPUS groups were significantly lower,with the pioglitazone group's levels significantly below those of the LIPUS group.Conclusion LIPUS and pioglitazone may promote anti-inflammatory action and COL2 synthesis in chondrocytes through the PPARγ/ NF-κB/iNOS pathway and play a protective role,at least in rabbits.
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Objective To observe the effect of low intensity pulsed ultrasound (LIPUS) on chondrocytes co-cultured with infrapatellar fat pads.Methods Twenty-four infrapatellar fat pads and cartilages from female knee trauma patients aged between 25 and 35 were cut and stained using hematoxylin-eosin staining.Chondrocytes were isolated from part of the integrated surface of the cartilages to be cultured in vitro.They were then randomly divided into a normal chondrocyte group (the control group),a normal chondrocyte+FCM (fat conditioned medium) group (the model group),a normal chondrocyte+ FCM + LIP US group (the treatment group),and a normal chondrocyte+ FCM + LIPUS + GRGDSP (an integrin inhibitor) group (the inhibited group).The treatment group and inhibited group received LIPUS at 40 mW/cm2 for 20 min once a day,while the other groups received sham LIPUS treatment.Five days later,the cells were collected and western blotting was used to examine the expression of type Ⅱ collagen (COL2),aggrecan (Acan),matrixmetalloproteinase (MMP)-13,integrin β1,phosphorylation-focal adhesion kinase (p-FAK) and phosphorylation p38 (p-p38).Results Western blotting showed that compared with the control group,the expression of COL2 and Acan was significantly lower in the model group,but that of MMP-13,integrin β1,p-FAK and p-p38 was significantly higher.As compared with the model group,the expression of COL2,Acan,integrin β1 and p-FAK was significantly higher,and that of MMP-13 and p-p38 was significantly lower in the treatment group.The expression of COL2,Acan,MMP-13,integrin β1,p-FAK and p-p38 showed no significant difference between the inhibited and model groups,but that of COL2,Acan,MMP-13,integrin β1,p-FAK and p-p38 was significantly different between the control and treatment groups.Conclusions LIPUS provides a protective effect on chondrocytes through inhibiting the expression of MMP-13 induced by adipokines and the degradation of COL2 and Acan through activating the integrin-FAK-p38 MAPK pathway.
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Objective To observe any effect of low intensity pulsed ultrasound (LIPUS) on the expression of integrin-focal adhesion kinase (FAK)-mitogen-activated protein kinase (MAPK) mechanochemical transduction pathway-related proteins in the chondrocytes of rabbits with knee osteoarthritis (OA).Methods Of the 18 New Zealand white rabbits selected for the study,twelve received knee anterior cruciate ligament transection to model OA.The remaining 6 rabbits served as normal controls.At the 4th week after modeling the rabbits were sacrificed and chondrocytes were isolated and cultured in vitro.All the cultured cells were randomly divided into three groups:a normal control group (NC),an OA model group (OA) and an OA model plus LIPUS group (OA + LIPUS).When the cells had been cultured to the 2nd passage,the NC group and OA group cells had received no treatment.The OA + LIPUS group cells were exposed to 40 mW/cm2 of LIPUS for 20 min,once a day for 6 days.The expression of collagen protein type Ⅱ,aggrecan,MMP-13,integrin β1 p-FAK and p-p38,p-ERK1/2 and p-JNK Mapks were detected by Western blotting.Results Compared with the NC group,the expression of collagen type Ⅱ and aggrecan was significantly lower in the OA and OA + LIPUS groups,with more significantly lower expression of collagen type Ⅱ and aggrecan in the OA group than in the OA + LIPUS group.Compared with the NC group,the expression of MMP-13 was significantly higher in the OA and OA + LIPUS groups,with a significantly larger increase in the OA group.Compared with the NC group,the expression of integrin β1 and p-FAK was also significantly higher in the OA and OA + LIPUS groups,with a significantly larger increase in the OA + LIPUS group.Compared with the NC group,the expression of p-p38,p-ERK1/2 and p-JNK was also significantly higher in the OA group,but compared with the OA group,the expression of those kinases was,on average,significantly lower in the OA + LIPUS group.Conclusions LIPUS can inhibit the degradation of collagen type Ⅱ and aggrecan,and inhibit the expression of MMP-13,p-p38,p-ERK1/2 and p-JNK in OA chondrocytes,at least in vitro.At the same time,LIPUS can increase the expression of integrin β1 and p-FAK.The results show that LIPUS may activate an integrin-FAK-MAPK mechanochemical transduction pathway to induce changes in the extracellular matrix of chondrocytes.