Sepsis is a multiple organ dysfunction syndrome caused by a dysregulated host response to infection. The mortality rate remains high under current treatment methods, and there is an urgent need to explore new therapeutic targets. Ubiquitination modification, as a key posttranslational regulation mechanism of proteins, plays a central role in the occurrence and development of sepsis and multiple organ damage by regulating key pathological processes such as inflammatory response, cell death and barrier function. This article aims to systematically elucidate the molecular mechanism of ubiquitination modification system in sepsis related organ damage, summarize the latest research progress on treatment strategies targeting the ubiquitination pathway, and explore the challenges and future transformation directions faced in this field. Through comprehensive analysis of existing research, this review aims to provide new ideas and theoretical basis for precise treatment of sepsis.
Valosin-containing protein is a membrane-bound protein that is highly conserved and widely existed in cells. Valosin-containing protein and it’s cofactors jointly participate in various cell functions such as protein degradation, gene replication, and cell cycle regulation, which maintain cellular homeostasis. Valosin-containing protein also exists in tumor cells, and its expression is closely related to the occurrence and development of tumors, but the specific mechanism needs to be further clarified. The application of valosin-containing protein inhibitors in tumor treatment has been continuously and in-depth researched. This article will explore several aspects of the molecular basis of valosin-containing protein, valosin-containing protein and cell homeostasis, tumors, and targeted therapy with valosin-containing protein. It aims to provide a certain basis for the clinical application of valosin-containing protein inhibitors.
ObjectiveTo observe expre with ssions of insulin receptor substrate 1(IRS-1) and ubiquitin-protein in skeletal muscle of non-obese rats with type 2 diabetes mellitus following gastric bypass operation (GBP), and to investigate possible mechanism of GBP in improving insulin resistance. MethodsMale GK rats were randomly divided into diabetic operation group (DO group), diabetic sham operation group (DSO group), and diabetic control group (DC group), 8 rats in each group; besides 8 male Wistar rats were served as normal control group (NC group). Fasting body weight (FBW), fasting plasma glucose (FPG), and fasting insulin (FINS) were measured respectively before operation and on week 1, 2, 4 and 8 after operation. Homeostasis model-insulin resistant (HOMA-IR) index was calculated respectively before operation and on week 8 after operation. The expressions of IRS-1 protein and ubiquitin-protein in skeletal muscle were detected by using Western blot method on week 8 after operation. Results① Compared with the preoperative levels, the FBWs on week 1, 2, and 4 after operation markedly decreased (P < 0.05), but it recovered to the preoperative level on week 8 after operation (P > 0.05) in the DO group; which in the DSO group decreased on week 1 after operation (P < 0.05) and then increased on week 4 after operation (P < 0.05); which in the DC group or the NC group increased continuously and had a significant difference on week 8 after operation (P < 0.05).② The FPGs in the DO, DSO and DC groups were significantly higher than those of the NC group before operation (P < 0.05), which in the DO group decreased from (9.10±0.98) mmoL/L before operation to (5.70±0.91) mmol/L on week 8 after operation (P < 0.05) and were significantly lower than those of the DSO group or the DC group on week 2, 4, and 8 after operation (P < 0.05); which in the DC group, DSO group and NC group had no obviously changes between before and after operations (P > 0.05). ③ The FINS had no significant differences among these four groups before operation (P > 0.05), which in the DO group obviously increased[(9.64±1.59) mU/L] on week 2 after operation (P < 0.05) and then obviously decreased[(6.58±1.05) mU/L] on week 8 after operation (P < 0.05) and significantly lower than those of the DSO group or the DC group on week 8 after operation (P < 0.05), while which had no significant difference between before and after operations in the DSO group, the DC group, or the NC group (P > 0.05). ④ The HOMA-IR index in the DO, DSO or DC group was significantly higher than that of the NC group before operation (P < 0.05), which in the DO group markedly decreased from 3.18±0.50 before operation to 1.96±0.63 on week 8 after operation (P < 0.05) and significantly lower than that of the DSO group or the DC group on week 8 after operation (P < 0.05), while which had no significant difference between before and after operations in the DSO group, the DC group, or the NC group (P > 0.05). ⑤ The expression of IRS-1 protein in the DO group was significantly higher than that in the DSO group (P < 0.05) or the DC group (P < 0.05) on week 8 after operation. While there was no significant difference between the DSO and the DC group after operation (P > 0.05). ⑥ Compared with the NC group, the expression of ubiquitin-protein was significantly increased in the DO group, the DSO group, or the DC group (P < 0.05). Compared with the DSO group or the DC group, the expression of ubiquitin-protein was significantly decreased in the DO group on week 8 after operation (P < 0.05), especially it was most obvious near the molecular weight of 180×103. While there was no significant difference between the DSO group and the DC group after operation (P > 0.05). ConclusionsExpression of IRS-1 protein in skeletal muscle insulin signaling pathway in type 2 diabetes mellitus rats following GBP is increased, it might be associated with decreasing ubiquitin-protein level in skeletal muscle, thus reduces the IRS-1 ubiquitin-degradation, increase insulin sensitivity, and improve insulin resistance of skeletal muscle.
