[Influence of reactive oxygen species responsive self-assembled nanomicelle loaded with pyroptosis inhibitor on full-thickness skin defects in diabetic rats].

Objective: To investigate the influence of reactive oxygen species (ROS) responsive self-assembled nanomicelle loaded with pyroptosis inhibitor on full-thickness skin defects in diabetic rats. Methods: Experimental research methods were employed. A nucleotide-binding oligomerization domain (NOD) 1/2 inhibitor (NOD-IN-1) was encapsulated with nanomicelle polyethylene glycol-block-polypropylene sulfide (PEG-b-PPS), and the resulting product was called PEPS@NOD-IN-1. The morphology and hydration particle size of PEG-b-PPS and PEPS@NOD-IN-1 were observed by transmission electron microscope and particle size analyzer, respectively, and the encapsulation rate and drug loading rate of PEPS@NOD-IN-1 to NOD-IN-1 and the cumulative release rate of NOD-IN-1 by PEPS@NOD-IN-1 in phosphate buffer solution (PBS) alone and hydrogen peroxide-containing PBS within 40 h were measured and calculated by microplate reader, and the sample number was 3. Twenty-four male Sprague-Dawley rats aged 6-7 weeks were injected with streptozotocin to induce type 1 diabetes mellitus. Six full-thickness skin defect wounds were made on the back of each rat. The injured rats were divided into PBS group, NOD-IN-1 group, PEG-b-PPS group, and PEPS@NOD-IN-1 group with corresponding treatment according to the random number table, with 6 rats in each group. The wound healing was observed on post injury day (PID) 3, 7, and 12, and the wound healing rate was calculated. The ROS levels in wound tissue were detected by immunofluorescence method on PID 3. On PID 7, the granulation tissue thickness in wound was assessed by hematoxylin-eosin staining, the mRNA expressions of NOD1 and NOD2 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction, and the protein expressions of NOD1, NOD2, and GSDMD-N terminals were detected by Western blotting. Six wounds from different rats in each group were taken for detection of the above indicators. Wound tissue (3 samples per group) was taken from rats in PBS group and PEPS@NOD-IN-1 group on PID 7, and transcriptome sequencing was performed using high-throughput sequencing technology platform. Differentially expressed genes (DEGs) significantly down-regulated in PEPS@NOD-IN-1 group as compared with PBS group were screened, and the enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed. The DEG heatmap of the NOD-like receptor pathway, a pyroptosis-related pathway, was made. Protein-protein interaction (PPI) analysis of DEGs in heatmap was performed through the STRING database to screen key genes of PEPS@NOD-IN-1 regulating the NOD-like receptor pathway. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey test. Results: PEG-b-PPS and PEPS@NOD-IN-1 were in spherical structures of uniform size, with hydration particle sizes of (134.2±3.3) and (143.1±2.3) nm, respectively. The encapsulation rate of PEPS@NOD-IN-1 to NOD-IN-1 was (60±5)%, and the drug loading rate was (15±3)%. The release of NOD-IN-1 from PEPS@NOD-IN-1 in PBS alone was slow, and the cumulative release rate at 40 h was only (12.4±2.3)%. The release of NOD-IN-1 from PEPS@NOD-IN-1 in hydrogen peroxide-containing PBS within 10 h was very rapid, and the cumulative release rate at 10 h reached (90.1±3.6)%. On PID 3 and 7, the wounds of rats in the four groups were gradually healed, and the healing in PEPS@NOD-IN-1 group was better than that in the other three groups. On PID 12, the wound scab area in PBS group was large, the wound epithelialization in NOD-IN-1 group and PEG-b-PPS group was obvious, and the wound in PEPS@NOD-IN-1 group was close to complete epithelialization. Compared with those in PBS group, NOD-IN-1 group, and PEG-b-PPS group, the wound healing rates on PID 7 and 12 in PEPS@NOD-IN-1 group were significantly increased (P<0.05), the level of ROS in wound tissue on PID 3 was significantly decreased (P<0.05), the thickness of granulation tissue in wound on PID 7 was significantly thickened (P<0.05), and the mRNA expressions of NOD1 and NOD2 and the protein expressions of NOD1, NOD2, and GSDMD-N terminals in wound tissue on PID 7 were significantly decreased (P<0.05). KEGG pathway analysis showed that DEGs significantly down-regulated in PEPS@NOD-IN-1 group as compared with PBS group were significantly enriched in NOD-like receptors, hypoxia-inducible factors, mitogen-activated protein kinases, and tumor necrosis factor (TNF) pathways. In the DEG heatmap of NOD-like receptor pathway, the genes regulating pyroptosis mainly involved NOD1, NOD2, NOD-like receptor thermoprotein domain-related protein 3, Jun, signal transduction and transcriptional activator 1 (STAT1), TNF-α-induced protein 3. The PPI results showed that NOD1, NOD2, and STAT1 were the key genes of PEPS@NOD-IN-1 regulating the NOD-like receptor pathway. Conclusions: PEPS@NOD-IN-1 can down-regulate the level of local ROS in wounds and the expression of NOD1, NOD2, and GSDMD-N terminals, the key regulators of pyroptosis, thereby promoting the repair of full-thickness skin defect wounds in diabetic rats. PEPS@NOD-IN-1 can also significantly down-regulate the pyroptosis, inflammation, and hypoxia-related pathways of wounds, and regulate NOD-like receptor pathways by down-regulating key genes NOD1, NOD2, and STAT1.

