Regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts / 中华烧伤杂志

Objective: To investigate the regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts (HSFbs). Methods: The experimental research methods were used. The 4th-6th passage of HSFbs from human skin were used for the following experiments. HSFbs were co-cultured with sodium ferulate at final mass concentrations of 1, 1×10-1, 1×10-2, 1×10-3, 1×10-4, 1×10-5, and 1×10-6 mg/mL for 48 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and linear regression was used to analyze the half lethal concentration (LC50) of sodium ferulate (n=6). HSFbs were co-cultured with sodium ferulate at final mass concentrations of 0.1, 0.2, 0.3, and 0.4 mg/mL for 24, 48, 72, and 96 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and the cell proliferation inhibition rate was calculated (n=3). According to the random number table, the cells were divided into 0.300 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, 0.003 mg/mL sodium ferulate group treated with sodium ferulate at corresponding final mass concentrations, and negative control group without any treatment. After 72 hours of culture, the cell absorbance values were determined by methyl thiazolyl tetrazolium method (n=5), the microscopic morphology of cells was observed by transmission electron microscope (n=3), the cell apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and the apoptosis index was calculated (n=4), the protein expressions of B lymphocystoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cysteine aspartic acid specific protease-3 (caspase-3) were determined by immunohistochemistry (n=4), and the protein expressions of transformed growth factor β1 (TGF-β1), phosphorylated Smad2/3, phosphorylated Smad4, and phosphorylated Smad7 were detected by Western blotting (n=4). Data were statistically analyzed with one-way analysis of variance and Dunnett test. Results: The LC50 of sodium ferulate was 0.307 5 mg/mL. After being cultured for 24-96 hours, the cell proliferation inhibition rates of cells treated with sodium ferulate at four different mass concentrations tended to increase at first but decrease later, which reached the highest after 72 hours of culture, so 72 hours was chosen as the processing time for the subsequent experiments. After 72 hours of culture, the cell absorbance values in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were 0.57±0.06, 0.53±0.04, 0.45±0.05, respectively, which were significantly lower than 0.69±0.06 in negative control group (P<0.01). After 72 hours of culture, compared with those in negative control group, the cells in the three groups treated with sodium ferulate showed varying degrees of nuclear pyknosis, fracture, or lysis, and chromatin loss. In the cytoplasm, mitochondria were swollen, the rough endoplasmic reticulum was expanded, and local vacuolation gradually appeared. After 72 hours of culture, compared with that in negative control group, the apoptosis indexes of cells were increased significantly in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group (P<0.05 or P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expressions of Bcl-2 of cells in 0.300 mg/mL sodium ferulate group was significantly decreased (P<0.01), the protein expressions of Bax of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.05), and the protein expression of caspase-3 of cells in 0.300 mg/mL sodium ferulate group was significantly increased (P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expression levels of TGF-β1, phosphorylated Smad2/3, and phosphorylated Smad4 of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly decreased (P<0.05 or P<0.01), and the protein expression levels of phosphorylated Smad7 of cells in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.01). Conclusions: Sodium ferulate can inhibit the proliferation of HSFbs of human skin and promote the apoptosis of HSFbs of human skin by blocking the expression of key proteins on the TGF-β/Smad signaling pathway and synergistically activating the mitochon- drial apoptosis pathway.

Synergy with the active ingredients of toad venom and sorafenib suppresses hepatocellular carcinoma HepG2 cells proliferation through down-regulating Akt/NF-κB signaling pathways / 中国药理学通报

Aim To investigate the effect of the active ingredients of toad venom (bufalin and cinobufagin) combined with sorafenib on the growth of hepatocellular carcinoma HepG2 cells,and to explore the possible mechanism.Methods The rates of inhibition after treated with drugs 12,24,48 h were detected by MTT assay.The changes of cell morphology were detected by Hoechst 33342 fluorescent staining.The changes of cell cycle were detected by flow cytometry.The expressions of proteins such as Akt,p-Akt (Ser473),IκB,NF-κB,p-NF-κB p65,Bcl-2,Bax,cyclin A,PCNA were detected by Western blot.Results Bufalin,cinobufagin and sorafenib could inhibit the proliferation of HepG2 cells,presenting a dose-and time-dependent manner.Meanwhile,it could significantly increase the inhibitory rate of cells compared with those of single treatment,and they performed a synergistic activity in sorafenib combined with cinobufagin or bufalin by Jin Formula after 24 h treatment (P ＜ 0.01).The results of fluorescence staining showed the observation of the morphological features of nuclear condensation.Sorafenib induced the cell cycle G0/G1 phase arrest (P ＜0.01),and bufalin,cinobufagin and the combination treatment generated the cell cycle S phase arrest (P ＜0.01).The results of Western blot showed that the expressions of Akt,NF-κB were not obviously changed between control and all other treatment.The expression levels of p-Akt (Ser473),p-NF-κB p65,Bcl-2,PC-NA and cyclin A in combination treatment significantly decreased,and the expression levels of IκB and Bax significantly increased compared to those in single treatment (P ＜ 0.01).Conclusion The active ingredients of toad venom (bufalin and cinobufagin) combined with sorafenib performs a synergetic effect on the anti-cancer of HepG2 cells by down-regulating Akt/ NF-κB signaling pathway.

