USP15 promotes the apoptosis of degenerative nucleus pulposus cells by suppressing the PI3K/AKT signalling pathway

ObjectivesDegenerative disc disease is characterized by an enhanced breakdown of its existing nucleus pulposus (NP) matrix due to the dysregulation of matrix enzymes and factors. Ubiquitin‐specific protease 15 (USP15) is reported to be abnormal in certain human diseases. However, its role in NP degeneration remains unclear. Therefore, we aimed to explore the function of USP15 in degenerative NP cell specimens.MethodsWe induced gene silencing and overexpression of USP15 in degenerative NP cells using RNA interference (RNAi) and a lentiviral vector, respectively. qRT‐PCR and Western blotting were used to determine gene and protein expression levels. Cell apoptosis was analysed via flow cytometry. Protein interaction was examined by performing a co‐immunoprecipitation assay. Furthermore, the PI3K inhibitor LY294002 and agonist IGF‐1 were used to investigate the link between USP15 and AKT in NP degeneration.ResultsWe found that USP15 was up‐regulated in degenerative NP cells and that its overexpression accelerated the process of apoptosis. Moreover, USP15 expression levels negatively correlated with AKT phosphorylation in degenerative NP cells. Furthermore, targeting and silencing USP15 with miR‐338‐3p and studying its interaction with FK506‐binding protein 5 (FKBP5) revealed enhancement of FKBP5 ubiquitination, indicating that USP15 is a component of the FKBP5/AKT signalling pathway in degenerative NP cells.ConclusionsOur results show that USP15 exacerbates NP degradation by deubiquitinating and stabilizing FKBP5. This in turn results in the suppression of AKT phosphorylation in degenerative NP cells. Therefore, our study provides insights into the understanding of USP15 function as a potential molecule in the network of NP degeneration.     

METTL3 promotes IL‐1β–induced degeneration of endplate chondrocytes by driving m6A‐dependent maturation of miR‐126‐5p

METTL3 is an important regulatory molecule in the process of RNA biosynthesis. It mainly regulates mRNA translation, alternative splicing and microRNA maturation by mediating m6A‐dependent methylation. Interleukin 1β (IL‐1β) is an important inducer of cartilage degeneration that can induce an inflammatory cascade reaction in chondrocytes and inhibit the normal biological function of cells. However, it is unclear whether IL‐1β is related to METTL3 expression or plays a regulatory role in endplate cartilage degeneration. In this study, we found that the expression level of METTL3 and methylation level of m6A in human endplate cartilage with different degrees of degeneration were significantly different, indicating that the methylation modification of m6A mediated by METTL3 was closely related to the degeneration of human endplate cartilage. Next, through a series of functional experiments, we found that miR‐126‐5p can play a significant role in IL‐1β–induced degeneration of endplate chondrocytes. Moreover, we found that miR‐126‐5p can inhibit the PI3K/Akt signalling pathway by targeting PIK3R2 gene, leading to the disorder of cell vitality and functional metabolism. To further determine whether METTL3 could regulate miR‐126‐5p maturation, we first confirmed that METTL3 can bind the key protein underlying pri‐miRNA processing, DGCR8. Additionally, when METTL3 expression was inhibited, the miR‐126‐5p maturation process was blocked. Therefore, we hypothesized that METTL3 can promote cleavage of pri‐miR‐126‐5p and form mature miR‐126‐5p by combining with DGCR8.     

Isoliquiritigenin ameliorates caerulein‐induced chronic pancreatitis by inhibiting the activation of PSCs and pancreatic infiltration of macrophages

Chronic pancreatitis (CP) is characterized by persistent inflammation of the pancreas that results in progressive loss of the endocrine and exocrine compartment owing to atrophy and/or replacement with fibrotic tissue. Currently, the clinical therapeutic scheme of CP is mainly symptomatic treatment including pancreatic enzyme replacement, glycaemic control and nutritional support therapy, lacking of specific therapeutic drugs for prevention and suppression of inflammation and fibrosis aggravating in CP. Here, we investigated the effect of isoliquiritigenin (ILG), a chalcone‐type dietary compound derived from licorice, on pancreatic fibrosis and inflammation in a model of caerulein‐induced murine CP, and the results indicated that ILG notably alleviated pancreatic fibrosis and infiltration of macrophages. Further in vitro studies in human pancreatic stellate cells (hPSCs) showed that ILG exerted significant inhibition on the proliferation and activation of hPSCs, which may be due to negative regulation of the ERK1/2 and JNK1/2 activities. Moreover, ILG significantly restrained the M1 polarization of macrophages (RAW 264.7) via attenuation of the NF‐κB signalling pathway, whereas the M2 polarization was hardly affected. These findings indicated that ILG might be a potential anti‐inflammatory and anti‐fibrotic therapeutic agent for CP.     

