Mechanism of intestinal flora in process of knee osteoarthritis in mice alleviated by Jiawei Duhuo Jisheng Mixture / 中国药理学通报

Aim To investigate the potential mechanism of Jiawei Duhuo Jisheng Mixture regulating intestinal flora in the treatment of knee osteoarthritis(KOA)by 16S rRNA sequencing.Methods Eight-week-old male C57 mice were randomly divided into three groups:sham group,DMM group,and model+Jiawei Duhuo Jisheng Mixture group(Mixture group),6 mice per group.KOA model was induced by destabilization of medial meniscus surgery.16.25 mL·kg-1 dose mixture was given daily to the mixture group,and normal saline was given to the sham and DMM group.After eight weeks,the knee joints and colons of mice were collected,and the knee joints were prepared into paraffin sections,and the cartilage changes were observed with Safranin O-Fast Green and immunohistochemistry staining.16S rRNA sequencing of intestinal contents was performed to observe the changes of intestinal flora.Results Compared with model group,Jiawei Duhuo Jisheng Mixture could significantly reduce cartilage wear and OARSI score(P=0.033 5,P=0.029 5).16S rRNA sequencing showed that Jiawei Duhuo Jisheng Mixtrue could change the intestinal flora richness of KOA model mice,and improve the Alpha diversity(Chao1,Simpson)and Beta diversity(PCoA,NMDS).LefSe analysis showed that there were species with significant difference in abundance among the three groups(P=0.001),mainly including Lactobacillus,Firmicutes,Proteobacteria and other species.MetaCyc analysis indicated that Jiawei Duhuo Jisheng Mixture had effects on various metabolic pathways such as fatty acid,sugar and amino acid of intestinal flora(P<0.05).Conclusions Jiawei Duhuo Jisheng Mixtrue can effectively protect the articular cartilage and delay the progression of KOA.The mechanism may be through regulating the intestinal flora structure,protecting the intestinal barrier and reducing the inflammatory response.

Spatial-temporal distribution of glycogen synthase kinase 3beta and adenomatous polyposis coli protein are involved in the injury and repair of airway epithelial cells induced by scratching / 生理学报

To investigate the roles of glycogen synthase kinase 3beta (GSK3beta) and adenomatous polyposis coli (APC) protein in wound repair of airway epithelial cells (AECs), we established a wound model of airway epithelium in vitro. Then the following tests were undertaken: (1) Western blot was used to detect the levels of total GSK3beta and phosphorylated GSK3beta in human bronchial epithelial (16HBE) cells; (2) The localizations of APC protein was observed by using immunofluorescence technique; (3) Immunoprecipitation was used to investigate the relationship between APC protein and GSK3beta during the repair of 16HBE cells. The results were as follows: (1) The level of phosphorylated GSK3beta increased 0.5 h after scratching (P<0.05), reached a maximum at 6 h (P<0.05), and maintained until 12 h, while the total level of GSK3beta remained constant; (2) Results of immunofluorescence study showed that APC protein clustered with tubulin in the region of the migrating leading cells 6 h after scratching, which was dissimilar with that in the cells 0 h after scratching; (3) GSK3beta and APC protein were immunoprecipitated and analysed on SDS-PAGE. We found that GSK3beta and APC protein were precipitated, indicating that the two proteins existed in a complex. After scratching, dissociation of the two proteins occurred. Taken together, we conclude that scratching caused a decrease in phosphorylation of GSK3beta, and that reduced phosphorylation of GSK3beta promoted APC protein to bind to the plus ends of microtubules and stabilize the growing ends. These observations suggest that APC protein and GSK3beta may synergistically play an important role in the repair of airway epithelium.

Glycogen synthase kinase 3beta induces cell cycle arrest in a cyclin D1-dependent manner in human lung adenocarcinoma cell line A549 / 生理学报

The effect of glycogen synthase kinase 3beta (GSK3beta) has been repeatedly implicated in cell proliferation, but studies on the effect of GSK3beta in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3beta on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3beta mutant plasmids, constitutively active form S9A-GSK3beta and dominant negative form KM-GSK3beta. Twenty-four hours later, cell counting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3beta activity caused a decrease in cell number, as well as a higher percentage of cells at G(1) phase. Further, the expression of cyclin D1 was down-regulated by GSK3beta. Taken together, our observations suggest that GSK3beta may induce G(1) cell cycle arrest in a cyclin D1-dependent fashion and therefore possibly plays a growth-inhibitory role in A549 cells.

