A study on the correlation between M2 macrophages and regulatory T cells in the progression of colorectal cancer.

BACKGROUND: M2 macrophages and regulatory T cells (Tregs) can promote tumors and development by inhibiting the anti-tumor immune response. This study investigated the effect of CD163-positive M2 macrophages and Foxp3-positive Tregs in the progression of colorectal cancer and lymph node metastasis. It also investigated the correlation between M2 macrophages and Tregs. METHODS: Postoperative tissue specimens and clinical data were collected from 197 patients with colorectal cancer who underwent initial surgical treatment in The Second Ward of Colorectal Surgery of the First Affiliated Hospital of Jinzhou Medical University from March 2020 to December 2020. Immunohistochemical methods were used to detect the expression levels of CD163 protein-labeled M2 macrophages and Foxp3 protein-labeled Tregs in colorectal cancer tissues, matched paracancer tissues, and lymph node tissues. The correlation between CD163 and Foxp3 in cancer tissues and lymph node tissues were analyzed, as well as the relationship between clinicopathological characteristics and preoperative tumor markers. RESULTS: M2 macrophages and Tregs were importantly positively correlated in cancer and lymph node tissues, which significantly increased in cancer and metastatic lymph node tissues. Interestingly, M2 macrophages in non-metastatic lymph nodes also increased significantly in patients with metastatic lymph nodes. In addition, both CD163 and Foxp3 were upregulated with increasing tumor node metastasis stage, depth of infiltration, and lymphatic metastasis; and both were positively correlated with carcinoembryonic antigen. CONCLUSION: CD163 may be a good predictor of pre-metastatic status of colorectal cancer lymph nodes. carcinoembryonic antigen affects the distribution of M2 macrophages and Tregs in colorectal cancer. There is a certain correlation between the two types of cells. It is possible that M2 macrophages, together with suppressor Tregs cells, promote an immunosuppressive environment.

M2-Type Macrophages Induce Tregs Generation by Activating the TGF-ß/Smad Signalling Pathway to Promote Colorectal Cancer Development.

PURPOSE: The increase of both M2-type macrophages and Tregs is closely associated with the development of colorectal cancer. However, the mechanism of their interaction is still unclear. In this study, we investigated the correlation of M2-type macrophages with Tregs and the possible mechanisms between them. METHODS: Using immunohistochemistry, we analysed Smad3 (a key protein in the TGF-ß/Smad signalling pathway) expression in colorectal cells, as well as infiltrating numbers of CD163 (a marker for M2-type macrophages), Foxp3 (a marker for Tregs) in 250 surgically resected colorectal cancer tissues, matched normal and paracancerous tissues. The relation of CD163 and Foxp3 was investigated in CRC with clinicopathological characteristics and preoperative tumour markers. RESULTS: CD163, Foxp3 and Smad3 were upregulated in CRC tissues compared to matched normal and paracancerous tissues. Interestingly, CD163 and Foxp3 were significantly positively correlated in CRC, and both were significantly positively correlated with Smad3. Both CD163 and Foxp3 were upregulated with increasing tumour TNM staging, increasing number of lymph node metastases and increasing vascular invasion. Additionally, CD163 was upregulated with increasing depth of infiltration. The number of M2-type macrophages and the expression levels of preoperative CEA, CA19-9 and CA72-4 were significantly positively correlated. The number of Tregs was significantly positively correlated with the expression levels of preoperative CEA and CA19-9. CONCLUSION: M2-type macrophages may induce Tregs generation through activation of the TGF-ß/Smad signalling pathway, which can promote the development of colorectal cancer.

Protective effect of 2-deoxy-D-glucose on the brain tissue in rat cerebral ischemia-reperfusion models by inhibiting Caspase-apoptotic pathway.