[Current consensus and controversy in neoadjuvant therapy for pancreatic cancer].

Pancreatic cancer is malignant and has a poor prognosis.At present, the treatment mode has changed from "Surgery First" to systemic therapy under multi-disciplinary team, but surgical resection is still the only way to cure pancreatic cancer. In systemic treatment of pancreatic cancer, the effect of postoperative adjuvant therapy is significant, and preoperative neoadjuvant therapy has gradually attracted widespread attention. Neoadjuvant therapy can improve the rate of R0 resection in patients with pancreatic cancer.There is a consensus on neoadjuvant therapy for patients who with borderline resectable and locally advanced, but for the patients who with resectable remains controversial.

Inhibition of EGFR pathway signaling and the metastatic potential of breast cancer cells by PA-MSHA mediated by type 1 fimbriae via a mannose-dependent manner.

To identify more therapeutic targets and clarify the detailed mechanisms of Pseudomonas aeruginosa-mannose-sensitive hemagglutinin (PA-MSHA) on breast cancer cells both in vitro and in vivo. PA-MSHA was administered to epidermal growth factor receptor (EGFR)-positive human breast cancer cell lines MDA-MB-231HM and MDA-MB-468 in vitro and to mice bearing tumor xenografts. The mannose cocultured test was used to detect the effect of mannose on PA-MSHA-induced cell proliferation, cell cycle arrest, apoptosis, and EGFR pathway signaling. We found that cells stimulated with PA-MSHA exhibited a downregulation of EGFR signaling. The addition of mannose partially inhibited the PA-MSHA-stimulated cell anti-proliferative effect, cell apoptosis, cell cycle arrest, activation of apoptosis-associated caspases, and even downregulation of the EGFR signaling pathway. In vivo, PA-MSHA treatment significantly suppressed mammary tumorigenesis in xenografts in mice and decreased lung metastasis in MDA-MB-231HM cell-transplanted mice. Tumor sample analyses confirmed inhibition of the EGFR pathway in the PA-MSHA-treated mice. In conclusion, this study showed that the involvement of the mannose-mediated EGFR pathway has a critical function in the preclinical rationale for the development of PA-MSHA for the treatment of human breast cancer. It also suggests the potentially beneficial use of PA-MSHA in adjuvant therapy for breast tumors with EGFR overexpression.

Enhanced expression of Duffy antigen receptor for chemokines by breast cancer cells attenuates growth and metastasis potential.

In addition to the role in regulating leukocyte trafficking, chemokines recently have been shown to be involved in cancer growth and metastasis. Chemokine network in tumor neovascularity may be regulated by decoy receptors. Duffy antigen receptor for chemokines (DARC) is a specific decoy receptor binding with the angiogenic CC and CXC chemokines. To investigate the effects of DARC on the tumorigenesis and the metastasis potential of human breast cancer cells, human DARC cDNA was reintroduced into the MDA-MB-231 and MDA-MB-435HM cells which have a high capability of spontaneous pulmonary metastasis. We demonstrated that DARC overexpression induced inhibition of tumorigenesis and/or metastasis through interfering with the tumor angiogenesis in vivo. This inhibition is associated with decreasing CCL2 protein levels, and MVD and MMP-9 expression in xenograft tumors. In human breast cancer samples, we also demonstrated that low expression of the DARC protein is significantly associated with estrogen receptor (ER) status, MVD, lymph node metastasis, distant metastasis and poor survival. Our results suggest for the first time that DARC is a negative regulator of growth in breast cancer, mainly by sequestration of angiogenic chemokines and subsequent inhibition of tumor neovascularity.

Micrometastases detected by cytokeratin 19 expression in sentinel lymph nodes of patients with early-stage cervical cancer.

