[Role of CTGF and PI3K/Akt signaling pathway in paraquat-induced mesenchymal changes in alveolar epithelial cells].

Objective: To investigate the role of connective tissue growth factor (CTGF) and PI3K/Akt signaling pathways in paraquat (PQ) -induced alterations in alveolar epithelial cell mesenchymalization (EMT) . Methods: In February 2023, RLE-6TN cells were divided into 2 groups, which were set as uncontaminated group and contaminated group (200 µmol/L PQ), and cellular EMT alteration, CTGF and PI3K/Akt signaling pathway related molecules expression were detected by cell scratch assay, qRT-PCR and western-blot assay. Using shRNA interference technology to specifically inhibit the expression of CTGF, RLE-6TN cells were divided into four groups: control group, PQ group (200 µmol/L PQ), interference group (transfected with a plasmid with shRNA-CTGF+200 µmol/L PQ), and null-loaded group (transfected with a plasmid with scramble- CTGF+200 µmol/L PQ), qRT-PCR and western blot were used to examine the alteration of the cellular EMT and the expression of molecules related to the activity of PI3K/Akt pathway. The PI3K/Akt signaling pathway was blocked by the PI3K inhibitor LY294002, and the expression of EMT-related molecules in cells of the control group, PQ group (200 µmol/L PQ), and inhibitor group (200 µmol/L PQ+20 µmol/L LY294002) was examined by qRT-PCR and western blot.The t-test was used to compare the differences between the two groups, while the analysis of variance (ANOVA) was applied to compare the differences among multiple groups. For further pairwise comparisons, the Bonferroni method was adopted. Results: The results of cell scratch test showed that compared with the uncontaminated group, RLE-6TN cells in the contaminated group had faster migration rate, lower mRNA and protein expression levels of E-Cadherin, and higher mRNA and protein expression levels of α-SMA, CTGF, PI3K and Akt, with statistical significance (P<0.05). After specific inhibition of CTGF expression, the mRNA and protein expression of CTGF, PI3K, Akt, and α-SMA in the cells of the interference group were significantly lower than that of the PQ group and the null-loaded group (P<0.05/6), whereas that of E-Cadherin was higher than that of the PQ group and the null-loaded group (P<0.05/6). Specifically blocking the PI3K/Akt signaling pathway, the mRNA and protein expression of PI3K, Akt and α-SMA in the cells of the inhibitor group was decreased compared with that of the PQ group (P<0.05/3), while the expression of E-Cadherin was elevated compared with that of the PQ group (P<0.05/3) . Conclusion: CTGF may promote PQ-induced alveolar epithelial cell EMT through activation of the PI3K/Akt signaling pathway. Inhibition of CTGF expression or blockade of PI3K/Akt signaling pathway activity can alleviate the extent of PQ-induced alveolar epithelial cell EMT.

[Analysis of occupational health status of radiation workers in Guangdong Province].

Objective: To discuss the effect of low-dose ionizing radiation on the health of radiation workers, and provide a basis for occupational health risk assessment of radiation workers. Methods: In January 2020, 3165 radiation workers who performed radiation occupational health examinations in Guangzhou Prevention and Treatment Hospital for Occupational Disease from January 2017 to December 2019 were selected as the research objects, and compared and analyzed the health status of radiation workers with different examination types (pre-job, in-job and off-job) , types of work, gender, and length of service. Results: The off-job occupational radiological health examination was rare at 2.3% (74/3165) . The abnormal detection rate of chest radiographs, renal function, thyroid function, and blood routine of the radiation workers in-job group was higher than that of the pre-job group (P<0.05) . No statistical difference was found in the abnormal detection rate of the examination items during the in-job group and the off-job group (P>0.05) . The blood routine abnormality detection rate of medical application group and industrial application group were higher than those of nuclear fuel group (P<0.05) . The abnormal detection rate of blood pressure and renal function of male radiation workers was higher than that of females, while the abnormal detection rate of blood routine of females was higher than that of males (P<0.05) . The abnormal detection rate of electrocardiogram, chest radiograph, blood pressure, renal function, thyroid function, and blood routine of radiation workers increased with increasing working age (P<0.05) . Conclusion: Occupational health status of radiation workers is not optimistic. Radiation occupational health monitoring should be strengthened, special attention should be paid to off-job radiation occupational health examination, focusing on the sensitive indicators of sensitive personnel, improving radiation protection conditions, and effectively protecting the occupational health of radiation workers.