We observed the effect of 2-deoxy-D-glucose (2-DG) on the brain tissue in rat cerebral ischemia-reperfusion (I/R) and explored its mechanism. After observing the effect of 2-DG on endoplasmic reticulum stress (ERS), rats were randomly divided into sham-operation group, I/R group and I/R+2-DG group (each group with 60 rats). I/R models were prepared by middle cerebral artery occlusion. In I/R+2-DG group, each rat was given intraperitoneal 2-DG of 100 mg/kg once a day for 7 days before brain ischemia. According to different time points (3h, 6h, 12h, 24h and 48h) after I/R, each group was divided into 5 subgroups (each subgroup with 12 rats). Nerve cell apoptosis, and the expressions of mRNA and protein of glucose regulated protein 78 (GRP78), cleaved-caspase-9 and cleaved-caspase-3 were determined with TUNEL, Western blotting and RT-PCR, respectively, in rat cerebral hippocampal CA1 area at each time point. TUNEL-positive cells were significantly less in I/R+2-DG group than in I/R group at each time point (all P<0.01). In I/R and I/R+2-DG groups, the expressions of mRNA and protein of GRP78 reached the maximum 12 h after I/R, and cleaved-caspase-9 and cleaved-caspase-3 reached the maximum 24 h after I/R. Compared with sham-operation group, the expressions of mRNA and protein of GRP78, cleaved-caspase-9 and cleaved-caspase-3 were all significantly increased (all P<0.01) in I/R and I/R+2-DG groups. However, the expressions of mRNA and protein of GRP78 were significantly higher in I/R+2-DG group than in I/R group (all P<0.05), but the expressions of mRNA and protein of cleaved-caspase-9 and cleaved-caspase-3 were all significantly lower in I/R+2-DG group than in I/R group (all P<0.05). We conclude that 2-DG has a neuroprotective effect on the brain tissue in rat cerebral ischemia-reperfusion models. The mechanism may be that 2-DG starts ERS followed by up-regulation of mRNA and protein of GRP78 and down-regulation of mRNA and protein of cleaved-caspase-9 and cleaved-caspase-3, which blocks the apoptotic pathway.

Receptor-interacting protein kinase 3-mediated programmed cell necrosis in rats subjected to focal cerebral ischemia-reperfusion injury.

In the current study, the activation of tumor necrosis factor-α receptor 1 (TNFR1) and receptor-interacting protein kinase 3 (RIP3) were investigated following cerebral ischemia-reperfusion injury (CIRI). Healthy SD rats were randomly divided into 3 groups: Sham operation group, model group and inhibitor group. The model group and inhibitor group were further divided into 4 subgroups of 6, 12, 24 and 72 h following CIRI. Using right middle cerebral artery embolization, the CIRI model was generated. To confirm that the CIRI model was established, neurological scores, TTC staining and brain water content measurements were conducted. Immunohistochemistry and western blotting were conducted to investigate the expression of TNFR1 and RIP3 in the cerebral cortex. It was observed that nerve cell necrosis occurred following 6 h of CIRI. The appearance of necrotic cells was gradually increased with increasing CIRI duration. TNFR1 and RIP3 were positively expressed following 6 h of CIRI. With increasing durations of CIRI, the protein expression levels of TNFR1 and RIP3 were significantly increased. Pre­administration with Z-VAD-FMK (zVAD) significantly increased the protein level of RIP3, however, had no effect on the levels of TNFR1, and was accompanied by a reduction in necrosis. In conclusion, RIP3­mediated cell necrosis was enhanced by caspase blockade zVAD and the function of zVAD was independent of TNFR1 signaling following IR.

Combination of ginsenoside Rg1 and bone marrow mesenchymal stem cell transplantation in the treatment of cerebral ischemia reperfusion injury in rats.

