| ObjectiveThe research was observe the TLR-NF-kB signal pathway, to investigate the influencing mechanisms in the development of blood stasis syndrome(BSS) associated with hypertension disease, probe the pharmacological mechanism of APS and Pae treatment BSS associated with hypertension disease, confirmation vascular endothelial cells(VECs) injury model of BSS associated with hypertension disease.Methods1. Research of BSS associated with hypertension disease:(1).The human umbilical vein endothelial cells (HUVEC-Cs) were intervened by the 10% serum of the patient of BSS associated with hypertension disease for 24h to establish VECs injury model of BSS associated with hypertension disease(the blood stasis syndrome control group), the non-blood stasis syndrome control group,the normal control group and the blank control group were intervened by the 10% serum of the patient of non-blood stasis syndrome (NBSS) associated with hypertension disease,the 10% serum of the healthy human and non-serum medium separately. The activeness of the HUVEC-Cs was determined by MTT assay and the change of cells was observed by the inverse phase contrast microscope.(2).The HUVEC-Cs were effected by the 10% serum of the patient of BSS associated with hypertension disease and different dosage APS or Pae during the same time for 24h. The activeness of the HUVEC-Cs was determined by MTT assay and the change of cells was observed by the inverse phase contrast microscope.(3).The TLR4mRNA expression in HUVEC-Cs of the blood stasis syndrome control group,the non-blood stasis syndrome control group and the normal control group was detected by real time PCR.(4).The level of TLR4 expression(24h) and NF-kB activation(2h) in lipopolysaccharide(LPS) activated HUVEC-Cs were effected by different dosage APS or Pae during the same time. TLR4 mRNA and NF-kB mRNA were detected by real time PCR, TLR4 protein and NF-kB protein were detected by western blot. The NF-kB expression reflected with IkBα.2. Research of hypertension disease with different graduation:(1).The HUVEC-Cs of the normal control group,the grade 1 essential hypertension control group,the grade 2 essential hypertension control group,the grade 3 essential hypertension control group were intervened by the 10% serum of the healthy human,the patient of grade 1 essential hypertension disease,the patient of grade 2 essential hypertension disease,the patient of grade 3 essential hypertension disease separately for 24h. The activeness of the HUVEC-Cs was determined by MTT assay and the change of cells was observed by the inverse phase contrast microscope.(2).The TLR4 expression level in the HUVEC-Cs of the normal control group,the grade 1 essential hypertension control group,the grade 2 essential hypertension control group,the grade 3 essential hypertension control group was detected by real time PCR,Results1. After the serum acted on the HUVEC-Cs for 24h, the HUVEC-Cs activeness was reduced according to the order of the normal control group(0.3697±0.0548),the non-blood stasis syndrome control group(0.3196±0.0479),the blood stasis syndrome control group(0.2120±0.0317),the blank control group (0.1853±0.0129), there was a significant difference (P＜0.01 or P＜0.001); the HUVEC-Cs of the blood stasis syndrome control group shrank, the intercellular space increased, dark granules existed in the cytoplasm.2. APS and Pae had the protective function of lighten the HUVEC-Cs Shape change effected by the serum the patient of BSS associated with hypertension disease; The activeness of the HUVEC-Cs was increased on dose-dependent rule, The OD of each APS groups was: the normal control group(0.4419±0.0216),the blood stasis syndrome control group(0.2243±0.0298),the APS low dose group(0.2340±0.0376),the APS middle dose group(0.2656±0.0233),the APS high dose group(0.3487±0.0469), the difference is distinct(P<0.01 or P<0.001); he OD of each Pae groups was: the normal control group(0.5290±0.0946),the blood stasis syndrome control group(0.2888±0.0319),the Pae low dose group(0.3227±0.0371),the Pae middle dose group(0.3732±0.0736),the Pae high dose group(0.4510±0.0909), the difference is distinct(P<0.01 or P<0.001)。