The purpose of this study was to detect micrometastases in sentinel lymph nodes (SLNs) by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) analyses for cytokeratin 19 (CK19) expression in early-stage cervical cancer. One hundred twenty-five SLNs were collected from 46 patients with early-stage cervical cancer. Conventional histopathologic techniques revealed 14 metastatic SLNs from 11 out of 46 patients. CK19 expression was detected by RT-PCR and IHC in all the 125 SLNs. Cervical cancer tissues from nine patients and five pelvic lymph nodes from the patients without tumor were utilized as positive and negative controls, respectively. All the metastastic SLNs on conventional histopathologic techniques were positive by either RT-PCR or IHC analyses, while all the positive controls were positive and all the negative controls were negative as expected. Of 35 patients without metastatic SLNs on conventional histopathologic techniques, the detection rate of micrometastases was 42.85% by RT-PCR and 20% by IHC analyses. RT-PCR and IHC were more sensitive to identify micrometastases in SLNs of patients with early-stage cervical cancer than routine pathology. These findings demonstrated that micrometastasis could be identified by molecular technique such as RT-PCR and IHC analyses for CK19 expression. RT-PCR was more sensitive to detect micrometastases in SLNs than IHC in patients with early-stage cervical cancer. Therefore, molecular assessment of the SLNs may be a valuable tool to complement routine histologic examination of cervical cancer. The importance of micrometastases in SLNs is under close clinical observation to determine whether it can be used as a predicting factor to help us make decision whether to proceed with whole-pelvic lymph node dissection or as a prognostic factor for clinical outcome.

Anisodamine inhibits shiga toxin type 2-mediated tumor necrosis factor-alpha production in vitro and in vivo.

Cytokines, in particular tumor necrosis factor (TNF), appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection. In this study we examined the effect of anisodamine, a vasoactive drug, on TNF-alpha production in Shiga toxin type 2 (Stx2)-stimulated human monocytic cells in vitro and in Stx2-injected mice sera in vivo. Human monocytes and THP-1 cells were stimulated by Stx2 (1-100 ng/ml) with or without anisodamine addition (1-400 micrograms/ml). For in vivo evaluations, C57BL/6 mice were given a single intraperitoneal injection of anisodamine (6-50 mg/kg) or saline after intraperitoneal injection of Stx2 (50 ng/kg). The results showed that anisodamine suppressed Stx2-induced TNF-alpha production in a dose- and time-dependent manner. Anisodamine also suppressed Stx2-induced TNF-alpha mRNA expression. Further study showed that endogenous prostaglandin E2 may be involved in this inhibitory effect. In contrast to TNF-alpha mRNA, anisodamine at concentrations as high as 400 micrograms/ml did not decrease Stx2-induced IL-1 beta and IL-8 mRNA levels. In addition, anisodamine (> 50 micrograms/ml) increased Stx2-stimulated THP-1 cell viability. Levels of TNF-alpha in anisodamine-treated mice sera were significantly lower than those in the saline-treated group 1.5 and 24 hr after Stx2 injection. Anisodamine induced a lower percentage of death in Stx2-injected mice. Taken together, our results indicate that anisodamine has an important regulatory effect on Stx2-induced TNF-alpha production in vitro and in vivo. The present study suggested that this drug should be further investigated for its effects on Stx2-mediated diseases in humans.

Protective effect of anisodamine against Shiga toxin-1: inhibition of cytokine production and increase in the survival of mice.

The purpose of this study was to investigate whether anisodamine could inhibit Shiga toxin-1 (Stx1)-induced cytokine production and increase the survival of Stx1-treated mice. Human monocytic cells were stimulated by Stx1 (1 to 100 ng/mL) with or without anisodamine addition (1 to 400 microg/mL). For in vivo evaluations, C57BL/6 mice were given a single intraperitoneal injection of anisodamine (1 mg) or saline solution after intraperitoneal injection of Stx1 (2.75 microg/kg). The results showed that anisodamine significantly suppressed Stx1-induced tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-8 production. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that anisodamine suppressed Stx1-mediated TNF-alpha mRNA expression. Further study showed that this TNF-alpha inhibitory effect was via a prostaglandin E2-dependent mechanism. Anisodamine treatment prolonged the survival time of mice and decreased the lethality of Stx1 (94.5% to 44%). Because cytokines, in particular TNF-alpha, contribute to the pathologic process in Stx-producing Escherichia coli (STEC) infection, this study suggested that anisodamine could be a potential drug for treatment of STEC infection.

Effective methylprednisolone dose in experimental crescentic glomerulonephritis.