BACKGROUND/AIMS: The present study aims to explore the protective role and mechanism of ginsenoside Rg1 combined with bone marrow mesenchymal stem cell (BMSC) transplantation for cerebral ischemia reperfusion injury (CIRI) in rat brain. METHODS: One hundred twenty male SD rats were randomly divided into a sham group, an Ischemia Reperfusion (IR) group, an IR group treated with BMSC transplantation (IR+BMSCs), an IR group treated with Rg1 (IR+Rg1), and an IR group treated with BMSC transplantation and Rg1 (IR+Rg1+BMSCs). To establish a CIRI model, right middle cerebral artery embolization was used. The neurological score, 2,3,5-triphenyltet-razolium chloride monohydrate (TTC) staining and brain water content were detected to assess the treatment efficiency. HE staining and TUNEL were used to explore the pathologic changes and apoptosis. To explore the protein levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), immunofluoresence was utilized. Western blotting was used to explore apoptosis-related proteins such as Bcl-2 and Bax. RESULTS: Compared with the sham group, the IR group demonstrated obvious ischemic changes, such as significant neurologic defects and enhanced brain water content. The Rg1 treatment resulted in an obvious decrease in cell apoptosis and improved ischemic conditions. By BMSC transplantation, the transplanted cells could be differentiated into neurons and glial cells, which also improved cerebral ischemia. More importantly, the IR+Rg1+BMSCs group showed the best treatment efficiency with reduced cell apoptosis and better cerebral recovery. CONCLUSIONS: The Rg1 treatment resulted in an obvious decrease in cell apoptosis, while the transplanted cells could be differentiated into neurons and glial cells, which also improved cerebral ischemia.

Activation of autophagy in rat brain cells following focal cerebral ischemia reperfusion through enhanced expression of Atg1/pULK and LC3.

The present study aimed to investigate the activation of Atg1/pULK, and LC3 in the cerebral cortex following focal cerebral ischemia reperfusion (CIR) injury, thereby examining its effect on autophagy in brain cells. Rat CIR models were established using the technique of middle cerebral artery occlusion. The neurological function score, TTC staining and the water content of brain tissue were used to evaluate the CIR model. Levels of autophagy in the brain cells were examined at different time­points following CIR damage using electron microscopy. Immunohistochemistry and western blot analysis were also used for the qualitative and quantitative detection of levels of Atg1/pULK and LC3 in the cerebral cortex. Autophagy was observed in the early stage of CIR, and the expression of Atg1/pULK and LC3 were observed 1 h following CIR in the rats and reached peak expression levels after12 h, which following which the they gradually decreased. These results suggested Atg1/pULK and LC3 are key in the regulation of autophagy following CIR in the rat brain.

Nicotinamide Mononucleotide Adenylyltransferase 1 Protects Neural Cells Against Ischemic Injury in Primary Cultured Neuronal Cells and Mouse Brain with Ischemic Stroke Through AMP-Activated Protein Kinase Activation.

Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) is a nicotinamide adenine dinucleotide biosynthetic enzyme. It has been shown to be neuroprotective against neonatal excitotoxicity-induced brain injury, but its role in ischemic stroke is unclear. In this study, the role of NMNAT1 in oxygen-glucose deprivation (OGD)-induced primary cultured neuronal cell injury and mouse middle cerebral artery occlusion-induced cerebral ischemic injury and its regulation on AMP-activated protein kinase (AMPK) activation were evaluated. The results showed that NMNAT1 overexpression reduced cell death and apoptosis both in vitro and in vivo. Conversely, NMNAT1 knockdown exacerbated cell death and apoptosis. Furthermore, NMNAT1 overexpression regulated neuron survival via AMPK activation, as NMNAT1 overexpression enhanced AMPK activity in OGD-treated cortical neurons, and AMPK inhibitor blocked LV-NMNAT1-induced neuroprotection in OGD-treated cortical neurons. In addition, NMNAT1 overexpression could reduce brain infarction size and improve behavioral outcomes in mice with ischemic stroke. These results suggested that up-regulation of NMNAT1 could induce neuroprotection against ischemic injury through AMPK activation and indicated that NMNAT1 is a potential therapeutic target for stroke.

[Effect of echinacoside-containing serum in promoting mesenchymal stem cell osteogenic differentiation and ZHX3 expression in rats].