Objective To study the role of ubiquitin-proteasome pathway in diaphragm of COPD rats. Mathods Thirty rats were divided into a normal control group and a COPD group. COPD model was established by exposure to cigarette smoke for three months. The protein levels of E2-14k and proteasome subunit C8 in diaphragms were measured by Western blot. The mRNA levels of ubiquitin and proteasome subunit C2 in diaphragms were measured bymeans of realtime polymerase chain reaction( RT-PCR) . Results Compared with the control group, the protein expression of E2-14k increased significantly in the COPD group ( 0. 81 ±0. 28 vs 0. 50 ±0. 25, P lt;0. 05) , but C8 protein level was not significantly different between the two groups( P gt;0. 05) . The mRNA expression of ubiquitin increased significantly in the COPD group( 0. 89 ±0. 20 vs 0. 50 ±0. 15, P lt;0. 05) , but C2 mRNA level was not significantly different between the two groups ( P gt; 0. 05 ) . Conclusions The mRNA and protein expressions of ubiquitin-proteasome pathway in diaphragmincreased significantly in COPD rats, suggesting that the activity of ubiquitin-proteasome pathwayincreased, which lead to an increase of protein degradation.
ObjectiveTo compare the different effects of ubiquitin(UB) on human umbilical vein endothelial cells (HUVECs) and macrophages under normal circumstances,and analyze whether UB could protect HUVECs from lipopolysaccharide(LPS) induced injury. MethodsThe morphologic changes of HUVECs in vitro with up-rising concentrations of UB interventions were observed. HUVECs and human macrophages in vitro were divided into 4 groups according to UB concentration (0.01 μg/mL,0.1 μg/mL, 1 μg/mL, and 10 μg/mL). Supernatant and cells of each group were collected in 24 h after UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA while NF-κB protein level in cells was detected by Western blot. HUVECs were divided into a LPS group(LPS 10 μg/mL) and an UB+LPS group(UB 0.1 μg/mL,LPS 10 μg/mL). The supernatant of the two groups were collected in 8,16 and 24 h after LPS and UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA. ResultsThe injury of HUVECs got worse with the ascending concentrations of UB.At the concentration of 50 μg/mL,UB induced HUVECs got ballooned and died massively. With the increase of UB concentration,the levels of TNF-α and VCAM-1 in HUVECs' supernatant ascended firstly and then descended,while those in human macrophages' supernatant ascended gradually. zHowever,the tendency of the NF-κB protein level in the two kinds of cells was similar when the concentration of UB increased.At the consentration of 0.1 μg/mL or 1 μg/mL,ubiquitin induced NF-κB protein level obviously increased.At the concentration of 0.01 μg/mL or 10 μg/mL,UB induced the protein level was similar with those of the control group and even decreased slightly. There was no significant difference in TNF-α or VCAM-1 levels at each time point between the LPS group and the UB+LPS group. ConclusionsUB injuries HUVECs obviously at a low concentration but injuires human macrophages at much higher concentraton. UB can not protect HUVECs from LPS-induced injury in vitro.
