Effects of anesthetic depth on perioperative T lymphocyte subsets in patients undergoing laparoscopic colorectal cancer surgery: a prospective, parallel-controlled randomized trial.

BACKGROUND: During the perioperative period, the surgical stress response induced by surgical trauma tends to cause a decrease in peripheral lymphocytes. Anesthetics could reduce the stress response during surgery and prevent sympathetic nerve overexcitation. The goal of this study was to investigate how BIS-guided anesthetic depth affected peripheral T lymphocytes in patients undergoing laparoscopic colorectal cancer surgery. METHODS: A total of 60 patients having elective laparoscopic colorectal cancer surgery were randomly assigned and analyzed (n = 30 for deep general anesthesia, BIS 35, n = 30 for light general anesthesia, BIS 55). Blood samples were collected immediately before anesthesia induction and immediately after operation, 24 h and 5 days postoperatively. The CD4+/CD8 + ratio, T lymphocyte subsets (including CD3 + T cells, CD4 + T cells, and CD8 + T cells), and natural killer (NK) cells were analyzed by flow cytometry. Serum interleukin-6 (IL-6), interferon -É£ (IFN-É£), and vascular endothelial growth factor-α (VEGF-α) were also measured. RESULTS: The CD4+/CD8 + ratio decreased 24 h after surgery in two groups, but the reduction did not differ between the two groups (P > 0.05). The concentration of IL-6 and the numerical rating scale (NRS) score in the BIS 55 group were significantly higher than that in the BIS 35 group 24 h after surgery (P = 0.001). There were no intergroup differences in CD3 + T cells, CD4 + T cells, CD8 + T cells, NK cells, VEGF-α, or the IFN-É£. Statistical analyses showed no differences between the two groups in the incidence of fever and surgical site infection during hospitalization. CONCLUSIONS: Despite the fact that patients in deep general anesthesia group had low levels of the IL-6 24 h after surgery, the deep general anesthesia was not associated to a positive effect on patients' peripheral T lymphocytes during colorectal cancer surgery. We found no evidence that peripheral T lymphocyte subsets and natural killer cells were affected by the targeting a BIS of either 55 or 35 in patients undergoing laparoscopic colorectal cancer surgery in this trial. TRIAL REGISTRATION: ChiCTR2200056624 ( www.chictr.org.cn ).

Co-culture with osteoblasts up-regulates glycolysis of chondrocytes through MAPK/HIF-1 pathway.

It is well recognized that the neighbor location between cartilage layer and subchondral bone facilitates the intercellular communication and material exchange. However, the evidence that demonstrates the influence of direct communication between cartilage and subchondral bone on their cell behaviors are still partially unknown. In the current study, we established a co-culture system of chondrocytes and osteoblasts aiming to explore the changes of intracellular metabolism of chondrocytes induced by osteoblasts. By using lactate assay kit, RNA sequencing, qRT-PCR and western blot, we found that osteoblasts enhanced the glycolysis in chondrocytes by characterizing the changes of lactate secretion and cytoplasmic expression, and gene expressions including glucose-6-phosphate isomerase 1 (Gpi1), phosphofructokinase, liver type (Pfkl), lactate dehydrogenase A (Ldha), aldolase, fructose-bisphosphate C (Aldoc), phosphoglycerate kinase 1 (Pgk1), glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and triosephosphate isomerase 1 (Tpi1). The enhanced glycolysis might be due to the activation of HIF-1 signaling and its downstream target, pyruvate dehydrogenase kinase1 (PDK1), by qRT-PCR, western blot and immunofluorescence. We also detected the up-regulation of ERK and p38/MAPK upstream signaling in chondrocytes induced by osteoblasts by western blot and immunofluorescence. The enhanced glycolysis in chondrocytes induced by osteoblasts could help us to better understand the intracellular metabolic mechanism of chondrocytes and cartilage disease occurrence.

PDGF-AA promotes gap junction intercellular communication in chondrocytes via the PI3K/Akt pathway.

BACKGROUND: Gap junction intercellular communication (GJIC) plays an important role in cell growth, development and homeostasis. Connexin 43 (Cx43) is an important half-channel protein responsible for gap junction formation. Platelet-derived growth factor AA (PDGF-AA) regulates the proliferation, migration, metabolism, apoptosis and cell cycle of chondrocytes. However, the role of PDGF-AA in gap junction intercellular communication in chondrocytes is not fully understood. In the current study, we performed experiments to explore the effect of PDGF-AA on GJIC and its underlying biomechanical mechanism. METHODS: qPCR was performed to determine the expression of PDGF, PDGFR and connexin family genes in chondrocytes and/or cartilage. A scrape loading/dye transfer assay was used to determine GJIC. Western blot analysis was applied to detect the expression of Cx43 and PI3K/Akt signaling pathway proteins. Immunofluorescence staining was utilized to examine protein distribution. Scanning electron microscopy was used to delineate the morphology of chondrocytes. RESULTS: Expression of PDGF-A mRNA was highest among the PDGF family in chondrocytes and cartilage tissues. PDGF-AA promoted functional GJIC formation in chondrocytes by upregulating the expression of Cx43. Enhanced functional GJIC formation in chondrocytes induced by PDGF-AA occurred through the activation of PI3K/Akt signaling and its nuclear accumulation. CONCLUSION: For the first time, this study provides evidence demonstrating the role of PDGF-AA in cell-to-cell communication in chondrocytes through mediating Cx43 expression.

