Organellophagy regulates cell death:A potential therapeutic target for inflammatory diseases.

BACKGROUND: Dysregulated alterations in organelle structure and function have a significant connection with cell death, as well as the occurrence and development of inflammatory diseases. Maintaining cell viability and inhibiting the release of inflammatory cytokines are essential measures to treat inflammatory diseases. Recently, many studies have showed that autophagy selectively targets dysfunctional organelles, thereby sustaining the functional stability of organelles, alleviating the release of multiple cytokines, and maintaining organismal homeostasis. Organellophagy dysfunction is critically engaged in different kinds of cell death and inflammatory diseases. AIM OF REVIEW: We summarized the current knowledge of organellophagy (e.g., mitophagy, reticulophagy, golgiphagy, lysophagy, pexophagy, nucleophagy, and ribophagy) and the underlying mechanisms by which organellophagy regulates cell death. KEY SCIENTIFIC CONCEPTS OF REVIEW: We outlined the potential role of organellophagy in the modulation of cell fate during the inflammatory response to develop an intervention strategy for the organelle quality control in inflammatory diseases.

LncRNA GAS5 inhibits miR-579-3p to activate SIRT1/PGC-1α/Nrf2 signaling pathway to reduce cell pyroptosis in sepsis-associated renal injury.

Sepsis is a life-threatening condition that can lead to several organ failures including kidney. In this study, we investigated the roles of GAS5 and miR-579-3p in regulating cell pyroptosis in the sepsis-induced renal injury model. Lipopolysaccharide (LPS) treatment or cecal ligation and puncture (CLP) surgery was used to create the in vitro and in vivo sepsis-induced renal injury model. The interactions between GAS5 and miR-579-3p, and miR-579-3p and SIRT1 were determined by bioinformatic prediction, luciferase reporter assay, and RIP assay. In vitro cell pyroptosis was examined by flow cytometry marked with active caspase-1 and PI. The protein levels of IL-1ß and IL-18 induced by cell pyroptosis were quantified using ELISA assay. In vivo renal injuries were evaluated with HE and TUNEL stainings, bacterial load in serum and creatinine, and blood urea nitrogen content analyses. Expression levels of GAS5, miR-579-3p, pyroptosis, and SIRT1/PGC-1a/Nrf2 pathway-related molecules were evaluated by qRT-PCR or Western blot. GAS5 and SIRT1 were downregulated, whereas miR-579-3p was upregulated in in vitro and in vivo sepsis-induced renal injury models. GAS5 negatively and directly regulated miR-579-3p to reduce cell pyroptosis via the activation of SIRT1/PGC-1a/Nrf2 pathway. In addition, miR-579-3p suppressed PGC-1a/Nrf2 pathway to induce cell pyroptosis by directly targeting SIRT1. What's more, overexpression of GAS5, or knockdown of miR-579-3p, enhanced SIRT1 expression that led to the improved survival rate, reduced the weight loss, and relieved renal injuries in septic mice. Overexpression of GAS5 demonstrated protective effects against sepsis-induced renal injury via downregulating miR-579-3p and activating SIRT1/PGC-1α/Nrf2 pathway to inhibit cell pyroptosis.

Prevalence and genotype distribution of HPV infection among 214,715 women from Southern China, 2012-2018: baseline measures prior to mass HPV vaccination.

