Comparison of the major cell populations among osteoarthritis, Kashin-Beck disease and healthy chondrocytes by single-cell RNA-seq analysis

Chondrocytes are the key target cells of the cartilage degeneration that occurs in Kashin-Beck disease (KBD) and osteoarthritis (OA). However, the heterogeneity of articular cartilage cell types present in KBD and OA patients and healthy controls is still unknown, which has prevented the study of the pathophysiology of the mechanisms underlying the roles of different populations of chondrocytes in the processes leading to KBD and OA. Here, we aimed to identify the transcriptional programmes and all major cell populations in patients with KBD, patients with OA and healthy controls to identify the markers that discriminate among chondrocytes in these three groups. Single-cell RNA sequencing was performed to identify chondrocyte populations and their gene signatures in KBD, OA and healthy cells to investigate their differences as related to the pathogenetic mechanisms of these two osteochondral diseases. We performed immunohistochemistry and quantitative reverse-transcription PCR (qRT-PCR) assays to validate the markers for chondrocyte population. Ten clusters were labelled by cell type according to the expression of previously described markers, and one novel population was identified according to the expression of a new set of markers. The homeostatic and mitochondrial chondrocyte populations, which were identified by the expression of the unknown markers MT1X and MT2A and MT-ND1 and MT-ATP6, were markedly expanded in KBD. The regulatory chondrocyte population, identified by the expression of CHI3L1, was markedly expanded in OA. Our study allows us to better understand the heterogeneity of chondrocytes in KBD and OA and provides new evidence of differences in the pathogenetic mechanisms between these two diseases

Differential expression of cyclins CCNB1 and CCNG1 is involved in the chondrocyte damage of kashin-beck disease

The purpose of this study was clarify the relationship between the differential expression of cyclins CCNB1 and CCNG1 and chondrocyte damage in Kashin-Beck disease. Systematic review and high-throughput sequencing of chondrocytes derived from Kashin-Beck disease patients were combined to identify the differentially expressed cyclins and cyclin-dependent kinase genes. In parallel, weaned SD rats were treated with low selenium for 4 weeks and then T-2 toxin for 4 weeks. Knee cartilage was collected to harvest chondrocytes for gene expression profiling. Finally, the protein expression levels of CCNB1 and CCNG1 were verified in knee cartilage tissue of Kashin-Beck disease patients and normal controls by immunohistochemical staining. The systematic review found 52 cartilage disease-related cyclins and cyclin-dependent kinase genes, 23 of which were coexpressed in Kashin-Beck disease, including 15 upregulated and 8 downregulated genes. Under the intervention of a low selenium diet and T-2 toxin exposure, CCNB1 (FC = 0.36) and CCNG1 (FC = 0.73) showed a downward expression trend in rat articular cartilage. Furthermore, compared to normal controls, CCNB1 protein in Kashin-Beck disease articular cartilage was 71.98% and 66.27% downregulated in the superficial and middle zones, respectively, and 12.06% upregulated in the deep zone. CCNG1 protein was 45.66% downregulated in the superficial zone and 12.19% and 9.13% upregulated in the middle and deep zones, respectively. The differential expression of cyclins CCNB1 and CCNG1 may be related to articular cartilage damage in Kashin-Beck disease

Dynamic Analysis of Gear Rattling of a Certain Type of Dual-Clutch Transmission

In order to accurately explore the transmission rattling phenomenon and the influence of different factors on the dynamic characteristics of the gear rattling of the dual-clutch transmission under the condition of preselected gears, this paper establishes the gear rattling dynamics model of the transmission with the 1st gear without preselection and the preselected 4th gear, respectively; The model takes into account factors such as time-varying mesh stiffness, mesh damping, nonlinear oil film force, nonlinear backlash, and the drag torque generated by the clutch in the unengaged state. In addition, the feasibility of the dynamic model was verified by the bench test. On this basis, we took the gear meshing power and system power loss as quantitative indexes to analyze the influence of the preselected gear state and different parameters on the rattle vibration of the transmission. The results show that the pre-selected gear will not have a significant effect on the gears that have been rattled in the non-pre-selected state, and the torque fluctuation of the non-power flow shaft is aggravated by the influence of the transmission power flow branch and transmission ratio at different levels, which makes the overall rattling strength increase. In order to improve the transmission efficiency of the gear, the torque fluctuation of the input end of the system should be reduced as much as possible, and a larger lubricant viscosity can be appropriately selected, the inertia of the empty gear can be properly reduced, and the tooth clearance can be relaxed for selection