Synchronous dynamic research on respiratory and intestinal microflora of chronic bronchitis rat model / 中国结合医学杂志

<p><b>OBJECTIVES</b>To investigate the mechanism of the Chinese medicine theory that Fei (Lung) and Dachang (Large Intestine) are exteriorly and interiorly related via synchronous observation on the dynamic changes of the respiratory and intestinal microflora.</p><p><b>METHODS</b>Forty specific pathogen free Sprague-Dawley rats were selected and randomly divided into blank (10 rats) and chronic bronchitis model groups (30 rats). The blank group rats were put into the smoke-free environment and the model group rats were put into the smoke environment in order to establish pulmonary disease (chronic bronchitis) model. Then the corresponding changes of the respiratory and intestinal microflflora of the model on 20th, 50th and 70th days were synchronously observed.</p><p><b>RESULTS</b>The respiratory tract microflflora showed an increase in the total aerobic and Staphylococcus aureus and reduced anaerobic amount signifificantly on 20th day in the respiratory tract microflflora (P<0.05 or 0.01). On 50th day, total aerobic, total anaerobic amount and bififidobacterium signifificantly increased (P<0.05). On 70th day, Staphylococcus aureus reduced and lactobacillus increased signifificantly (P<0.01). The intestinal microflflora showed an increase in the total aerobic, Clostridium perfringens, enterobacter and enterococcus significantly increased on 20th day (P<0.05 or 0.01). Staphylococcus aureus on 50th day increased significantly (P<0.05). Total aerobic and enterococcus increased, total anaerobic and Clostridium perfringens reduced signifificantly on 70th day (P<0.05 or 0.01).</p><p><b>CONCLUSION</b>The microecosystem of respiratory tract and intestine of rat model during the pathological process showed a dynamic disorder, indicating an interaction between the lung and large intestine which may be one of the connotations as they exteriorly and interiorly related.</p>

Changes of CCK-8, CGRP, SP, and VIP in the colon and the lung tissue of allergic asthma model rats: an experimental observation / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe changes of cholecystokinin octapeptide (CCK-8), calcitonin gene related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP) in each tissue of the digestive system of allergic asthma (AA) model rats.</p><p><b>METHODS</b>The pulmonary disease (AA) rat model was duplicated by 1% ovalbumin. Its effect on the pathological morphology of the six main parts of the digestive system (stomach, duodenum, jejunum, ileum, colon and rectum) and related regulating factors such as CCK8, CGRP, SP, and VIP were observed.</p><p><b>RESULTS</b>The pathological morphology of the lung was synchronously changed as that of the colon of model rats. But there was no obvious change in the stomach, duodenum, jejunum, ileum, or rectum. Significant changes occurred in CCK8 (79 961.4 +/- 12 577.9, 48 519.5 +/- 12 240.7), CGRP (41 950.1 +/- 12 600.1, 38 059.8 +/- 11 942.4), and SP (88 243.9 +/- 32 177.2, 47 417.8 +/- 16 462.4), and VIP (20 711.4 +/- 7 334.6, 43 208.1 +/- 13 433.8) of the lung tissue and the colon tissue of model rats (P < 0. 05, P < 0.01). But there was no significant change in the aforesaid substances of the stomach, duodenum, jejunum, ileum and rectum (P > 0.05).</p><p><b>CONCLUSIONS</b>Pulmonary disease might affect the colon, inducing pathological changes of the colon tissue and changes of related regulating factors such as CCK8, CGRP, SP, and VIP. It showed no significant effect on the stomach, duodenum, jejunum, ileum and rectum.</p>