OBJECTIVE: To observe the effect of acupuncture on chondrocyte autophagy in rats of knee osteoarthritis (KOA) and explore its underlying mechanisms. METHODS: Forty male SPF-grade SD rats were randomized into a blank group, a model group, a suspension group, an acupuncture group, and a combined therapy group, 8 rats in each one. Except the blank group, KOA model was prepared by the injection with papain. The suspension exercise therapy (10 min each time, three times daily), acupuncture (at "Yanglingquan" [GB34], "Zusanli" [ST36], and "Dubi" [ST35] on the right side, 30 min each intervention, once daily) and the combined therapy (the suspension exercise therapy combined with acupuncture) were delivered in the suspension group, the acupuncture group and the combined therapy group, respectively. The intervention of each group was performed continuously for 6 days, and 4 consecutive weeks, at the interval of 1 day. Before and after intervention, Lequesne MG score was assessed in the rats. After intervention, HE staining was adopted to observe the cartilaginous tissue morphology of the right knee joints, and Mankin score was evaluated; the serum levels of interleukin (IL)-1β, IL-6, and tumor neurosis factor-α (TNF-α) were measured using ELISA; the real-time PCR was provided to determine the mRNA expression of collagen protein type Ⅱ(COL2), collagen protein type Ⅹ (COL10), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and autophagy-regulated protein (Beclin-1) in the cartilaginous tissue of the right knee joint; Western blot was employed to detect the protein expression of PI3K, phosphorylated PI3K (p-PI3K), Akt, phosphorylated Akt (p-Akt), mTOR, phosphorylated mTOR (p-mTOR) and Beclin-1 in the cartilaginous tissue of the right knee joint. RESULTS: Compared with the blank group, the rats in the model group showed the higher Lequesne MG score (P<0.01), thinner cartilage of the right knee, reduced chondrocytes and disordered arrangement, and higher Mankin score (P<0.01). Besides, in the model group, the serum levels of IL-1β, IL-6 and TNF-α were elevated (P<0.01), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 decreased (P<0.01), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR increased (P<0.01) in the cartilaginous tissue of the right knee joint. Compared with the model group, in the suspension group, the acupuncture group and the combined therapy group, the Lequesne MG scores were reduced (P<0.01), the cartilage of the right knee was thickened, the arrangement of chondrocytes was improved, and the Mankin scores were lower (P<0.01). Besides, in these intervention groups, the serum levels of IL-1β, IL-6 and TNF-α were reduced (P<0.01), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 increased (P<0.05, P<0.01), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR decreased (P<0.05, P<0.01) in the cartilaginous tissue of the right knee joint. When compared with the suspension group and the acupuncture group, in the combined therapy group, the Lequesne MG score was reduced (P<0.01), and the Mankin score was reduced (P<0.05, P<0.01). Besides, the serum levels of IL-1β, IL-6 and TNF-α were reduced (P<0.05), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 increased (P<0.05), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR decreased (P<0.05, P<0.01) in the cartilaginous tissue of the right knee joint. CONCLUSION Acupuncture can promote cartilage regeneration of knee joint and autophagy in KOA rats, alleviate inflammation, so as to retard cartilage degeneration, which may be possibly associated with the PI3K/Akt/mTOR signaling pathway.
Animals ; Male ; Acupuncture Therapy ; Proto-Oncogene Proteins c-akt/genetics* ; TOR Serine-Threonine Kinases/genetics* ; Osteoarthritis, Knee/physiopathology* ; Phosphatidylinositol 3-Kinases/genetics* ; Rats ; Signal Transduction ; Rats, Sprague-Dawley ; Chondrocytes/metabolism* ; Humans ; Autophagy ; Acupuncture Points

Objective:To investigate the promotion effect of 4,5,6,7-tetrabromobenzotriazole (TBB)on the apoptosis of human colon cancer SW480 cells,and to explore its possible mechanism.Methods:The human colon cancer SW480 cells at logarithmic growth phase were divided into control group (0 μmol·L-1 TBB)and experiment group (1,3,10,30,and 100 μmol·L-1 TBB).The viability of cells was measured by MTT assay;the apoptotic rate of the SW480 cells and the level of intracellular reactive oxygen species (ROS)were analyzed by Annexin Ⅴ-FITC/PI double staining and flow cytometry.The expression levels of anti-apoptotic proteins p-Akt and Bcl-2,and pro-apoptotic proteins Bad, pro-caspase-9 and cleaved-caspase-3 were detected by Western blotting method. Results:The MTT results showed that the viabilities of SW480 cells in experiment group were decreased in a dose-dependent manner,which were lower than that in control group (P < 0.05).The Annexin Ⅴ-FITC/PI double staining and flow cytometry results showed that the apoptotic rates of SW480 cells in experiment group (3,6,12, and 24 h)were significantly higher than that in control group (0 h)(P <0.05).The flow cytometry results showed the levels of ROS in SW480 cells after treated with TBB for 3,6,12 and 24 h were higher than that in 0 h group (P <0.05).The Western blotting results showed that the expression levels of anti-apoptotic proteins p-Akt and Bcl-2 in SW480 cells in experiment group (3,6,12,and 24 h)were decreased obviously,whereas the expression levels of the pro-apoptotic proteins Bad and cleaved-caspase-3 were increased and the expression level of pro-caspase-9 was decreased compared with those in control group (0 h) (P < 0.05 ).Conclusion: TBB could inhibit the cell proliferation and induce the apoptosis of human colon cancer SW480 cells,and its mechanism may be related to the inhibition of the activity of Akt and the promotion of the level of intracellular ROS.

This study was aimed to investigate the efficacy ofTong-Bu San-Sheng(TBSS) Decoction to reduce the toxicity and side effects of chemotherapy, as well as prolong progression-free survival (PFS) for advanced lung squamous carcinoma patients who received chemotherapy. A total of 83 lung squamous carcinoma cases were divided into two groups by patients’ wishes. The control group contained 41 cases were treated by the chemotherapy of gemcitabine plus cisplatin (GP). The trial group contained 42 cases were treated by chemotherapy plus Chinese herbal medicine TBSS decoction. The toxicity and side effects of chemotherapy, as well as short-term outcome were evaluated. PFS of patient was recorded. The results showed that there were no differences on granulocytopenia (P = 0.115) or short-term outcome (P = 0.081) for patients of both groups after chemotherapy. The percentages of nausea, vomiting and thrombocytopenia in the trial group were lower than that in the control group (P = 0.037,P = 0.040). The PFS of patients in the trail group were prolonged compared to patients in the control group (4.31 ± 0.24 VS 3.78 ± 0.16 month;P = 0.043). It was concluded that Chinese herbal medicine TBSS decoction cannot reduce granulocytopenia caused by chemotherapy, or improve the tumor response rate (RR) of short-term outcome. However, it can prolong PFS, relieve nausea, vomiting and thrombocytopenia during chemotherapy.

10.3969/j.issn.1008-9691.2013.03.009