Objective:To explore if there is premature senescence of chondrocytes in patients with Kashin-Beck disease (KBD) and osteoarthritis.Methods:Five knee cartilage samples of KBD, osteoarthritis and control groups were collected, respectively, from the Second Affiliated Hospital of Xi'an Jiaotong University. DNA was then extracted from cartilage samples and DNA methylation was analyzed by Illumina Infinium HumanMethylation450 BeadChip. At the same time, based on genome-wide methylation data, the online DNA methylation aging clock calculator (https://dnamage.genetics.ucla.edu/home) was used to calculate the DNA methylation age (DNAm age) of samples, and the results were compared with their actual ages.Results:In the comparison between KBD group and control group, 1 212 differentially methylated CpG sites were found, including 497 hypermethylated CpG sites and 715 hypomethylated CpG sites, corresponding to 264 hypermethylated genes and 368 hypomethylated genes, respectively. In the comparison between osteoarthritis group and control group, 656 differentially methylated CpG sites were found, including 343 hypermethylated CpG sites and 313 hypomethylated CpG sites, corresponding to 177 hypermethylated genes and 174 hypomethylated genes, respectively. In the above comparison, 367 overlapped CpG sites (corresponding to 182 genes) were found, which were differentially methylated in both KBD and control groups and osteoarthritis and control groups. The results of DNA methylation aging clock showed that the average age acceleration differences between DNAm age and actual age of KBD, osteoarthritis and control groups were 2.549, 0.017, and - 5.364 years, respectively, the DNAm ages of KBD and osteoarthritis groups were greater than the actual ages.Conclusion:The chondrocytes show premature senescence in both KBD and osteoarthritis.

Objective To evaluate the role of nuclear factor kappa B (NF-κB) in cognitive decline in aged mice with sepsis.Methods Forty-five SPF healthy aged female C57BL/6 mice,aged 10-12 months,weighing 20-30 g,were assigned into 3 groups (n=15 each) using a random number table:control group (group C),sepsis group (group Sep) and NF-κB selective inhibitor pyrrolidine dithiocarbamate (PDTC) group (group PDTC).Lipopolysaccharide 250 μg/kg was injected intraperitoneally once a day for 7 consecutive days in Sep and PDTC groups,and in addition PDTC 50 mg/kg was injected intraperitoneally at 30 min before lipopolysaccharide injection once a day for 7 consecutive days in group PDTC.The equal volume of normal saline was given in group C.Five mice in each group were sacrificed at 2 h after the last administration,cardiac puncture was performed and blood samples were collected,and then the mice were sacrificed and hippocampi were harvested for determination of the levels of tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β) and IL-6 in plasma and hippocampal tissues using enzyme-linked immunosorbent assay.Cognitive function was assessed using open field,elevated plus maze and Morris water maze tests at 24 h after the last administration in the other mice left in each group.Results Compared with group C,the levels of TNF-α,IL-1β and IL-6 in plasma and hippocampal tissues were significantly increased,the time of movement at the central region was shortened,the percentage of time spent in the open arms and number of entries into the open and closed arms were decreased,the escape latency was prolonged,the time of staying at the original platform quadrant was shortened,and the frequency of crossing the platform was decreased in group Sep (P＜0.05).Compared with group Sep,the levels of TNF-α,IL-1β5 and IL-6 in plasma and hippocampal tissues were significantly decreased,the time of movement at the central region was prolonged,the percentage of time spent in the open arms and number of entries into the open and closed arms were increased,the escape latency was shortened,and the time of staying at the original platform quadrant was prolonged in group PDTC (P＜0.05).Conclusion NF-κB is involved in cognitive decline in aged mice with sepsis.