BACKGROUND: The epidemiology on the human papillomavirus (HPV) among females in Southern China is not well-established. Baseline data on the prevalence of HPV infection in China prior to mass prophylactic HPV vaccination would be useful. Thus, this study aims to determine the type-specific HPV prevalence and distribution among females from Southern China prior to mass HPV vaccination. METHODS: A retrospective cross-sectional study employing 214,715 women attending ChenZhou NO.1 People's Hospital for cervical screening during 2012-2018 was conducted prior to widespread HPV vaccination. HPV genotype was detected using nucleic acid molecular diversion hybridization tests. The overall prevalence, age-specific prevalence, type distribution, and annual trend were analyzed. RESULTS: The overall HPV prevalence was 18.71% (95% confidence interval [CI], 18.55-18.88%) among Southern China females. During 2012-2018, the prevalence of HPV infection showed a downward tendency, from 21.63% (95% CI, 21.07-22.20%) in 2012 to 18.75% (95% CI, 18.35-19.16%) in 2018. Age-specific HPV distribution displayed a peak at young women aged less than 21 years (33.11, 95% CI, 31.13-35.15%), 20.07% (95% CI, 19.70-20.44%) among women aged 21-30 years, 17.29% (95% CI, 17.01-17.57%) among women aged 31-40 years, 17.23% (95% CI, 16.95-17.51%) among women aged 41-50 years, 21.65% (95% CI, 21.11-22.20%) among women aged 51-60 years, and 25.95% (95% CI, 24.86-27.07%) among women aged over 60 years. Of the 21 subtypes identified, the top three prevalent high-risk HPV (HR-HPV) genotypes were HPV52 (5.12%; 95% CI, 21.11-22.20%), - 16 (2.96%; 95% CI, 2.89-3.03%), and - 58 (2.51%; 95% CI, 2.44-2.58%); the predominant low-risk HPV (LR-HPV) genotypes were HPV81 (1.86%; 95%CI, 1.80-1.92%) and - 6 (0.69%; 95% CI, 0.66-0.73%) respectively. Incidence of HR-HPV only, LR-HPV only and mixed LR- and HR-HPV were 15.17, 2.07 and 1.47% respectively. Besides, single HPV infection accounted for 77.30% of all positive cases in this study. CONCLUSIONS: This study highlights 1) a high prevalence of HPV infection among females with a decreasing tendency towards 2012-2018, especially for young women under the age of 21 prior to mass HPV vaccination; 2) HPV52, - 16 and - 58 were the predominant HPV genotypes, suggesting potential use of HPV vaccine covering these HPV genotypes in Southern China.

The interaction between C/EBPß and TFAM promotes acute kidney injury via regulating NLRP3 inflammasome-mediated pyroptosis.

Sepsis-induced inflammatory damage is a crucial cause of acute kidney injury (AKI), and AKI is an ecumenical fearful complication in approximately half of patients with sepsis. CCAAT/enhancer-binding protein ß (C/EBPß) plays roles in regulating acute phase responses and inflammation. However, the role and mechanism of C/EBPß in AKI are unclear. LPS combined with ATP-treated renal epithelial cells HK2 and cecal ligation-peferation (CLP)-mice were used as models of AKI in vitro and in vivo. Cell damage, the secretion of interleukin-1 beta (IL-1ß), IL-18 and cysteinyl aspartate specific proteinase 1 (caspase-1) activity were tested by LDH, ELISA assay and flow cytometry analysis, respectively. The expression levels of TFAM, C/EBPß, and pyroptosis-related molecules were tested by qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assessed the interaction between C/EBPß with TFAM. Hematoxylin-Eosin (H&E) staining detected pathological changes of kidney tissues, and immunohistochemistry measured TFAM and C/EBPß in mice kidney tissues. C/EBPß or TFAM were up-regulated in LPS combined with ATP -induced HK2 cells. Knockdown of C/EBPß could suppress cell injury and the secretion of IL-1ß and IL-18 induced by LPS combined with ATP. Furthermore, C/EBPß up-regulated the expression levels of TFAM via directly binding to TFAM promoter. Overexpression of TFAM reversed the effects of C/EBPß deficiency on pyroptosis. Knockdown of C/EBPß could inhibit NLRP3 inflammasome-mediated caspase-1 signaling pathway by inactivating TFAM/RAGE pathway. It was further confirmed in the AKI mice that C/EBPß and TFAM promoted AKI by activating NLRP3-mediated pyroptosis. The interaction of between C/EBPß and TFAM facilitated pyroptosis by activating NLRP3/caspase-1 signal axis, thereby promoting the occurrence of AKI.

Involvement of phosphatase and tensin homolog-induced putative kinase 1-Parkin-mediated mitophagy in septic acute kidney injury.

