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

Background and aims:Primary sclerosing cholangitis(PSC)is an autoimmune liver disease characterized by complex pathogenesis and limited available therapeutic options.The mechanisms underlying the development and progression of PSCs remain unclear.Liver X receptor beta(LXR-β)is recognized to modulate lipid metabolism and immune response,but its specific involvement in the PSC has not been elucidated.Here,we explored the role and mechanism of LXR-β in PSC induced by 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-collidine(DDC).Methods:CRISPR-Cas9 technology was applied to generate Abcb4(coding MDR2,next named as Mdr2),Nr1h2(coding LXR-β,next named as Lxrβ),and Rag2(coding RAG2)knockout mice.DDC was used to induce PSC.Hematoxylin and eosin and Sirius red staining were used to assess the extent of hepatic injury and fibrosis.Flow cytometry was used to observe immune cell subsets.Results:We observed a declining trend in hepatic Lxrβ in the PSC model.Unexpectedly,Lxrβ knockout failed to modulate DDC-induced PSC pathogenesis.Concomitantly,assessment of the influence of Rag2 deficiency on PSC progression revealed the absence of aggravated or alleviated hepatic injury or fibrosis in the Rag2-/-DDC mice.However,Lxrβ depletion intensified DDC-induced PSC in the Rag2-/-mice,with more abundant infiltrative inflammatory cells and more severe liver fibrosis.Compared with Rag2-/-DDC mice,Lxrβ-/-Rag2-/-DDC mice had higher serum ALT and AST levels and mRNA expression of proinflammatory and profibrotic genes.Flow cytometry showed that LXR-β deficiency resulted in a diminished population of hepatic innate immune cells.Conclusion:This study indicated innate immune cell LXR-β deficiency can exacerbate hepatic injury and fibrosis in murine models of PSC suggesting that LXR-β may regulate the function of innate immunity in the fibrotic advancement of PSC.

The outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. Currently, there is no specific viral protein-targeted therapeutics. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein remains unclear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the -sheet core. Complemented by binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.