Pulse methylprednisolone (MP) therapy improves the prognosis of crescentic glomerulonephritis, but the optimal dose is uncertain. We reported previously that treatment with MP at a dose of 30 mg/kg reduces glomerular crescents and infiltrating mononuclear cells and ameliorates the clinical abnormalities in an animal model of crescentic glomerulonephritis. In the present study, we assessed MP dose requirement for these beneficial effects in correlation with the effect on gene expression of chemokines, potential molecules responsible for recruitment and activation of leukocytes. Animals were treated with MP, 5 to 30 mg/kg/d, for 4 consecutive days after cellular crescents had been formed diffusely. The level of crescents and numbers of glomerular and interstitial monocytes/macrophages and T lymphocytes were reduced significantly by 5 mg/kg of MP, but maximal effect was obtained by 30 mg/kg of MP. Urinary protein was reduced significantly in a 30-mg/kg group but not in other groups. The gene expression of chemokines, MCP-1, MCP-3, TCA3, MIP-1alpha, MIP-1ss, RANTES, and lymphotactin, was enhanced in this model and was inhibited strongly by 5 mg/kg of MP. These results indicate that MP reduces the number of infiltrating mononuclear cells and crescents in the rat model in a dose-dependent fashion and that, despite the strong inhibition of chemokine expression at a lower dose, the beneficial effect of MP is maximal at a dose of 30 mg/kg.

Transient and sequential expression of chemokine mRNA in glomeruli in puromycin aminonucleoside nephrosis.

Chemokines are a large family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. We previously reported that among six chemokines, the expression of mRNAs for MCP-1, MCP-3, TCA3, and MIP-1alpha, but not for MIP-1beta and RANTES, was markedly elevated in the renal cortex of rats with puromycin aminonucleoside induced nephrosis. In this study we have determined the glomerular expression of the chemokine mRNAs in this model using quantitative competitive reverse-transcriptase polymerase chain reaction. After an injection of puromycin aminonucleoside, the number of monocytes/macrophages and CD4+ and CD8+ cells markedly increased by day 5 and increased thereafter until day 10. The levels of mRNAs for MCP-1, MCP-3, and lymphotactin increased on day 5 and returned to their normal levels by day 7. The level of TCA3 mRNA increased on day 3, and that of MIP-1alpha mRNA increased on day 7, but both returned to their normal levels within 2 days. No increase in the mRNAs of MIP-1beta or RANTES was observed until day 10. These results indicate that the expression pattern of the chemokine mRNAs in glomeruli resembles that in renal cortex, but is more transient and sequential.

Gene expression of CC chemokines in experimental acute tubulointerstitial nephritis.

The infiltration of mononuclear leukocytes into glomeruli or the interstitium is a feature in most forms of glomerular diseases. CC chemokines, mostly chemoattractants for mononuclear leukocytes, are molecules that are potentially responsible for the recruitment of these cells in the kidney. We previously reported that the gene expression of six CC chemokines-MCP-1, MCP-3, MIP-1alpha, MIP-1beta, RANTES, and TCA3-was enhanced in a rat model of crescentic glomerulonephritis, the most severe form of glomerulonephritis. In this study we analyzed their gene expression in a model of another type of kidney disease, acute nephrosis accompanied by tubulointerstitial lesions, which is induced by an injection of puromycin aminonucleoside. Because leukocyte infiltration in this model is much more prominent in the interstitium than in glomeruli, we analyzed their gene expression in the renal cortex. On day 3, when the level of urinary protein was slightly but significantly increased but the number of interstitial leukocytes was unchanged, the enhanced expression of mRNAs for MCP-1, MCP-3, and TCA3 was observed. On day 5, the numbers of interstitial monocytes and lymphocytes significantly increased, and the levels of the mRNA expression of the above chemokines were still higher than the control animals, whereas the levels of mRNAs for MIP- 1alpha, MIP-1beta, and RANTES were not higher or were only slightly higher than the control ones. These results suggest that multiple CC chemokines may play a role in the recruitment of leukocytes in this model and that the expression pattern of CC chemokines depends on the type of kidney injury.

Expression of lymphotactin mRNA in experimental crescentic glomerulonephritis.

Lymphotactin (LTN) is the sole member of C chemokine, the third subfamily of chemokines. LTN has been shown to be a chemoattractant specific for CD8+ cells and/or natural killer (NK) cells, and to be produced by CD8+ T cells, NK cells, and mast cells. However, there have been no reports describing its expression in clinical or experimental models of diseases so far. Since glomerular infiltration of CD8+ cells is prominent in an animal model of crescentic glomerulonephritis induced in WKY rats by an injection of anti-glomerular basement membrane antibody, we investigated the gene expression of LTN in this model. LTN mRNA was not detected in normal glomeruli but was detected at 0.5 h after the antibody injection, which detection preceded the infiltration of CD8+ cells. The expression of LTN mRNA peaked on day 3 and decreased thereafter. We next studied the expression of LTN mRNA in cultured glomerular and vascular cells, and found that glomerular mesangial and vascular endothelial cells could express LTN mRNA when stimulated with IL-1beta. These results indicate that the gene expression of LTN is enhanced in the animal model of glomerulonephritis and that intrinsic renal cells are the potential source of the gene expression of LTN in the kidney.