3. After the serum acted on the HUVEC-Cs for 24h, the HUVEC-Cs TLR4mRNA expression was increased according to the order of the normal control group(5.4160×10-4±0.2488×10-4),the non-blood stasis syndrome control group(6.4692×10-4±0.0956×10-4),the blood stasis syndrome control group(8.2598×10-4±0.2685×10-4), there was a significant difference(P<0.01).4. APS and Pae had the function of reduce the TLR4mRNA expression in LPS activated HUVEC-Cs on dose-dependent rule, The TLR4mRNA expression level of each groups was: the normal control group(5.4160×10-4±0.2488×10-4),the blood stasis syndrome control group (8.2598×10-4±0.2685×10-4),the LPS control group (97.3569×10-4±0.9599×10-4),the APS low dose group(33.4288×10-4±0.8112×10-4),the APS middle dose group(13.5707×10-4±0.6434×10-4),the APS high dose group(7.2484×10-4±0.0566×10-4),the Pae low dose group(10.7065×10-4±0.6657×10-4),the Pae middle dose group(7.1994×10-4±0.0939×10-4),the Pae high dose group(6.4403×10-4±0.1271×10-4).APS and Pae had the function of suppress the IκBmRNA degeneration in LPS activated HUVEC-Cs on dose-dependent, The NF-κBmRNA expression level of each groups was: the normal control group(0.4536±0.0164),the LPS control group (0.0665±0.0007),the APS low dose group(0.0873±0.0007),the APS middle dose group(0.1333±0.0005),the APS high dose group(0.1371±0.0004),the Pae low dose group(0.0973±0.0003),the Pae middle dose group(0.1357±0.0016),the Pae high dose group(0.1425±0.0012).5. APS and Pae had the function of reduce the TLR4 protein expression and suppress the IκB protein degeneration in LPS activated HUVEC-Cs.6. After the serum acted on the HUVEC-Cs for 24h, the HUVEC-Cs activeness was reduced according to the order of the normal control group(0.4609±0.0382),the grade 1 essential hypertension control group (0.4241±0.0270),the grade 2 essential hypertension control group(0.3330±0.0249),the grade 3 essential hypertension control group(0.2116±0.0222).There was a significant difference(P＜0.01) between the normal control group and the grade 2 essential hypertension control group,the grade 3 essential hypertension control group, but no significant difference(P > 0.05)between the normal control group and the grade 1 essential hypertension control group. The HUVEC-Cs of the grade 2 essential hypertension control group and the grade 3 essential hypertension control group shrank, the intercellular space increased, dark granules existed in the cytoplasm.7. After the serum acted on the HUVEC-Cs for 24h, the HUVEC-Cs TLR4mRNA expression was increased according to the order of the normal control group(5.4160×10-4±0.2488×10-4),the grade 2 essential hypertension control group (5.9333×10-4±0.3431×10-4),the grade 1 essential hypertension control group(8.0697×10-4±0.1249×10-4),the grade 3 essential hypertension control group (8.1314×10-4±0.0702×10-4).There was a significant difference(P<0.01) between the normal control group and the grade 1 essential hypertension control group,the grade 3 essential hypertension control group, but no significant difference(P > 0.05) between the normal control group and the grade 2 essential hypertension control group.Conclusions1. The VEC injury model of BSS associated with hypertension disease conforms to the morbidity factor of Traditional Chinese Medicine, explain the characteristic of "disease" and "syndrome" unify, and easy to duplicate, has the high rePaetability.2. APS and Pae can lighten the injury of the HUVEC-Cs that effected by the serum the patient of BSS associated with hypertension disease, increase cells activeness and maintenance the shape of the cells.3. Inflammatory reaction and immunity disorder initiated by TLR-NF-kB signal pathway is one of the influencing mechanisms in the development of blood stasis syndrome(BSS) associated with hypertension disease.4. APS and Pae can sterilization and adjustment immunity by reduce the TLR4 expression and suppress the IκBαdegeneration, it is possibly one of the pharmacological mechanism of APS and Pae with activating blood function.5. APS and Pae exert the protection function to HUVEC-Cs, sterilization and adjustment immunity by reduce the TLR4 expression and suppress the IkBαdegeneration.6. The TLR-NF-kB signal pathway initiates essential hypertension inflammatory reaction and immunity disorder, it possibly one of the influencing mechanisms of essential hypertension morbidity. |
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