BACKGROUND:Papain-induced rat knee osteoarthritis is a common modeling method, which can obtain a stable osteoarthritis model.
OBJECTIVE:To observe the change of ultrastructure of chondrocytes in the early process of papain-induced rat knee osteoarthritis under transmission electron microscope.
METHODS:A total of 18 Sprague-Dawley rats were randomly divided into three groups. Two rats were considered as a normal control group, without intervention. The mixture of papain and L-cysteine was injected in right knee joint cavity of 16 rats to induce osteoarthritis models (osteoarthritis model group). Physiological saline was injected in the left side (physiological saline control group). At 1, 2, 4 and 6 weeks after injection, samples were col ected. Transmission electron microscope was used to observe the change of cartilage ultrastructure of the medial femoral condyle joint.
RESULTS AND CONCLUSION:For the normal control group and physiological saline control group, their cytoplasm contained abundant rough endoplasmic reticulum and mitochondria. After 1 week of injection,
mitochondria vacuoles and light expanded rough endoplasmic reticulum were visible. Two weeks later, lipid droplets appeared, mitochondria degeneration was distinct, vacuolization was serious and its number was reduced, and rough endoplasmic reticulum expansion was obvious. Four weeks later, lipid droplets became increased, and the number of mitochondria decreased significantly. Most of the rough endoplasmic reticula were highly expanded, and part of the rough endoplasmic reticula were dissolved and fractured. Six weeks later, a number of lipid droplets were visible in cytoplasm, most of the mitochondria disappeared, only a smal number of mitochondria existed, and most of the rough endoplasmic reticula were dissolved and fractured. These results confirmed that cartilage ultrastructure changes gradual y in the early process of papain-induced rat knee osteoarthritis under transmission electron microscope.

Objective:To evaluate the relationship between methyltransferase-like 3(METTL3)-mediated RNA N6-Methyladenosine (m6A) methylation modification and silent information regulator factor 1 (SIRT1) during sevoflurane post-conditioning-induced mitigation of cognitive impairments in a mouse model of hemorrhagic shock and resuscitation(HSR).Methods:Forty clean-grade healthy male C57BL/6 mice, aged 8-10 weeks, with a body weight ranging from 22-26 g, were assigned into 5 groups ( n=8 each) using a random number table method: sham operation group, HSR group, sevoflurane post-conditioning + HSR group (SP+ HSR group), over-expression of METTL3 gene rAAV + sevoflurane post-conditioning + HSR group (METTL3+ SP+ HSR group), and over-expression of METTL3 gene rAAV negative control + sevoflurane post-conditioning + HSR group (NC+ SP+ HSR group). The HSR model was established by withdrawing 40% of the total blood volume from mice through the right carotid artery within 30 min, followed by reinfusion of the withdrawn blood over 30 min 1 h later. The SP+ HSR group underwent HSR modeling first and then inhaled sevoflurane (end-tidal concentration 2.4%) for 30 min starting from the time point immediately after blood transfusion. The Sham group and HSR group inhaled a mixture of 70% O 2 and 30% CO 2 for 30 min at the corresponding time points. In METTL3+ SP+ HSR group and NC+ SP+ HSR group, the corresponding virus 450 nl was injected into bilateral hippocampus at 4 weeks before establishing the model.Morris water maze and novel object recognition tests were conducted at 72 h after developing the model to assess the learning and memory abilities. After the end of behavioral tests, the expression of METTL3 and SIRT1 in hippocampal tissues was detected using Western blot, the expression of SIRT1 mRNA was measured using qRT-PCR, and the methylation of RNA m6A was detected using Dot blot. Results:Compared to Sham group, the escape latency was significantly prolonged at 1-6 days, the time spent in the target quadrant was shortened, the number of crossing the original platform was decreased, the novel object recognition index was decreased, the expression of METTL3 was up-regulated, the expression of SIRT1 protein and mRNA was down-regulated, and the methylation of RNA m6A was increased in HSR group( P<0.05). Compared to HSR group, the escape latency was significantly shortened at 1-6 days, the time spent in the target quadrant was prolonged, the number of crossing the original platform was increased, the novel object recognition index was increased, the expression of METTL3 was up-regulated, the expression of SIRT1 protein and mRNA was down-regulated, and the methylation of RNA m6A was increased, the novel object recognition index was increased, the expression of METTL3 was down-regulated, the expression of SIRT1 protein and mRNA was up-regulated, and the methylation of RNA m6A was decreased in SP+ HSR group( P<0.05). Compared to SP+ HSR group, the escape latency was significantly prolonged at 2-6 days, the time spent in the target quadrant was shortened, the number of crossing the original platform was decreased, the novel object recognition index was decreased, the expression of METTL3 was up-regulated, the expression of SIRT1 protein and mRNA was down-regulated, and the methylation of RNA m6A was increased in METTL3+ SP+ HSR group( P<0.05), and no significant change was found in the aforementioned indicators in NC+ SP+ HSR group ( P>0.05). Conclusions:The mechanism by which sevoflurane post-conditioning alleviates cognitive dysfunction is associated with down-regulation of METTL3 expression, reduction of RNA m6A methylation, and up-regulation of SIRT1 expression in HSR mice.