BACKGROUND: Studies have reported mitophagy activation in renal tubular epithelial cells (RTECs) in acute kidney injury (AKI). Phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and E3 ubiquitin-protein ligase Parkin are involved in mitophagy regulation; however, little is known about the role of PINK1-Parkin mitophagy in septic AKI. Here we investigated whether the PINK1-Parkin mitophagy pathway is involved in septic AKI and its effects on cell apoptosis in vitro and on renal functions in vivo. METHODS: Mitophagy-related gene expression was determined using Western blot assay in human RTEC cell line HK-2 stimulated with bacterial lipopolysaccharide (LPS) and in RTECs from septic AKI rats induced by cecal ligation and perforation (CLP). Autophagy-related ultrastructural features in rat RTECs were observed using electron microscopy. Gain- and loss-of-function approaches were performed to investigate the role of the PINK1-Parkin pathway in HK-2 cell mitophagy. Autophagy activators and inhibitors were used to assess the effects of mitophagy modulation on cell apoptosis in vitro and on renal functions in vivo. RESULTS: LPS stimulation could significantly induce LC3-II and BECN-1 protein expression (LC3-II: 1.72 ±â€Š0.05 vs. 1.00 ±â€Š0.05, P < 0.05; BECN-1: 5.33 ±â€Š0.57 vs. 1.00 ±â€Š0.14, P < 0.05) at 4 h in vitro. Similarly, LC3-II, and BECN-1 protein levels were significantly increased and peaked at 2 h after CLP (LC3-II: 3.33 ±â€Š0.12 vs. 1.03 ±â€Š0.15, P < 0.05; BECN-1: 1.57 ±â€Š0.26 vs. 1.02 ±â€Š0.11, P < 0.05) in vivo compared with those after sham operation. Mitochondrial deformation and mitolysosome-mediated mitochondria clearance were observed in RTECs from septic rats. PINK1 knockdown significantly attenuated LC3-II protein expression (1.35 ±â€Š0.21 vs. 2.38 ±â€Š0.22, P < 0.05), whereas PINK1 overexpression markedly enhanced LC3-II protein expression (2.07 ±â€Š0.21 vs. 1.29 ±â€Š0.19, P < 0.05) compared with LPS-stimulated HK-2 cells. LPS-induced proapoptotic protein expression remained unchanged in autophagy activator-treated HK-2 cells and was significantly attenuated in PINK1-overexpressing cells, but was remarkably upregulated in autophagy inhibitor-treated and in PINK1-depleted cells. Consistent results were observed in flow cytometric apoptosis assay and in renal function indicators in rats. CONCLUSION: PINK1-Parkin-mediated mitophagy might play a protective role in septic AKI, serving as a potential therapeutic target for septic AKI.

Upregulation of Mitochondrial Transcription Factor A Promotes the Repairment of Renal Tubular Epithelial Cells in Sepsis by Inhibiting Reactive Oxygen Species-Mediated Toll-Like Receptor 4/p38MAPK Signaling.

BACKGROUND: Mitochondrial transcription factor A (TFAM) plays multiple pathophysiologic roles in mitochondrial DNA (mtDNA) maintenance. However, the role of TFAM in sepsis-induced acute kidney injury (AKI) remains largely unknown. METHODS: Lipopolysaccharide (LPS) treatment of HK-2 cells mimics the in vitro model of AKI inflammation. pcDNA3.1 plasmid was used to construct pcDNA3.1-TFAM. sh-TFAM-543, sh-TFAM-717, sh-TFAM-765, sh-TFAM-904 and pcDNA3.1-TFAM were transfected into HK-2 cells using Lipofectamine 2000. MtDNA transcriptional levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was performed to assess the cell viability. Changes in reactive oxygen species (ROS) and mitochondrial membrane potential in HK-2 cells were detected using the corresponding kits. Immunofluorescence experiment was used to investigate the displacement of TFAM. mRNA and protein expression levels of TFAM and its related genes were measured by qRT-PCR and western blot respectively. Mice in sepsis were administered cecal ligation and puncture surgery. RESULTS: LPS treatment was a non-lethal influencing factor, leading to the upregulation of ROS levels and downregulation of mtDNA copy number and NADH dehydrogenase subunit-1 (ND1) expression, and caused damage to the mitochondria. As the LPS treatment time increased, TFAM was displaced from the periphery of the nucleus to cytoplasm. TFAM reduced ROS and P38MAPK levels by inhibiting toll-like receptor 4 (TLR4) expression, ultimately inhibiting inflammation and repairing mtDNA. CONCLUSIONS: Our results indicate that TFAM repairs mtDNA by blocking the TLR4/ROS/P38MAPK signaling pathway in inflammatory cells, thereby repairing septic tubular epithelial cells, and TFAM may serve as a new target for sepsis therapy.

