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Chinese Journal of Experimental Traditional Medical Formulae ; (24): 41-49, 2022.
Article in Chinese | WPRIM | ID: wpr-940286


ObjectiveTo observe the preventive and control effects of Danggui Niantongtang against adjuvant arthritis differentiated into wind-damp-heat impediment in rats and its influences on the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3), homolog of yeast Atg6 (Beclin1) and p62. MethodThe six-week-old male SD rats were randomly divided into the normal group, wind-damp-heat impediment model group, low-, medium-, and high-dose Danggui Niantongtang (5.67, 11.34, 22.68 g·kg-1) groups, and methotrexate (MTX, 1.35 mg·kg-1) group, with 10 rats in each group. A rat model of adjuvant arthritis was established by subcutaneous injection of inactivated Mycobacterium tuberculosis into the tail root, followed by exposure to the manual climatic box for 16 d for inducing the wind-damp-heat impediment. The drugs were administered intragastrically on the day of immunization for 28 d. The general conditions of rats were observed and the swelling degree of toes and arthritis index (AI) were detected. The pathological changes in the synovial tissues of the knee joints were observed by hematoxylin-eosin (HE) staining. The mRNA expression levels of LC3, Beclin1, and p62 in the synovial tissues were measured by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR), followed by the assay of their protein expression by Western blot and immunohistochemistry. ResultCompared with the normal group, the wind-damp-heat impediment model group exhibited significantly increased swelling degree of toes (P<0.01), increased AI (P<0.01), proliferated synovial cells (P<0.01), up-regulated LC3 and Beclin1 protein and mRNA expression (P<0.01), and down-regulated p62 protein and mRNA expression (P<0.01) after 16, 20, 24, 28-d medication. Compared with the wind-damp-heat impediment model group, each medication group displayed alleviated toe swelling and synovial hyperplasia to different degrees, decreased mRNA and protein expression levels of LC3 and Beclin1 (P<0.01), and increased p62 mRNA and protein expression (P<0.05,P<0.01), with the best outcomes observed in the medium-dose Danggui Niantongtang group. ConclusionDanggui Niantongtang effectively relieves adjuvant arthritis due to wind-damp-heat impediment in rats, which may be related to its regulation of the expression of autophagy-related proteins LC3, Beclin1, and p62 and the inhibition of autophagy.

Chinese Journal of Experimental Traditional Medical Formulae ; (24): 98-104, 2021.
Article in Chinese | WPRIM | ID: wpr-905069


Objective:To observe the effect of Qiyu Sanlong decoction (QYSL) on the expressions of key molecules in signal axis of mammalian rapamycin target protein (mTOR)/yeast Atg6 homologous (Beclin1)/ microtubule-associated protein1 light chain3 (LC3) in A549 cells. Method:With A549 cells as the research object, the effect of QYSL medicated serum on cell viability of A549 cells were detected by cell counting kit-8 (CCK-8) method. The effect of QYSL decoction on A549 cell apoptosis, autophagosome formation and the expression of autophagy markers were detected by Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method, transmission electron microscope (TEM), Real-time polymerase chain reaction (Real-time PCR) and Western blot. Result:QYSL medicated serum could inhibit the viability of A549 cells in a concentration-dependent manner. Compared with the blank serum group, the number of apoptotic A549 cells in the QYSL medicated serum group was significantly increased (P<0.01), and the formation of autophagosome was significantly increased. Compared with the blank serum group, the mRNA and protein expressions of mTOR in A549 cells in the QYSL serum group were significantly decreased (P<0.01), while mRNA and protein expressions of Beclin-1, autophagy related genes 5 (ATG5), autophagy related genes 13 (ATG13) were significantly increased (P<0.01). Conclusion:QYSL decoction can induce autophagy in A549 cells, and its specific mechanism may be related to the down-regulation of mTOR expression, the up-regulation of Beclin1, ATG5, ATG13 and LC3 expression, and the promotion of LC3Ⅰ conversion to LC3Ⅱ.