Dynamic Analysis of Gear Rattling of a Certain Type of Dual-Clutch Transmission

In order to accurately explore the transmission rattling phenomenon and the influence of different factors on the dynamic characteristics of the gear rattling of the dual-clutch transmission under the condition of preselected gears, this paper establishes the gear rattling dynamics model of the transmission with the 1st gear without preselection and the preselected 4th gear, respectively; The model takes into account factors such as time-varying mesh stiffness, mesh damping, nonlinear oil film force, nonlinear backlash, and the drag torque generated by the clutch in the unengaged state. In addition, the feasibility of the dynamic model was verified by the bench test. On this basis, we took the gear meshing power and system power loss as quantitative indexes to analyze the influence of the preselected gear state and different parameters on the rattle vibration of the transmission. The results show that the pre-selected gear will not have a significant effect on the gears that have been rattled in the non-pre-selected state, and the torque fluctuation of the non-power flow shaft is aggravated by the influence of the transmission power flow branch and transmission ratio at different levels, which makes the overall rattling strength increase. In order to improve the transmission efficiency of the gear, the torque fluctuation of the input end of the system should be reduced as much as possible, and a larger lubricant viscosity can be appropriately selected, the inertia of the empty gear can be properly reduced, and the tooth clearance can be relaxed for selection

Alterations in the gut microbiota and metabolite profiles of patients with Kashin-Beck disease, an endemic osteoarthritis in China

Kashin-Beck disease (KBD) is a severe osteochondral disorder that may be driven by the interaction between genetic and environmental factors. We aimed to improve our understanding of the gut microbiota structure in KBD patients of different grades and the relationship between the gut microbiota and serum metabolites. Fecal and serum samples collected from KBD patients and normal controls (NCs) were used to characterize the gut microbiota using 16S rDNA gene and metabolomic sequencing via liquid chromatography-mass spectrometry (LC/MS). To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria in the KBD patients, metagenomic sequencing of fecal samples from grade I KBD, grade II KBD and NC subjects was performed. The KBD group was characterized by elevated levels of Fusobacteria and Bacteroidetes. A total of 56 genera were identified to be significantly differentially abundant between the two groups. The genera Alloprevotella, Robinsoniella, Megamonas, and Escherichia_Shigella were more abundant in the KBD group. Consistent with the 16S rDNA analysis at the genus level, most of the differentially abundant species in KBD subjects belonged to the genus Prevotella according to metagenomic sequencing. Serum metabolomic analysis identified some differentially abundant metabolites among the grade I and II KBD and NC groups that were involved in lipid metabolism metabolic networks, such as that for unsaturated fatty acids and glycerophospholipids. Furthermore, we found that these differences in metabolite levels were associated with altered abundances of specific species. Our study provides a comprehensive landscape of the gut microbiota and metabolites in KBD patients and provides substantial evidence of a novel interplay between the gut microbiome and metabolome in KBD pathogenesis

Comparative analysis of the gut microbiota composition between knee osteoarthritis and Kashin-Beck disease in Northwest China

Background: Osteoarthritis (OA) and Kashin-Beck disease (KBD) both are two severe osteochondral disorders. In this study, we aimed to compare the gut microbiota structure between OA and KBD patients. Methods: Fecal samples collected from OA and KBD patients were used to characterize the gut microbiota using 16S rDNA gene sequencing. To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria between OA and KBD groups, metagenomic sequencing of fecal samples from OA and KBD subjects was performed. Results: The OA group was characterized by elevated Epsilonbacteraeota and Firmicutes levels. A total of 52 genera were identified to be significantly differentially abundant between the two groups. The genera Raoultella, Citrobacter, Flavonifractor, g__Lachnospiraceae_UCG-004, and Burkholderia-Caballeronia-Paraburkholderia were more abundant in the OA group. The KBD group was characterized by higher Prevotella_9, Lactobacillus, Coprococcus_2, Senegalimassilia, and Holdemanella. The metagenomic sequencing showed that the Subdoligranulum_sp._APC924/74, Streptococcus_parasanguinis, and Streptococcus_salivarius were significantly increased in abundance in the OA group compared to those in the KBD group, and the species Prevotella_copri, Prevotella_sp._CAG:386, and Prevotella_stercorea were significantly decreased in abundance in the OA group compared to those in the KBD group by using metagenomic sequencing. Conclusion: Our study provides a comprehensive landscape of the gut microbiota between OA and KBD patients and provides clues for better understanding the mechanisms underlying the pathogenesis of OA and KBD