[The experiment study of treatment of infectious acute lung injury by intravenous administration of adenovirus borne inhibitor of nuclear factor-ΚB gene in rat].

OBJECTIVE: To observe the effects of adenovirus borne IΚB gene, an inhibitor of nuclear factor-ΚB (NF-ΚB), infused via central vein, to treat infectious acute lung injury (ALI) in rats. METHODS: According to random number table method, 30 pathogen-free Sprague-Dawley (SD) rats were randomly divided into three groups: sham group, ALI model group, IΚB gene treatment group, with 10 rats in each group. The rats of IΚB gene treatment group were infused 1 ml adenovirus borne IΚB gene (titre: 1×10(9)pfu ), the rats of sham group and ALI model group were infused 1 ml normal saline through central vein. Subsequently, the rats of ALI model group and the IΚB gene treatment group were given 1 ml lipopolysaccharide (LPS, 5 ml/kg) through tail vein to reproduce model of ALI. On the other hand, the rats of sham group were given 1 ml normal saline through tail vein. Blood gas analysis, the ratio of wet to dry weight (W/D) of lung, plasma contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and protein expression of NF-ΚBp65 in lung tissue were determined, the pathobiological changes in lung tissue were microscopically observed and the scores of lung injury were calculated after 7 days. RESULTS: The rats in three groups survived, except 1 rat died in ALI model group.Blood pH and partial pressure of arterial carbon dioxide (PaCO(2)) showed no obviously differences among three groups. Partial pressure of arterial oxygen (PaO(2) ) was highest in sham group and the lowest in ALI model group. The plasma content of TNF-α (µg/L) and IL-6 (ng/L ) in ALI model group were obviously higher than those in sham group (TNF-α: 5.20±1.09 vs. 3.01±0.46; IL-6: 540.28±100.78 vs. 214.45±61.37, both P<0.05). The plasma content of TNF-α and IL-6 in IΚB gene treatment group were obviously lower than those in ALI model group (TNF-α: 3.70±0.96 vs. 5.20±1.09, IL-6: 356.49±60.58 vs. 540.28±100.78, both P<0.05), and TNF-α content had restored to the level observed in sham group. The ratio of W/D of lung was lowest in sham group (4.49±0.36) and highest in ALI model group (5.78±0.43), and that of IΚB gene treatment group (5.33±0.38) was lower than that of ALI group. The score of lung injury was lowest in sham group (0.17±0.41) and highest in ALI model group (2.29±0.76), and that of IΚB gene treatment group (1.57±0.53) was lower than that of ALI group. The scale of NF-ΚBp65 immunohistochemistry was lowest in sham group (1.00±0.89) and highest in ALI model group (9.43±1.13), and that of IΚB gene treatment group (4.00±1.15) was lower than the latter. The differences of all the above parameters in three groups were statistically significant (all P<0.05 ). CONCLUSION: Increased expression of IΚB gene by an infusion of adenovirus borne IΚB gene through central vein can lower the levels of pro-inflammatory factors, such as TNF-α and IL-6, restrain the NF-ΚB activation, reduce lung water, alleviate alveolar collapse and lung consolidation in ALI in rats, thus lung injury is ameliorated.

[The enhancing effect of "Xuebijing injection" on lipopolysaccharide-induced apoptosis of regulatory T cells and mediation of polarization of helper T cells].