Objective:To evaluate the role of silent information regulator-1 (SIRT1)/nucleotide-binding domain (NOD)-like receptor protein-3 (NLRP3) signaling pathway in sevoflurane postconditioning-induced attenuation of oxygen-glucose deprivation and restoration (OGD/R) injury in mouse hippocampal neuronal cell line (HT22) cells.Methods:The HT22 cells were seeded in a culture plate (96-well plate, 100 μl/well; 6-well plate, 2 ml/well) at the density of 5×10 4 cells/ml or in a culture dish (6 cm in diameter) and then divided into 4 groups ( n=24 each) using a random number table method: control group (Control group), OGD/R group, sevoflurane postconditioning group (SPC group), and SIRT1 small interfering RNA group (si-SIRT 1 group). In Control group, cells were cultured at 37 ℃ in normal culture atmosphere. In OGD/R group, the culture medium was replaced with glucose-free serum-free culture medium, and cells were exposed to 95% N 2+ 5% CO 2 for 4 h in an incubator at 37 ℃, and then the glucose-free serum-free culture medium was replaced with the primary culture medium, and cells were cultured for 24 h at 37 ℃ in normal culture atmosphere. In SPC group, the glucose-free serum-free culture medium was replaced with the primary cell culture medium after 4-h oxygen and glucose deprivation, the cells were put into the hypoxia incubator chamber which was filled with 2% sevoflurane immediately after start of reoxygenation, then the chamber was placed in an incubator and the cells were cultured for 1 h at 37 ℃ in normal culture atmosphere, and finally the cells were removed from the chamber and cultured for 23 h at 37 ℃ in normal culture atmosphere. In si-SIRT1 group, SIRT1 small interfering RNA 150 pmol was added at 24 h before surgery, cells were then incubated, and the other procedures were the same as those previously described in group SPC. The cell survival rate was determined using MTT assay. TUNEL assay was used to detect cell apoptosis, and the apoptosis rate was calculated. The expression of SIRT1, NLRP3, IL-1β and IL-18 mRNA was determined using polymerase chain reaction. The expression of SIRT1, NLRP3, interleukin-1beta (IL-1β) and IL-18 was detected using Western blot. Results:Compared with Control group, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of SIRT1 protein and mRNA was down-regulated, and the expression of NLRP3, IL-1β and IL-18 protein and mRNA was up-regulated in OGD/R group ( P<0.05). Compared with OGD/R group, the cell survival rate was significantly increased, the apoptosis rate was decreased, the expression of SIRT1 protein and mRNA was up-regulated, and the expression of NLRP3, IL-1β and IL-18 protein and mRNA was down-regulated in SPC group ( P<0.05). Compared with SPC group, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of SIRT1 protein and mRNA was down-regulated, and the expression of NLRP3, IL-1β and IL-18 protein and mRNA was up-regulated in si-SIRT1 group ( P<0.05). Conclusions:Activation of SIRT1-NLRP3 signaling pathway is involved in sevoflurane postconditioning-induced attenuation of OGD/R injury in HT22 cells.