Objective To observe the degradation regulation of ubiquitinproteasome inhibitor nuclear factor kappa;B（NF-kappa;B）and its inhibitory signal protein Ikappa;B kinase in earlier period diabetic retinopathy（DR）,and the effects on retinal ganglion cells (RGC) apoptosis.Methods Forty healthy adult Wistar rats were randomly divided into control (group A）,DR（group B）,DR+lowconcentration MG132 treated （group C）and DR+high concentration MG132 treated（group D）groups,10 rats in each group.After 6 and 8 weeks,the results of body masses and fasting blood glucose (FBG) were detected,the expression of NF-kappa;B and Ikappa;B were observed by immunohistochemistry respectively.RGC apoptosis was assessed by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labelling (TUNEL) method.Results The expression of NF-kappa;B was upregulated in group B compared with group A,its expression decreased in group D compared with group B; but the expression of Ikappa;B was contrary to NF-kappa;B; RGC apoptosis was followed a similar pattern with the expression of NF-kappa;B; the differences among them were statistically significant (P＜0.01).Compared the expression of NF-kappa;B，Ikappa;B and RGC apoptosis in group C and D, there were no statistically significant differences(P＞0.05).Conclusion Ubiquitin-proteasome inhibitor MG132 can block the activation of NF-kappa;B,inhibit ubiquitination of Ikappa;B degradation and RGC apoptosis.
Objective To investigate the influence of proteasome inhibitorMG-132 on inflammatory factors in COPD rats and its potential mechanism. Methods The COPD rat model was established by instillation of lipopolysaccharide and exposure to cigarette smoke. Then the rats were randomly divided into 4 groups( n = 12 in each group) , ie. a COPD model group, a COPD + MG-132 low concentration group ( 0. 05 mg·kg- 1·d - 1 ) , a COPD + MG-132 high concentration group( 0. 1 mg· kg- 1 · d - 1 ) , and a normal control group. The rats were injected intraperitoneally with different dose of MG-132 or normal saline. After 1 week and 4 weeks, 6 rats in each group were sarcrificed. Then the following parameters were determined including histopathological changes of lung tissue, and the concentrations of IL-1β, IL-6, IL-8 in serum and diaphragm via ELISA. Results The lung histopathological examination showed obvious emphysema and inflammatory infiltration in the COPD rats. These pathological changes were obviously ameliorated in two MG-132 treatment groups. The IL-1β, IL-6, and IL-8 levels in serumand diaphragmin the COPD model group were all significantly increased from1 week and 4 week than those in the normal control group( P lt;0. 05) .MG-132 down-regulated the expression of these inflammatory factors in a time-and dosedependent manner. The IL-1β, IL-6, and IL-8 levels in serum and diaphragm in the MG-132 low concentration group and high concentration group were all decreased compared with the COPD model group ( P lt; 0. 05) . Conclusion COPD is a systemic inflammatory disease which can be inhibited by the proteasome inhibitor MG-132 through suppressing inflammatory factors.
Objective To study the effect of tumor necrosis factor-α( TNF-α) onhypermetabolism of skeletal muscle protein in rats with chronic obstructive pulmonary disease ( COPD) and explore its underlying mechanism. Methods Forty-five SD rats were randomly divided into a normal control group, a COPD group and a COPD + TNF-α group, with 15 rats in each group. COPD model was established by passive cigarette smoking in COPD group and COPD + TNF-αgroup. Then the extensor digitorium longus muscles ( EDL) were dissected and incubated in vitro muscle incubation system with adequate oxygen supply. The EDL were either cultured with or without recombinant rat TNF-α( 10 μg/L) . The mRNA and protein expressions of proteasome subunit C2 in EDL were quantified by real-time quantitative PCR and Western blot analysis, respectively. Results The mRNA and protein expressions of proteasome subunit C2 were both significant higher in the COPD group and COPD + TNF-αgroup than those in the normal control group( P lt;0. 01 or 0. 05) . The COPD+TNF-αgroup had higher expression of proteasome subunit C2 mRNA than that in the COPD group( P lt; 0. 01) , whereas the protein expression was not significantly different( P gt; 0. 05) . Conclusion Incresed proteolytic metabolism in skeletal muscle in COPD might be regulated by TNF-αactivated ubiquitin-dependent pathway.