Chinese Journal of Experimental Traditional Medical Formulae ; (24): 29-35, 2020.
Article in Chinese | WPRIM | ID: wpr-873310


Objective::To study the effect of Qiyu Sanlong decoction on the growth of subcutaneous tumor in lung cancer mice and the expressions of key autophagy molecule, yeast Atg6 homologous (Beclin1), autophagy related genes5 (Atg5), and microtubule-associated protein1 light chain3 (LC3B). Method::Lewis lung carcinoma cells (LLC) were used to reproduce the lung cancer mice transplanted model. After the modeling, the mice were randomly divided into model group, Qiyu Sanlong decoction group, chemotherapy group and combination group, with 18 transplanted mice in each group. In model group, mice were fed with 0.9% saline 20 mL·kg-1 daily. In Qiyu Sanlong decoction group, mice were fed with Qiyu Sanlong decoction 80.48 g·kg-1 daily. The chemotherapy group was intraperitoneally injected with 0.4 mL cisplatin solution (DDP) at the 1st, 3rd and 5th day. The combination group was orally given the drugs at the concentration of 80.48 g·kg-1, and 0.4 mL DDP solution was intraperitoneally injected at the 1st, 3rd and 5th day. After 21 days of continuous treatment, tumor tissue was exfoliated and weighed, and the tumor inhibition rate was calculated. Hematoxylin-eosin (HE) staining was used to observe the histological changes of tumor. The expressions and localizations of Beclin1 and LC3B in tumor tissues were detected by immunohistochemical staining. Protein expressions of Beclin1, Atg5, LC3B-Ⅰand LC3B-Ⅱ were determined by Western blot, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated. The transcription levels of Beclin1, Atg5 mRNA in tumor tissues were detected by Real-time PCR. Result::Qiyu Sanlong decoction had a mild inhibitory effect on transplanted tumor, with an inhibitory rate of 31.2%. Under microscope, patchy necrotic tumor cells were observed in the tumor tissues of Qiyu Sanlong decoction group. Immunohistochemical staining and Western blot analysis showed that Qiyu Sanlong decoction could up-regulate the expressions of Beclin1, Atg5 and LC3B protein (P<0.01), and promote the conversion from LC3B-Ⅰ into LC3-Ⅱ compared with the model group. Real-time PCR results showed that Qiyu Sanlong decoction could promote the transcription of Beclin1 mRNA and Atg5 mRNA compared with the model group (P<0.01). Conclusion::Qiyu Sanlong decoction has a mild inhibitory effect on lung tumors, and its mechanism may be related to up-regulating the expressions of autophagy key proteins Beclin1, Atg5 and LC3B, and promoting the conversion from LC3B-Ⅰ to LC3B-Ⅱ.

Journal of Third Military Medical University ; (24)2003.
Article in Chinese | WPRIM | ID: wpr-566748


Objective In the heart,autophagy is important for the turnover of organelles at low basal levels under normal conditions,but is upregulated in response to stresses such as ischemia/reperfusion and in cardiovascular diseases such as heart failure. It can prevent the heart from injury under stress conditions. This study was designed to test the hypothesis that autophagy would be upregulated in the myocardium of children with cyanotic congenital cardiac defects. Methods Eighteen children with cyanotic (n=10) or acyanotic cardiac defects (n=8) who were admitted in our hospital from October 2008 to April 2009 and received surgical treatment were investigated. Samples from the right ventricular myocardium taken immediately after aortic clamping were morphologically studied with transmission electron microscopy for the ultrastructure. Microtubule-associated protein 1 light chain 3 (LC-3) was detected by Western blot analysis in the obtained samples. Results Children with cyanotic cardiac defects had higher oxyhemoglobin saturation before operation than those with acyanotic cardiac defects. Electron microscopy showed that the former group had more mitochondria,disordered arrangement,swollen endoplasmic reticulum and typical autophagosomes in the myocardial cytosol. LC3-II were significantly elevated in patients with cyanotic compared with those with acyanotic congenital cardiac defects (P