Formononetin,J1 and J2 have different effects on endothelial cells via EWSAT1‐TRAF6 and its downstream pathway

Formononetin is a natural isoflavone compound found mainly in Chinese herbal medicines such as astragalus and red clover. It is considered to be a typical phytooestrogen. In our previous experiments, it was found that formononetin has a two‐way regulatory effect on endothelial cells (ECs): low concentrations promote the proliferation of ECs and high concentrations have an inhibitory effect. To find a specific mechanism of action and provide a better clinical effect, we performed a structural transformation of formononetin and selected better medicinal properties for formononetin modifier J1 and J2 from a variety of modified constructs. The MTT assay measured the effects of drugs on human umbilical vein endothelial cell (HUVEC) activity. Scratch and transwell experiments validated the effects of the drugs on HUVEC migration and invasion. An in vivo assessment effect of the drugs on ovariectomized rats. Long‐chain non‐coding RNA for EWSAT1, which is abnormally highly expressed in HUVEC, was screened by gene chip, and the effect of the drug on its expression was detected by PCR after the drug was applied. The downstream factors and their pathways were analysed, and the changes in the protein levels after drug treatment were evaluated by Western blot. In conclusion, the mechanism of action of formononetin, J1 and J2 on ECs may be through EWSAT1‐TRAF6 and its downstream pathways.     

Antibacterial potential of Forsythia suspensa polysaccharide against resistant Enterobacter cloacae with SHV‐12 extended‐spectrum β‐lactamase (ESBL)

In this study, a homogenous polysaccharide (FSP), with an average molecular weight of 9.08 × 10⁴ Da, was isolated from Forsythia suspense and its antibacterial potential against Enterobacter cloacae producing SHV‐12 ESBL was investigated. Growth kinetics, in vitro competition and biofilm formation experiments demonstrated that SHV‐12 ESBL contributed to a fitness benefit to E cloacae strain. The antibacterial activity of FSP (2.5, 5.0 and 10.0 μg/mL) was tested against E cloacae bearing SHV‐12 ESBL gene using bacterial sensitivity, agar bioassay and agar well diffusion assays. It was found that the addition of FSP demonstrated potent antibacterial activities against this bacterial as showed by the decrease of bacterial growth and the increase of the inhibition zone diameter. Furthermore, SHV‐12 ESBL gene expression was decreased in E cloacae strain following different FSP treatment in a concentration‐dependent manner. In conclusion, these data showed that FSP exhibited potent good antibacterial activity against E cloacae producing SHV‐12 ESBL via inhibition of SHV‐12 ESBL gene expression, which may promote the development of novel natural antibacterial agents to treat infections caused by this drug‐resistant bacterial pathogen.     

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway

Microtubule actin cross‐linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3‐E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast‐specific Osterix (Osx) promoter‐driven Macf1 conditional knockout mice (Macf1^f/fOsx‐Cre). The Macf1^f/fOsx‐Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1^f/fOsx‐Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1^f/fOsx‐Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1^f/fOsx‐Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease.     

Linc01014 regulates gefitinib resistance in oesophagus cancer via EGFR‐PI3K‐AKT‐mTOR signalling pathway

This study aimed to explore the underlying mechanism of linc01014 in oesophagus cancer gefitinib resistance. Gefitinib‐resistant oesophagus squamous cell carcinoma (ESCC gefitinibR) cell lines were constructed by using different gefitinib treatment in FLO‐1, KYAE‐1, TE‐8 and TE‐5 cell lines and confirmed by MTS50 and proliferation assays. Expression of linc01014 was overexpressed/silenced in FLO‐1 cells followed by gefitinib treatment, and then, the apoptosis‐associated markers Bax and Bcl‐2, and PI3KCA in PI3K signalling pathway were determined using Western blotting. MST50 and morphology analyses showed that ESCC gefitinibR cell lines presented obvious gefitinib resistance than their parental ESCC cell lines. ESCC gefitinibR cell lines showed significantly higher proliferation abilities than their parental ESCC cell lines after treating with gefitinib. Overexpression of linc01014 significantly inhibited the apoptosis of FLO‐1 cells induced by gefitinib and silencing linc01014 obviously promoted the apoptosis of FLO‐1 cells induced by gefitinib. Silencing linc01014 could significantly increase the gefitinib chemotherapy sensitivity of oesophagus cancer via PI3K‐AKT‐mTOR signalling pathway.