The quality and flavor of Liuyang Douchi are usually closely related to the metabolites compostion. This work described the metabolic profiles of Liuyang douchi during fermentation. Obvious hydrolysis of carbohydrates, proteins and slight lipids degradation were observed. Notably, the qu-making and pile-fermentation stage of douchi could be easily distinguished according to their metabolites profile, and pile-fermentation stage showed the most abundant metabolites. Specifically, organic acid, such as succinic acid and lactic acid, accumulated during pile-fermentation, as well as amino acids and derivatives. Especially glutamate (Glu), which contributed to the umami taste, increased form 0.82 mg/g to 15.90 mg/g after fermentation. Meanwhile, metabolisms related to amino acids were also the main enrichment metabolic pathways. Among them, some flavor compunds such as phenylacetaldehyde might drived from phenylalanine metabolism. These results could provide a new understanding on the metabolic characteristics during Liuyang douchi fermentation.
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To investigate whether mechanical stretch stimulation affects the expression of the immediate early gene c-fos mRNA in rat Achilles-derived tendon stem cells (TSCs) in vitro.TSCs were isolated from the Achilles tendons of 8 weeks old male Sprague Dawley rats by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated by a uniaxial cyclic stretching loading system under the condition of 1 Hz, respectively with 4% or 8% stretch intensity for 0, 5, 15, 30, 60, and 120 minutes. At each time point, TSCs were collected to detect c-fos mRNA expressions and to find the best time-point T max by real-time fluorescence quantitative PCR. Then, TSCs were simulated with 2%, 4%, 6%, 8%, or 12% stretch intensity for T max to observe the relative expressions of c-fos mRNA under different stretch intensities. Next, TSCs were stretched for 0, 5, or 15 minutes respectively and followed by incubation at relax status up to T max to observe the changes of c-fos mRNA expressions after short period stimulation. Finally, TSCs were stimulated with 4% or 8% stretch intensity respectively for 0, T max, or 120 minutes to detect the expressions of the tenogenic differentiation related genes [collagen type I, tenomodulin (TNMD)], the osteogenic differentiation related genes [runt related transcription factor 2 (Runx2), distal-less homeobox 5 (Dlx5)], and the adipogenic differentiation related gene [fatty acid binding protein 4 (FABP4)].Under 4% or 8% stretch intensity, the relative expressions of c-fos mRNA significantly increased at 15 minutes ( P<0.05), reached the maximum at 30 minutes ( P<0.05), and returned to baseline at 60 minutes ( P>0.05) when compared with expression at 0 minute. Therefore, T max was 30 minutes. The stretch intensity of 2% was enough to cause the expression of c-fos mRNA at 30 minutes, and the expression was significantly higher under the stretch intensity of 6%, 8%, and 12% than 2% and 4% ( P<0.05). Even for a short period stimulation of 5 minutes, c-fos mRNA expression could still significantly increase at 30 minutes ( P<0.05). The relative expressions of differentiation related genes at 30 and 120 minutes showed no significant difference when compared with the expression at 0 minute under 4% stretch intensity ( P>0.05); but the relative expression of Runx2 gene significantly increased at 30 minutes, and the relative expressions of collagen type I, TNMD, Dlx5, and Runx2 increased at 120 minutes under 8% stretch intensity ( P<0.05).Mechanical stretch stimulation can affect the relative expression of the immediate early gene c-fos mRNA of rat Achilles-derived tendon stem cells in vitro, and there is time- and intensity-dependence. It is suggested that the mechanical stimulation with different time or intensity may affect the differentiation of TSCs at early stage. This study is meaningful for the further study on TSCs intracellular mechanical signal transfer mechanism.探讨机械刺激对大鼠跟腱来源肌腱干细胞（tendon stem cells，TSCs）立早基因 c-fos 表达的影响。.取 8 周龄雄性 SD 大鼠跟腱组织，用酶消化法分离、培养 TSCs，取第 3 代细胞用于实验。利用单拉循环牵伸载荷模型在1 Hz条件下，分别用 4% 和 8% 牵拉强度牵拉细胞，并在牵拉 0、5、15、30、60、120 min 后收集细胞进行实时荧光定量 PCR 检测，观察 c-fos mRNA 相对表达量随牵拉时间的变化，并找到表达最高时间点T max。然后分别用 2%、4%、6%、8% 和 12% 的牵拉强度，在牵拉 T max 时收集细胞，观察c-fos mRNA相对表达量随牵拉强度的变化。接着分别用 2%、4%、6%、8% 和 12% 的牵拉强度，对 TSCs 经 0、5、15 min 短时间牵拉后静置至 T max，观察 c-fos mRNA 相对表达量经短时刺激后的变化情况。最后分别用 4% 和 8% 牵拉强度牵拉细胞 0、T max、120 min，检测成肌腱分化标志基因Ⅰ型胶原、腱调蛋白（tenomodulin，TNMD），成骨分化标志基因 Runx2、远端缺失基因 5（distal-less homeobox 5，Dlx5），成脂肪分化标志基因脂肪酸结合蛋白4（fatty acid binding protein 4，FABP4）的表达情况。.与 0 min 相比，在 4% 和 8% 牵拉强度下，c-fos mRNA 相对表达量在牵拉 15 min 时显著升高（ P<0.05），30 min 时出现峰值（ P<0.05），至 60 min 时表达量恢复至起始水平（ P>0.05）；故T max为 30 min。牵拉 30 min 时，2% 的牵拉强度即可使 c-fos mRNA 相对表达量升高，且 6%、8% 和 12% 牵拉强度较 2%、4% 牵拉强度进一步升高，差异均有统计学意义（ P<0.05）。经过 5 min 的短时间牵拉，并静置至 30 min 时即可使 c-fos mRNA 相对表达量升高（ P<0.05）。4% 牵拉强度下，在牵拉 30 min 和 120 min 时，各分化标志基因相对表达量与 0 min 比较差异均无统计学意义（ P>0.05）；而 8% 牵拉强度下，在牵拉 30 min 时，Runx2 基因相对表达量显著升高，在牵拉 120 min 时，Ⅰ型胶原、TNMD、Dlx5、Runx2 等基因相对表达量均升高，差异均有统计学意义（ P<0.05）。.循环牵拉能够影响大鼠跟腱来源 TSCs 立早基因 c-fos 的 mRNA 表达，并且呈时间和强度依赖性；提示机械刺激的时间和强度可能在作用早期即对 TSCs 的分化产生影响，对进一步研究 TSCs 机械-细胞内信号耦联机制具有重要意义。.