Objective To evaluate the role of group Ⅱ metabotropic glutamate receptors (mGluRs) in cognitive decline caused by multiple administrations of ketamine in mice and the relationship with hippocampal glycogen synthase kinase-3 beta (GSK-3β) expression.Methods Forty-five SPF healthy female C57BL/6 mice,aged 6-8 weeks,weighing 20-30 g,were randomized into 3 groups (n=15 each) using a random number table method:control group (group C),ketamine group (group K) and mGluR agonist LY354740 group (group L+K).In K and L+K groups,ketamine 30 mg/kg was intraperitoneally injected three times a day at an 30-min interval for 14 consecutive days.LY354740 was intraperitoneally injected at 30 min before the first injection of ketamine in group L+K.The equal volume of normal saline was given instead in group C.Morris water maze test was performed the day after the last administration.The mice were then sacrificed,and hippocampi were harvested to determine the expression of GSK3β,NR2A and postsynaptic density protein 95 (PSD95) by Western blot.Results Compared with group C,the escape latency was significantly prolonged,the time of staying at the original platform quadrant was shortened,the frequency of crossing the original platform was decreased,the expression of GSK3β3 and NR2A was up-regulated,and the expression of PSD95 was down-regulated in group K (P＜0.05),and no significant change was found in the parameters mentioned above in group L+K (P＞0.05).Compared with group K,the escape latency was significantly shortened,the time of staying at the original platform quadrant was prolonged,the frequency of crossing the original platform was increased,the expression of GSK3β and NR2A was down-regulated,and the expression of PSD95 was up-regulated in group L+K (P＜0.05).Conclusion Group Ⅱ mGluRs are involved in the process of cognitive decline caused by multiple administrations of ketamine in mice,which is associated with up-regulated expression of hippocampal GSK-3β.

Objective:To investigate the impact of matrix metalloproteinase 13 (MMP13) and low-density lipoprotein receptor-related protein 1 (LRP1) on autophagy of articular chondrocytes in patients with Kashin-Beck disease (KBD).Methods:Human articular cartilage samples obtained from 4 KBD patients and 4 control subjects were collected from Shaanxi Institute for Endemic Disease Prevention and Control, and the expression levels of MMP13 and LRP1 in cartilage tissue were determined using immunohistochemistry (IHC). Chondrocytes were extracted and cultured in vitro, the mRNA and protein expression levels of LRP1 and the autophagy related genes [Beclin 1 (BECN1), microtubule associated protein 1 light chain 3 (LC3)], cartilage injury related genes [MMP13, caspase-3 (CASP3)], chondrocyte differentiation related genes [collagen type Ⅱ alpha 1 chain (COL2A1), and SRY-box transcription factor 9 (SOX9)] were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot (WB), respectively. Chondrocytes from 3 KBD patients were extracted, and MMP13 gene silencing experiment was performed by RNA interference (RNAi) technology, the mRNA and protein expression levels of the above genes were detected by qRT-PCR and WB, respectively. In addition, the antagonist receptor associated protein (RAP) of LRP1 was used to block the LRP1 of human normal chondrocytes (C28/I2 cells), and qRT-PCR and WB were used to detect the mRNA and protein expression levels of LRP1, chondrocyte autophagy, differentiation and cartilage injury related genes, respectively. Results:The IHC results showed that the expression levels of MMP13 (1.67 ± 0.21, 0.59 ± 0.15, 0.51 ± 0.12) in the surface, middle, and deep layers of cartilage tissue of KBD patients were significantly higher than those of control subjects (0.25 ± 0.03, 0.26 ± 0.04, 0.06 ± 0.01), and the differences were statistically significant ( t = - 11.38, P < 0.001; t = - 3.82, - 6.26, P = 0.019, 0.003). The expression levels of LRP1 (0.10 ± 0.02, 0.03 ± 0.01, 0.17 ± 0.03) were significantly lower than those of control subjects (1.63 ± 0.40, 0.44 ± 0.12, 0.34 ± 0.08), and the differences were statistically significant ( t = 6.61, 5.61, 3.64, P = 0.003, 0.005, 0.022). The mRNA and protein expression levels of MMP13, CASP3, SOX9 in chondrocytes of KBD patients were significantly higher than those of control subjects, and the differences were statistically significant ( P < 0.05). The mRNA expression levels of LRP1, LC3, COL2A1 were significantly lower than those of control subjects, and the differences were statistically significant ( P < 0.05). After silencing the MMP13 gene in chondrocytes of KBD patients, there were no significant differences in the mRNA and protein expression levels of LRP1, BECN1, LC3, CASP3, COL2A1, and SOX9 ( P > 0.05). After blocking LRP1 with RAP, the protein expression levels of LRP1, BECN1, LC3, MMP13, COL2A1 and SOX9 in chondrocytes were significantly lower than those in control group ( P < 0.05). Conclusions:There is no direct correlation between MMP13 and abnormal autophagy of articular chondrocytes in KBD patients. After blocking LRP1, the expression of the autophagy related genes BECN1 and LC3 in chondrocytes is decreased.