[Effect of low dose ionizing radiation on peripheral blood cells of radiation workers in nuclear power industry].

Objective: To investigate the effects of long-term ionizing radiation on peripheral blood cells of nuclear power workers. Methods: In March 2019, a total of 530 radiation exposed workers in the nuclear power industry who underwent in-service radiation occupational health examination in Guangzhou occupational disease prevention and control hospital in 2018 and with service age ≥1 year were selected as the radiation group. At the same time, 545 workers in nuclear power industry were selected as control group. According to the methods and requirements of GBZ 235-2011 ＂technical specification for occupational health monitoring of radiation workers＂ and GBZ 98-2017 ＂health requirements for radiation workers＂, the occupational health monitoring data were collected, and the change rules of peripheral blood cells in the two groups were analyzed. Results: Compared with the control group, the total number of WBC, NEUT, LYMP, Hb, MCV and MCHC in radiation group were lower than those in control group (P<0.05) , and the difference was statistically significant (P<0.05) . The MPV increased significantly (P<0.05) . Compared with the control group, the abnormal rate of WBC and Hb in the radiation group was higher than that in the control group (P<0.01) , but there was no significant difference in the abnormal rate of RBC and PLT (P>0.05) . Conclusion: Low dose ionizing radiation has a certain cumulative damage effect on peripheral blood cells of radiation workers in nuclear power industry. The change rules of different cell subtypes are different, and the changes of WBC and PLT appear earlier.

[Effect of bone marrow mesenchymal stem cells on paraquat-induced pulmonary fibrosis in rats].

Objective: To study the effect of the injected bone marrow mesenchymal stem cells (BMSC) on rats with pulmonary fibrosis induced by paraquat (PQ) during different poisoning periods and explore the potential mechanism. Methods: From October to December 2018, BMSCs of SPF SD rats were isolated and purified by whole-bone marrow adherent culture method and cultured to the Third Generation (P3) . The surface antigens CD29, CD90, CD45 and CD34 of P3 BMSC were detected by Flow cytometry, the formation of alkaline phosphatase (ALP) , calcium nodules and fat droplets were observed by ALP, Alizarin Red staining and oil red O staining. At the same time, 36 SPF male rats were randomly divided into 6 groups: NC Group (Blank Control Group, injected with the same amount of saline) and PQ group (PQ model group, injected with 20% PQ solution 18 mg/kg intraperitoneally) , bMSC-A group, BMSC-B group, BMSC-C group and BMSC-D group were injected with BMSC suspension 1×10(6) cells/mice at 3 h、3 d、7 d and 14 d after PQ poisoning. After 28 days, the rats were killed, the lung organ coefficients were calculated, the hydroxyproline (HYP) content in lung tissue was calculated by alkaline hydrolysis, and the lung injury and fibrosis were observed by HE and Masson staining, serum TGF-1、TNF-α、MMP-9 and TIMP-1 were detected by Elisa. Results: High Purity BMSCs were successfully isolated and obtained. The P3 BMSC generation was positive expression of CD29、CD90、and negative expression of CD34、CD45, and had the potential of osteogenic and adipogenic differentiation. The results of HE staining and Masson staining showed that the alveolar structure in NC group was intact and homogeneous, in PQ group, the alveolar structure was severely damaged and a lot of collagen fibers and fibroblasts were deposited, and the degrees of lung injury in each BMSC intervention group were obviously less than in PQ group, in BMSC-A group and BMSC-B group, the degrees of reduction were obvious. Compared with NC group, the Lung organ coefficient, HYP content in lung tissue and TGF-ß1, TIMP-1 levels in serum were significantly higher in PQ group (P<0.05) , while TNF-α and MMP-9 had no significant difference (P>0.05) . Compared with PQ group, the lung organ Coefficients, HYP, TGF-1 and TIMP-ß1 in BMSC-A and BMSC-B groups were lower than those in PQ group (P<0.05) . The Lung organ coefficients, TGF-ß1 and TIMP-1 in BMSC-C and BMSC-D groups were lower than those in PQ group, there was no significant difference (P>0.05) . Conclusion: Early BMSC injecting can alleviate pulmonary fibrosis induced by PQ. The mechanism may be that BMSC can reduce pulmonary fibrosis through reducing the level of TGF-ß1 and regulating the balance of TIMP-1/MMP-9, threrby reducing inflammatory damage and increasing the degradation of extracellular matrix (ECM) .