To investigate the effect and possible mechanism of echinacoside-containing serum on the osteogenic differentiation in rat bone marrow mesenchymal stem cells. Rat bone marrow mesenchymal stem cells were cultivated by the whole bone marrow adherence method. The 3rd generation of cells were divided into 3 groups: the blank control group, the classic osteogenic-induced group and the 10% echinacoside-containing serum group. The expression of alkaline phosphatase and osteocalcin were detected by ELISA. The ex- pression of ZHX, protein was detected by Western blot technique. RT-PCR technique was used to detect the expression of ZHX3mRNA. According to the result, the expressions of the alkaline phosphatase and osteocalcin in the classic osteogenic-induced group and the 10% echinacoside-containing serum group were significantly higher than that of the blank control group (P <0. 01). And expressions of the alkaline phosphatase activity and osteocalcin in the 10% echinacoside-containing serum group were significantly higher than that in the classic osteogenic-induced group (P < 0.01). Meanwhile, the classic osteogenic-induced group and the 10% echinacoside-containing serum group showed obviously higher ZHX3 protain and mRNA expression than that of the black control group, with significant differences (P < 0.01); the 10% echinacoside-containing serum group showed obviously higher ZHX3 protain and mRNA expression than that of the classic osteogenic-induced group, with a significant difference (P < 0.01). In conclusion, 10% echinacoside-containing serum can promote the differentiation of the bone marrow mesenchymal stem cells cultured in vitro. Its mechanism may be correlated with the increase in the ZHX3expression.

Hepatocyte necroptosis induced by ischemic acute kidney injury in rats.

While ischemic acute kidney injury (IAKI) is known often to cause hepatic injury, little is known about necroptosis involved in the hepatic injury. The purposes of this study were to identify necroptosis involvement and observe morphological changes of hepatocytes in hepatic injury induced by IAKI in rats. Based on successfully established IAKI rat models, enzyme-linked immunosorbent assay illustrated a significant higher level of tumor necrosis factor a in serums of IAKI animals. Tumor necrosis factor receptor a (TNFRa) and receptor interacting protein kinase 3 (RIPk3) showed significant higher expressions in immunoblot analyses and positive hepatocytes of RIPk3 immunohistochemical staining were also evident in livers of IAKI rats. In addition, light microscopy revealed necrotic lesions that contain hepatocytes ongoing necroptosis besides necrotic cells in IAKI livers. Electron microscopy revealed at least three types of necrotic hepatocytes, they were edema necrosis, vacuolization necrosis, and necroptosis. Hepatocytes undergoing necroptosis had both necrosis and apoptosis morphological characteristics, they were necrosis cytoplasm and apoptosis-like nucleus. Among cellular organelles of hepatocyte with necrosis, membranous structures, such as cell membrane, endoplasmic reticular system, and mitochondria were more vulnerable to the stress of IAKI and deformed nucleuses varied in shape and lytic or pyknotic chromatin appearances were noted under insults of IAKI. In conclusion, hepatocyte undergoing necroptosis, RIPk3-mediated necroptosis partly contributes to hepatic necrosis induced by IAKI. Both membranous structures and nucleuses of hepatocyte were vulnerable to ischemic acute kidney injury.

Glutathione S-transferase P1 and DNA polymorphisms influence response to chemotherapy and prognosis of bone tumors.

Osteosarcoma is the most common primary bone malignancy in children and adolescents, and its clinical outcome is poor. We evaluated the influence of GSTP1, ERCC1 and ERCC2 polymorphisms on response to chemotherapy among osteosarcoma patients, and the significnace of these genes for prognosis. A total of 187 patients with osteosarcoma were administered methotrexate, cisplatin/adriamycin, actinomycin D, cyclophosphamide, or vincristine. GSTP1, ERCC1 and ERCC2 polymorphisms were genotyped by PCR-RFLP assay. The results showed the average survival time of 187 patients were 38.4 months. Some 97 patients showed response to neoadjuvant chemotherapy. The GSTP1 Val and ERCC2 A/A genotypes had significantly higher rates of response to chemotherapy, with adjusted OR (95% CI) of 2.19 (1.15-6.21) and 2.88 (1.14-13.3). Individuals with the ERCC2 A/A genotype were likely to have a lower risk of death from oseosarcoma, and the adjusted HR was 0.32 (0.13-0.95). Our study indicated that GSTP1 and ERCC2 Lys751Gln polymorphisms might be candidate pharmacogenomic factors to be explored in the future to identify osteosarcoma patients who might benefit from chemotherapy.