ObjectiveTo explore the effects of silencing the expression of ubiquitin-conjugating enzyme E2T (UBE2T) gene on proliferation, apoptosis, migration and invasion abilities of A549 cells.MethodsA549 cells were cultured in vitro. Three sets of shRNA-UBE2T plasmid vectors (UBE2T-shRNA1 group, UBE2T-shRNA2 group, UBE2T-shRNA3 group) and shRNA-NC (shRNA-NC group) were constructed, respectively. A549 cells were transfected with lipofection transfection. The cells transfected with empty vector were enrolled as control (control group). The transfection efficiency was detected by RT-PCR. The effects of silencing the expression of UBE2T gene on biological behaviors (proliferation, apoptosis, migration, and invasion) of lung cancer A549 cells were detected by clone formation assay, flow cytometry, Transwell assay and scratch test. The expression of proliferation and apoptosis related proteins, and expression of PI3K/AKT signaling pathway proteins were detected by Western blot. ResultsAfter transfection, expression level of UBE2T mRNA in UBE2T-shRNA1 group, UBE2T-shRNA2 group and UBE2T-shRNA3 group was significantly down-regulated (all P<0.05), whose down-regulation was the most significant in UBE2T-shRNA3 group (P<0.05). Compared with control group and shRNA-NC group, clone formation rate, number of invasion A549 cells, scratch healing rate, Ki67 expression, PCNA expression, p-PI3K/PI3K ratio and p-AKT/AKT ratio were significantly decreased in UBE2T-shRNA3 group (P<0.05), while A549 apoptosis rate, Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio were significantly increased (P<0.05). There were no significant differences in the above indexes between control group and shRNA-NC group (P>0.05). ConclusionsThe shRNA interfering with UBE2T is reliable to construct the model of A549 cells with stable low-expression UBE2T. Down-regulation of UBE2T expression can promote apoptosis of A549 cells, inhibit their proliferation, invasion and migration abilities. The mechanism may be related to inhibiting the activation of PI3K/AKT signaling pathway.
ObjectiveTo investigate the effect of ubiquitin specific peptidase 22 (USP22) on the occurrence and development of esophageal squamous cell carcinoma (ESCC) under hypoxic conditions, and its regulatory relationship with hypoxia inducible factor-1α (HIF-1α). MethodsWestern blotting and quantitative polymerase chain reaction (qPCR) were used to detect the differences in USP22 protein and mRNA expression between normal esophageal epithelial cells HEEC and ESCC cell lines KYSE30, KYSE150, EC9706, and TE-1 under normoxic (5% CO2, 20% O2, 75% N2) and hypoxic (5% CO2, 1% O2, 94% N2) conditions. By transfecting USP22 plasmid or siUSP22, ESCC cells were divided into a normoxia control group, a normoxia+USP22 group, a normoxia+siUSP22 group, a hypoxia control group, a hypoxia+USP22 group, and a hypoxia+siUSP22 group. The proliferation and migration abilities of cells in each group were detected. The expression of USP22 and HIF-1α under hypoxic conditions after up-regulating or down-regulating USP22 was detected, and their regulatory relationship was verified. The interaction between USP22 and HIF-1α was verified by co-immunoprecipitation (Co-IP) technique. ResultsCompared with HEEC cells, the expression of USP22 in ESCC cells was significantly increased (P<0.05). Up-regulation of USP22 expression promoted the proliferation and migration of ESCC cells, while silencing USP22 inhibited the proliferation and migration of ESCC cells (P<0.05). Under hypoxic conditions, the expression of USP22 and HIF-1α increased, and with the up-regulation of USP22 expression, the expression of HIF-1α also significantly increased (P<0.05). Co-IP experiment confirmed the binding between USP22 and HIF-1α. ConclusionUp-regulation of USP22 expression promotes the proliferation and migration of ESCC cells. Hypoxia microenvironment can induce the increase of USP22 expression in ESCC. USP22 may participate in the regulation of the occurrence and development of ESCC by directly binding to HIF-1α.