[Effects and mechanism of apolipoprotein A5 on adipogenic differentiation of human adipose-derived mesenchymal stem cells].

Objective: To investigate the effect and related mechanism of apolipoprotein A5 (ApoA5) on adipogenic differentiation of human adipose-derived mesenchymal stem cells (AMSC). Methods: Subcutaneous adipose tissue was obtained from 40 patients undergoing abdominal surgery at our hospital from February to July 2015. After induction of human AMSC by collagenase digestion, the adipose tissue was induced to differentiate into mature adipocytes and treated with ApoA5 at 600 and 1 200 ng/ml, respectively (ApoA5 intervention groups). Cells treated without ApoA5 protein were used as control group. The cells were harvested on the 7th and 14th day of differentiation, and the following assays were performed: (1) the effect of ApoA5 on TG content was measured by a TG assay kit; (2) RT-qPCR assay was used to detect the effect of ApoA5 on aP2 and FAS mRNA expression; (3) the effect of ApoA5 on the expression of CIDEC mRNA and protein was detected by RT-qPCR and Western blot; (4) the effect of ApoA5 on the expression of C/EBPß mRNA and protein was detected by RT-qPCR and Western blot; (5) using lentiviral transfection technique, we overexpressed the gene of CIDEC in AMSC and cells were divided into lentiviral negative control group, lentiviral over-expressed CIDEC group and lentiviral over-expressed CIDEC+ApoA5 intervention group (the ApoA5 intervention concentration was 1 200 ng/ml). Thereby, we examined the effect of ApoA5 on the above indicators in adipogenic differentiation of AMSC in the case of CIDEC overexpression. Results: (1) Effect of ApoA5 on TG content in AMSC: on the 7th and 14th day after the intervention, the TG levels were lower in ApoA5 600 and 1 200 ng/ml group AMSC than those in the control group (all P<0.05). (2) The effect of ApoA5 on the expression of aP2 and FAS mRNA in AMSC: on the 7th day after intervention, the expression levels of aP2 and FAS mRNA were significantly lower in ApoA5 600 and 1 200 ng/ml group than those in the control group (all P<0.05). On the 14th day after intervention, the expression levels of aP2 and FAS mRNA were lower in ApoA5 600 and 1 200 ng/ml group than those in the control group (all P<0.05). (3) The effect of ApoA5 on the mRNA and protein expression of CIDEC in AMSC: on the 7th day after intervention, the mRNA and relative protein expression levels of CIDEC were significantly lower in AMSC of ApoA5 600 and 1 200 ng/ml group than those of the control group (all P<0.05). On the 14th day after intervention, the mRNA and relative protein levels of CIDEC were further reduced in ApoA5 600 and 1 200 ng/ml AMSC groups than those in the control group (all P<0.05). (4) The effect of ApoA5 on C/EBPß mRNA and protein expression in AMSC: on the 7th day after intervention, C/EBPß mRNA and relative protein expression levels were significantly lower in ApoA5 600 and 1 200 ng/ml group than those in the control group (all P<0.05). On the 14th day after intervention, the levels of C/EBPß mRNA and relative protein were lower in ApoA5 600 and 1 200 ng/ml group than those in the control group (all P<0.05). (5) The effect of ApoA5 on the content of TG in AMSC after CIDEC overexpression: on the 7th and 14th day after intervention, the TG contents in AMSC were higher in the lentivirus over-expressed CIDEC group than in the lentivirus negative control group (both P<0.05). However, TG contents in AMSC were similar between the over-expressed CIDEC group and the CIDEC+ApoA5 over-expression group (both P>0.05). (6) The effect of ApoA5 on the expression of aP2 and FAS mRNA in AMSC after CIDEC overexpression: on the 7th day after intervention, the expression levels of aP2 and FAS mRNA in AMSC were higher in the lentivirus over-expressed CIDEC group than in the lentivirus negative control group (both P<0.05). On the 14th day after intervention, the expression level of aP2 mRNA in the AMSC was higher in the lentivirus over-expressed CIDEC group than in the lentivirus negative control group (P<0.05). On the 7th and 14th day after intervention, the expression levels of aP2 and FAS mRNA in AMSC were similar between the lentivirus over-expressed CIDEC group and the lentivirus over-expressed CIDEC+ApoA5 group (all P>0.05). (7) The effect of ApoA5 on the expression of C/EBPß mRNA and protein in AMSC after CIDEC overexpression: on the 7th day after intervention, the mRNA and relative protein expressions of C/EBPß in AMSC were higher in lentivirus-overexpressed CIDEC group than in lentivirus negative control group (both P <0.05). On the 14th day after intervention, C/EBPß mRNA and protein expression levels in AMSC were higher in the lentivirus over-expressed CIDEC group than in the lentivirus negative control group (both P<0.05). On the 7th and 14th day after intervention, the expressions of C/EBPß mRNA and protein in AMSC were similar between lentivirus over-expressed CIDEC group and lentivirus over-expression CIDEC+ApoA5 intervention group (all P>0.05). Conclusions: ApoA5 can inhibit the adipogenic differentiation of AMSC,and this effect may be mediated by down-regulating the expression of CIDEC. Furthermore, our results indicate that CIDEC could be considered as a key factor in adipogenic differentiation.