OBJECTIVE: To investigate the enhancing effect of Chinese medicine-Xuebijing injection on lipopolysaccharide (LPS) -induced apoptosis of CD4+ CD25+ regulatory T cells (Tregs) and polarization of helper T cells (Th). METHODS: CD4+ CD25+ Tregs collected from rat spleen in vitro by immunomagnetic beads assay were divided into the control group, anti-CD3/CD28 group, anti-CD3/CD28 + LPS group, anti-CD3/CD28 + "Xuebijing injection" group and anti-CD3/CD28 + LPS + "Xuebijing injection" group. Tregs apoptosis rate and expression of winged helix transcription factor (Foxp3) in Tregs were detected by flow cytometry on 3rd post culture day. CD4+ CD25- T cells were co-cultured with CD4+ CD25- Tregs (1:1) for 68 hours with canavalin A stimulation. Interferon gamma (gamma-IFN), interleukin (IL)-4 and IL-17 in supernatants, which respectively was secreted by Th1, Th2 and Th17, were measured by ELISA. RESULTS: Tregs apoptosis rate of anti-CD3/CD28 + LPS + "Xuebijing injection" group (45.1 +/- 2.7%) was significantly higher than that of anti-CD3/CD28 + LPS group (29.4 +/- 1.6%, P < 0.01). Meanwhile, Foxp3 expressions in Tregs in above 2 groups were 95 +/- 9 and 140 +/- 18 respectively, showing statistically significant difference between them (P < 0.01). Gamma-IFN levels secreted in anti-CD3/CD28 + LPS + "Xuebijing injection" group were significantly higher than those in anti-CD3/CD28 + LPS group (P < 0.01), while IL-4 levels had an opposite tendency compared with gamma-IFN (P < 0.05), resulting in a marked increase in the ra- tio of gamma-IFN/IL-4 in anti-CD3/CD28 + LPS + "Xuebijing injection" group (P < 0.01). In anti-CD3/ CD28 + "Xuebijing injection" group, IL-17 secretion levels were significantly decreased compared with anti-CD3/CD28 group (P < 0.05). CONCLUSIONS: Activation of CD4+ CD25+ Tregs induced by LPS may mediate Th1 shift to Th2 response. "Xuebijing injection" can effectively regulate immune function of T cells, increase the LPS-induced apoptosis of CD4+ CD25+ Tregs as well as enhance the polarization of Th2 to Th1, thereby abating the suppressive state of cell-mediated immunity.

[Effect of apoptosis of CD4+ CD25+ regulatory T cells on proliferation as well as secretion of effector T cells and interventional activity of Xuebijing injection in septic rats].

OBJECTIVE: To investigate the effect of apoptosis of CD4+ CD25+ regulatory T cells (Tregs) on proliferation as well as secretory function of effector T cells (Teff) and potential influence of Xuebijing injection on them in septic rats. METHODS: A sepsis model was reproduced by cecal ligation puncture (CLP), and Wistar rats were randomly divided into the control group (n = 8), sham-operated group (n = 8), CLP group (n = 8), and Xuebijing injection treatment group (n = 8). CD4+ CD25+ Tregs in each group were separated by immunomagnetic beads isolate system on day 3, the apoptosis rate, expression of forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) on Tregs were analyzed by flow cytometry, and secretion levels of interleukin (IL)-10 from Tregs were measured by ELISA. Following co-culture of CD4+ CD25+ Treg with CD4+ CD25- T cells (1:1) for 68 hours, proliferative activity of Teff was determined by MTT, and IL-2/sIL-2R alpha levels were measured by ELISA. RESULTS: The apoptosis rate of Tregs in control group was 12.03% +/- 0.89%, which was not significantly different from sham-operated group 9.48% +/- 2.17%. The apoptosis rate of Tregs in CLP group 5.87% +/- 0.44% was lower than that in control group (P < 0.01), and treatment with Xuebijing injection markedly enhanced the apoptosis of Tregs 27.29% +/- 2.48%. Compared to CLP group, expression of Foxp3, CTLA-4, and the secretion of IL-10 of Treg were significantly lowered in Xuebijing injection group (all P < 0.01). The Teff proliferative activity in response to ConA, and IL-2 levels of Teff in CLP group were significantly suppressed compared with control group (P < 0.01), and secretion of sIL-2R alpha in the supernatants was much higher than that of the control group. In comparison to the CLP group, inhibitory rate of Teff proliferative activity and the sIL-2R alpha levels were significantly decreased, while the secretion of IL-2 was increased in Xuebijing injection group (P <0.01). CONCLUSION: CD4+ CD25+ Tregs could markedly upregulate the suppressive function on Teff in sepsis, and treatment with Xuebijing injection effectively enhanced apoptosis of Tregs, thereby down-regulating the suppression on Teff.