[Effect of cisplatin on apoptosis of spiral ganglion cell and expression of caspase-3 in mouse cochlea].

OBJECTIVE: To establish a mice model of cisplatin-induced ototoxicity, and to investigate the effect of cisplatin on apoptosis of spiral ganglion cell and expression of caspase-3 in mouse cochlea. METHODS: Terminal deoxynucleotidyl transferase-mediated nick end labeling method (TUNEL) was used to monitor the apoptosis of spiral ganglion cell. Envision method of immunohistochemistry was applied to detect the expression of caspase-3 in cochlea. Auditory brainstem response (ABR) was measured to observe the change of hearing. RESULTS: The weight and hearing of mice in different dose of cisplatin groups were declined significantly as compared with those of control group (P < 0.05, P < 0.01), and the TUNEL positive cell number and expression of caspase-3 were greater remarkably with the more cisplatin injected. CONCLUSION: A mouse model of cisplatin-induced ototoxicity can be established. Cisplatin can lead to the apoptosis of spiral ganglion cells, and caspase-3 has participated in this apoptosis process, which approves further that apoptosis might be one of the mechanisms of cisplatin ototoxicity.

Grp78 promotes the invasion of hepatocellular carcinoma.

BACKGROUND: Glucose regulated protein 78 (Grp78) is involved in the invasion and metastasis in many human cancers including gastric cancer, breast cancer, prostate cancer. But the role of Grp78 in the invasion of human hepatocellular carcinoma has not been reported. In this article, we examined if Grp78 was associated with the invasion of hepatocellular carcinoma and explored the possible underlying mechanism. METHODS: The Grp78 and FAK expression levels in 44 patients with hepatocellular carcinoma were examined using immunohistochemistry. Grp78 overexpressing SMMC7721 cells were established by pcDNA3.1 (+)-Grp78 transfection and screened by G418. Grp78 and FAK levels in Grp78 overexpressing cells were down-regulated by siRNA transfection. The invasion status of tumor cells was evaluated by transwell assay in vitro, and chick embryo metastasis model in vivo. Cell spreading was determined by cell spreading assay, and quantitatively measured by Orisis software HUG. Grp78, pY397 FAK, pY576/577 FAK and FAK levels were detected by western blot. RhoA activity was detected by GST pulldown assay. The distribution of actin cytoskeleton was observed by fluorescent staining. RESULTS: Grp78 expression levels in 44 patients with hepatocellular carcinoma were negatively correlated with tumor grading, and positively correlated with portal invasion and intra-hepatic invasion. Overexpression of Grp78 in SMMC7721 cells promoted the invasion of cancer cells in vitro and in vivo, and this increase in tumor cell invasion was blocked by Grp78 siRNA knockdown. Our results also revealed that overexpression of Grp78 in SMMC7721 cells accelerated the process of cell spreading and promoted lamellipodia formation. Further analysis showed that overexpression of Grp78 in SMMC7721 cells increased pY397 and pY576/577 levels of FAK. Grp78 siRNA knockdown decreased FAK activation and activity. Our results also revealed that Grp78 overexpression in SMMC7721 cells decreased RhoA-GTP level, and Grp78 siRNA knockdown rescued RhoA-GTP level in Grp78 overexpressing cells, indicating Grp78 inhibited RhoA activity in hepatocellular carcinoma cells. Furthermore, overexpression of Grp78 in SMMC7721 cells increased phospho-p190RhoGAP level. FAK siRNA knockdown in Grp78 overexpressing cells reversed phospho-p190RhoGAP level. These data suggested that Grp78 inhibited RhoA activity by up-regulated phospho-p190RhoGAP level and Grp78 mediated p190RhoGAP phosphorylation is FAK dependent. CONCLUSION: Grp78 promoted the invasion of hepatocellular carcinoma both in vitro and in vivo. Overexpression of Grp78 in hepatocellular carcinoma cells enhanced the activation and activity of FAK which negatively regulated Rock kinase activity by promoting the phosphorylation of p190RhoGAP.