Dynamic changes of adenomatous polyposis coli protein and glycogen synthase kinase 3beta in the repair of the injured airway epithelial cells in smoking mice / 生理学报

To investigate the roles of adenomatous polyposis coli (APC) protein and glycogen synthase kinase 3beta (GSK3beta) of smoking murine model in the repair of the injured airway epithelial cells (AECs) in different stages, 30 male Kun-Ming mice were randomly divided into two groups, the control group and the smoking group. There were 24 mice in smoking group, and 6 animals were separately killed at the end of the 1st, 4th, 8th and 12th week after smoking. Then the following tests were undertaken: (1) HE staining of lung section to observe the morphological changes of the bronchi in the smoking mice. (2) Immunohistochemical staining of APC protein and GSK3beta in the AECs. (3) Western blot was used to detect the levels of APC protein, GSK3beta and phosphorated GSK3beta (p-GSK3beta) in pulmonary tissue. (4) Observing the localizations of APC protein and GSK3beta in the AECs by immunofluorescence technique. The results showed: (1) AECs showed changes of predominant injury (1-, 4-week), repair (8-week) and reinjury (12-week) along with smoking time prolonged. The experimental results indicated that the model of smoking mice was duplicated successfully. (2) Immunohistochemical results showed that the expression of APC protein in the AECs increased after 1-week smoking (0.458 +/- 0.062 vs 0.399 +/- 0.060, P< 0.05 vs control), but was significantly decreased at the end of the 4th week (0.339+/- 0.056, P<0.01 vs control) and increased at the end of the 8th and 12th week (0.387 +/- 0.041, 0.378 +/- 0.037, P<0.05 vs 4-week). The expression of GSK3beta in the AECs of smoking mice obviously decreased (P<0.01 or P<0.05 vs control). (3) Western blot showed that the expressions of APC protein and GSK3beta in lung tissue were consistent with the results of immunohistochemistry; and the levels of p-GSK3beta in all smoking models were higher than that in control. (4) The results of immunofluorescence showed that APC protein was localized mainly near the regions of epithelial cell membrane at the end of the 1st and 8th week after smoking, which were dissimilar with the localization in control, and this change was not seen in the location of GSK3beta. Taken together, these results demonstrate that the expressions and localizations of APC protein, GSK3beta and the activity of GSK3beta are dynamically changed in the AECs with experimental smoking injury at different phases, suggesting that APC protein and GSK3beta may be involved in the regulation of migration and proliferation of AECs, and play an important role in the process of repair of airway epithelium injury.

Clinical pathological feature of early tongue amyloidosis / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the clinical pathological feature and diagnostic criteria of tongue amyloidosis (AL).</p><p><b>METHODS</b>During 1992 to 2005, 25 patients pathologically diagnosed as tongue AL in our hospital were reviewed retrospectively, and all of them had no enlarged tongue. Haematoxylin and eosin (HE) and immunohistochemical staining were used to detect the amyloid deposition on the tongue.</p><p><b>RESULTS</b>Totally 84% (21/25) patients had symptoms of xerostomia and taste-blindness, 44% (11/25) patients complained of activity limitation of tongue. Macroscopic observation showed mucosa pallescence, punctuate hemorrhage, red grain particles, and ulcers on the tongue. HE staining indicated amyloid depositions in basement membrane, muscle cell, vessel wall, and nerve fiber. Immunohistochemical study demonstrated kappa light-chain deposition in 64% (16/25) cases, and lambda light-chain deposition in 36% (9/25) cases. They presented in vessel wall, nerve fiber, and muscle cells.</p><p><b>CONCLUSION</b>The biopsy is an important means for the diagnosis of early tongue AL, and the wide variety of amyloid light chain is helpful to differential diagnosis.</p>