[Effect of apoptosis of CD4+ CD25+ regulatory T lymphocytes on polarization of helper T lymphocytes and potential interventional influence of Xuebijing injection in septic rats].

OBJECTIVE: To evaluate the influence of apoptosis of CD4(+)CD25(+) regulatory T lymphocyte (Treg) on polarization of helper T lymphocyte (Th) and effect of Xuebijing injection in septic rats. METHODS: A sepsis model was reproduced by cecal ligation and puncture (CLP). Wistar rats were randomly divided into the normal group (n=8), sham operation group (n=8), model group (n=8) and Xuebijing injection treatment group (n=8). CD4(+)CD25(+)Tregs in each group were separated by immunomagnetic beads isolation system on day 3, the apoptotic rates, expression of forkhead/winged helix transcription factor p3 (Foxp3) as well as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) of Treg were analyzed by flow cytometry, and the secretion level of interleukin-10 (IL-10) of Tregs was determined by enzyme linked immunosorbent assay (ELISA) after culturing Tregs for 12 hours. Interferon-gamma (IFN-gamma), IL-4, and IL-17 levels, which were respectively secreted by Th1, Th2 and Th17, were measured by ELISA in the supernatant after CD4(+)CD25(+)Tregs were co-cultured with CD4(+)CD25(-)T lymphocytes for 68 hours. RESULTS: The apoptosis rate of the normal group was (12.03+/-0.89)%, which was not significantly different compared with the sham operation group [(9.48+/-2.17)%]. The apoptosis rate of model group [(5.87+/-0.44)%] was much lower than that of the normal and sham operation groups, while the Xuebijing injection treatment group [(27.29+/-2.48)%] had the highest apoptosis rate compared to others (all P<0.01) . The expression of Foxp3, CTLA-4, and the secretion of IL-10 of Treg were negatively correlated with the apoptosis rates, correlation coefficients (r) respectively were -0.878 (P=0.042), -0.877 (P=0.042), and -0.743 (P=0.010). The secretion of IFN-gamma, IL-4 and ratio of IFN-gamma/IL-4 in model group were significantly elevated compared with the normal group [IFN-gamma: (254.70+/-44.88) ng/L vs. (0.68+/-0.78) ng/L , IL-4: (8.82+/-0.61) ng/L vs. (3.48+/-0.98) ng/L, ratio of IFN-gamma/IL-4: 30.28+/- 4.87 vs. 0.23+/-0.30, all P<0.01], and secretion of IFN-gamma as well as ratio of IFN-gamma/IL-4 were markedly higher in Xuebijing injection treatment group [(491.54+/-84.28) ng/L, 45.31+/-8.01, respectively] than in model group (P<0.01 and P<0.05). No marked change in IL-17 levels was noted in various groups (all P>0.05) . CONCLUSION: Due to the apoptosis of Treg, the suppression function of CD4(+)CD25(+)Tregs on CD4(+)T lymphocyte appears to be abated, and treatment with Xuebijing injection could effectively enhance the apoptosis of Tregs, mediating the response of shifting Th2 to Th1.

[Research advances in Th17 cells].

CD4+ T cells have been divided into different subsets, essentially based on the cytokines they produce. These subsets include T helper cell (Th)1, Th2 and regulatory T cells (Treg). Recently, another subpopulation of T cells has been identified, and it was named as Th17, which is characterized by the release of interleukin (IL)-17. The discovery of Th17 cells is not only a challenge to some traditional theories, such as the differentiation of CD4+ T cells and the drift theory of Th1/Th2, but also provides new research approach to inflammatory and immunologic diseases, especially, the balance among Th1, Th2, Treg and Th17 cells may play an important role in maintaining the normal immune response.