To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation.TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot.The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch ( P<0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch ( P<0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch ( P<0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch ( P<0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch ( P<0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E ( P<0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch ( P<0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E ( P<0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E ( P>0.05).The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.探讨不同机械牵伸条件对肌腱干细胞（tendon stem cells，TSCs）分化的影响，寻求 TSCs 成肌腱分化、成骨分化以及成脂肪分化的最佳单轴循环牵伸载荷。.取 8 周龄雄性 SD 大鼠跟腱，采用酶消化法分离培养 TSCs。取第 3 代 TSCs，随机分为不同牵拉条件组（实验组 A～D 组）及静态培养组（对照组 E 组），其中 A 组牵拉强度 4%、频率 1 Hz，B 组牵拉强度 4%、频率 2 Hz，C 组牵拉强度 8%、频率 1 Hz，D 组牵拉强度 8%、频率 2 Hz。利用课题组自行研发的体外细胞单轴循环牵拉设备，沿培养皿长轴对 A～D 组细胞进行单轴循环机械牵伸，E 组细胞行静态培养。分别处理 12、24、48 h 后收集各组细胞，采用实时荧光定量 PCR 检测成腱分化相关基因 Scleraxis（SCX）、抗细胞黏合素 C（Tenascin C，TNC），成脂肪分化相关基因 CCAAT/增强子结合蛋白-α（CCAAT-enhancer-binding protein-α，CEBPα）、脂蛋白脂肪酶（lipoprteinlipase，LPL）及成骨分化相关基因 RUNX2、远端缺失基因 5（distal-less homeobox 5，DLX5）的表达；Western blot 检测 TNC、CEBPα 及 RUNX2 蛋白表达。.实时荧光定量 PCR 检测示：SCX、TNC mRNA 相对表达量在 B 组牵拉 24 h 时显著高于其余各组，差异有统计学意义（ P<0.05）；CEBPα、LPL mRNA 相对表达量在 D 组牵拉 48 h 时显著高于其余各组，差异有统计学意义（ P<0.05）；RUNX2、DLX5 mRNA 相对表达量在 C 组牵拉 24 h 时显著高于其余各组，差异有统计学意义（ P<0.05）。Western blot 检测示：B 组牵拉各时间点 TNC 蛋白表达均高于 E 组（ P<0.05），同时牵拉 24 h 与 E 组相比 CEBPα 表达有显著抑制作用（ P<0.05）；C 组牵拉 24 h RUNX2 蛋白表达显著高于 E 组（ P<0.05），同时牵拉 24、48 h TNC 蛋白表达显著低于 E 组（ P<0.05）；D 组牵拉 48 h CEBPα 蛋白表达显著高于 E 组（ P<0.05），TNC 蛋白表达显著低于 E 组（ P<0.05），RUNX2 蛋白表达与 E 组比较差异无统计学意义（ P>0.05）。.机械力学刺激可以促进 TSCs 发生分化，而且不同条件的牵拉载荷会引起不同方向分化。4%、2 Hz 牵拉 24 h 为成腱分化最佳条件，8%、1 Hz 牵拉 24 h 为成骨分化最佳条件，8%、2 Hz 牵拉 48 h 为成脂肪分化最佳条件。.
Citri Reticulatae Pericarpium (CRP) is a kind of important traditional Chinese medicine and food. Sulfur-fumigation may induce chemical transformation of CRPs with no difference in appearance, leading to harmful effects on human health. In this paper, an accurate and nondestructive detection method of sulfur-fumigated CRPs was established based on portable near-infrared spectroscopy (NIRS) and chemometrics. 389 normal CRPs and 350 sulfur-fumigated CRPs were collected, while the spectra of outer skin and inner capsule were obtained directly without destroying the samples. A new variable selection-linear discriminant analysis (LDA) and other existing four LDA based on pattern recognition methods were used to establish the identification models of sulfur-fumigated CRPs combined spectral pretreatment methods. The models were evaluated by using the validation set and the external validation set collected one month later. The results show that the identification accuracies of outer skin are better than those of inner capsule. The second-order derivative (2nd)-competitive adaptive reweighted sampling (CARS)-LDA model has the best identification ability with 98.92% accuracy for validation set and 99.46% accuracy for external validation set. Besides, the randomization test (RT)-LDA method developed in this paper combines the advantages of high robustness and accuracy.
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