[Co-Culturing of Osteoblasts and Chondrocytes Upregulates HIF-1 Pathway of Chondrocytes via MAPK Signaling].

OBJECTIVE: To study the effect of co-culturing chondrocytes with osteoblasts on hypoxia-inducible factor (HIF)-1 pathway of chondrocytes and its mechanism. METHODS: Chondrocytes and osteoblasts were separately extracted from the knee joint and skull of newborn mice by trypsin digestion. The co-culturing system of osteoblasts and chondrocytes was constructed by using Transwell inserts to culture the osteoblasts and 6-well plate to culture the chondrocytes. We used qRT-PCR to examine changes in the mRNA expression of HIFs and its target gene pyruvate dehydrogenase kinase 1 ( PDK1) in chondrocytes co-cultured for 24 h. Western blot was used to analyze changes in the protein expression of HIFs and PDK1 and the changes in the activation of mitogen activated protein kinase (MAPK) signaling pathway after the cells were co-cultured for 48 h. Reactive oxygen species (ROS) staining was done to show the changes of ROS production in chondrocytes co-cultured for 48 h. RESULTS: The results of qRT-PCR and Western blot showed upregulated levels of HIF-1α gene and protein expression ( P<0.05) in the chondrocytes after they were co-cultured with osteoblasts. The gene and protein expression levels of PDK1 , the target gene of HIF-1, were also upregulated ( P<0.05). ROS staining showed that co-culturing of chondrocytes with osteoblasts decreased ROS production in chondrocytes. Western blot revealed that extracellular signal-regulated kinase (ERK) 1/2 and p38 signaling of co-cultured chondrocytes were enhanced ( P<0.05). CONCLUSION: Co-culturing with osteoblasts enhanced the ERK1/2 and p38 signaling of chondrocytes and upregulated the HIF-1 pathway of chondrocytes.

Osteoblasts impair cholesterol synthesis in chondrocytes via Notch1 signalling.

OBJECTIVES: Previous reports have proposed the importance of signalling and material exchange between cartilage and subchondral bone. However, the specific experimental evidence is still insufficient to support the effect of this interdependent relationship on mutual cell behaviours. In this study, we aimed to investigate cellular lipid metabolism in chondrocytes induced by osteoblasts. METHODS: Osteoblast-induced chondrocytes were established in a Transwell chamber. A cholesterol detection kit was used to detect cholesterol contents. RNA sequencing and qPCR were performed to assess changes in mRNA expression. Western blot analysis was performed to detect protein expression. Immunofluorescence staining was conducted to show the cellular distribution of proteins. RESULTS: Cholesterol levels were significantly decreased in chondrocytes induced by osteoblasts. Osteoblasts reduced cholesterol synthesis in chondrocytes by reducing the expression of a series of synthetases, including Fdft1, Sqle, Lss, Cyp51, Msmo1, Nsdhl, Sc5d, Dhcr24 and Dhcr7. This modulatory process involves Notch1 signalling. The expression of ncstn and hey1, an activator and a specific downstream target of Notch signalling, respectively, were decreased in chondrocytes induced by osteoblasts. CONCLUSIONS: For the first time, we elucidated that communication with osteoblasts reduces cholesterol synthesis in chondrocytes through Notch1 signalling. This result may provide a better understanding of the effect of subchondral bone signalling on chondrocytes.

Lipid metabolism in cartilage and its diseases: a concise review of the research progress.

The homeostasis of the vertebrate body depends on anabolic and catabolic activities that are closely linked the inside and outside of the cell. Lipid metabolism plays an essential role in these metabolic activities. Although a large amount of evidence shows that normal lipid metabolism guarantees the conventional physiological activities of organs in the vertebrate body and that abnormal lipid metabolism plays an important role in the occurrence and deterioration of cardiovascular-related diseases, such as obesity, atherosclerosis, and type II diabetes, little is known about the role of lipid metabolism in cartilage and its diseases. This review aims to summarize the latest advances about the function of lipid metabolism in cartilage and its diseases including osteoarthritis, rheumatoid arthritis, and cartilage tumors. With the gradual in-depth understanding of lipid metabolism in cartilage, treatment methods could be explored to focus on this metabolic process in various cartilage diseases.