Genetic Variants and Protein Alterations of Selenium- and T-2 Toxin-Responsive Genes Are Associated With Chondrocytic Damage in Endemic Osteoarthropathy

The mechanism of environmental factors in Kashin-Beck disease (KBD) remains unknown. We aimed to identify single nucleotide polymorphisms (SNPs) and protein alterations of selenium- and T-2 toxin-responsive genes to provide new evidence of chondrocytic damage in KBD. This study sampled the cubital venous blood of 258 subjects including 129 sex-matched KBD patients and 129 healthy controls for SNP detection. We applied an additive model, a dominant model, and a recessive model to identify significant SNPs. We then used the Comparative Toxicogenomics Database (CTD) to select selenium- and T-2 toxin-responsive genes with the candidate SNP loci. Finally, immunohistochemistry was applied to verify the protein expression of candidate genes in knee cartilage obtained from 15 subjects including 5 KBD, 5 osteoarthritis (OA), and 5 healthy controls. Forty-nine SNPs were genotyped in the current study. The C allele of rs6494629 was less frequent in KBD than in the controls (OR = 0.63, p = 0.011). Based on the CTD database, PPARG, ADAM12, IL6, SMAD3, and TIMP2 were identified to interact with selenium, sodium selenite, and T-2 toxin. KBD was found to be significantly associated with rs12629751 of PPARG (additive model: OR = 0.46, p = 0.012; dominant model: OR = 0.45, p = 0.049; recessive model: OR = 0.18, p = 0.018), rs1871054 of ADAM12 (dominant model: OR = 2.19, p = 0.022), rs1800796 of IL6 (dominant model: OR = 0.30, p = 0.003), rs6494629 of SMAD3 (additive model: OR = 0.65, p = 0.019; dominant model: OR = 0.52, p = 0.012), and rs4789936 of TIMP2 (recessive model: OR = 5.90, p = 0.024). Immunohistochemistry verified significantly upregulated PPARG, ADAM12, SMAD3, and TIMP2 in KBD compared with OA and normal controls (p < 0.05). Genetic polymorphisms of PPARG, ADAM12, SMAD3, and TIMP2 may contribute to the risk of KBD. These genes could promote the pathogenesis of KBD by disturbing ECM homeostasis

Table1_Differential expression of cyclins CCNB1 and CCNG1 is involved in the chondrocyte damage of kashin-beck disease.DOCX

The purpose of this study was clarify the relationship between the differential expression of cyclins CCNB1 and CCNG1 and chondrocyte damage in Kashin-Beck disease. Systematic review and high-throughput sequencing of chondrocytes derived from Kashin-Beck disease patients were combined to identify the differentially expressed cyclins and cyclin-dependent kinase genes. In parallel, weaned SD rats were treated with low selenium for 4 weeks and then T-2 toxin for 4 weeks. Knee cartilage was collected to harvest chondrocytes for gene expression profiling. Finally, the protein expression levels of CCNB1 and CCNG1 were verified in knee cartilage tissue of Kashin-Beck disease patients and normal controls by immunohistochemical staining. The systematic review found 52 cartilage disease-related cyclins and cyclin-dependent kinase genes, 23 of which were coexpressed in Kashin-Beck disease, including 15 upregulated and 8 downregulated genes. Under the intervention of a low selenium diet and T-2 toxin exposure, CCNB1 (FC = 0.36) and CCNG1 (FC = 0.73) showed a downward expression trend in rat articular cartilage. Furthermore, compared to normal controls, CCNB1 protein in Kashin-Beck disease articular cartilage was 71.98% and 66.27% downregulated in the superficial and middle zones, respectively, and 12.06% upregulated in the deep zone. CCNG1 protein was 45.66% downregulated in the superficial zone and 12.19% and 9.13% upregulated in the middle and deep zones, respectively. The differential expression of cyclins CCNB1 and CCNG1 may be related to articular cartilage damage in Kashin-Beck disease.</p