[The effect of pulmonary injury in rats induced by paraquat].

Objective: To study the effect of sodium aescinate on the development process of lung injury induced by paraquat. Methods: Forty-five health adult male SD rats were randomly divided into control group, PQ group, sodium aescinate group, and 15 rats in each group. The PQ group and sodium aescinate group were given a one-time intraperitoneal injection of 18mg/kg body weight of rats PQ, the control group was given the same amout normal saline. Rats in sodium aescinate group were injected 2 mg/kg body weight sodium aescinate into abdominal cavity for 7 days continually, but the same volume of saline was injected into the other groups. Finally, at 7, 14 and 28 days after PQ poisoning, five rats were kills for measuring lung tissue pathological changes and the value of TGF-ß(1), TNF-α, hydroxyproline in each group. Results: The expression of TNF-α in serum of 7th day [ (17.03±0.82) ng/ml] and 14th day [ (15.74±0.91) ng/ml] of sodium aescinate group were lower than the corresponding period of PQ groups', and the difference had statistical significance (P<0.05) . The expression of TGF-ß(1) in serum of 7th day[ (225.93±8.33) ng/ml], 14th day [ (216.62±9.48) ng/ml] and 28th[ (181.41±6.10) ng/ml] of sodium aescinate group were lower than the corresponding period of PQ groups', and the difference had statistical significance (P<0.05) . Lung tissue pathological changes showed, compared with control group, inflammatory injury at 7th day and fibrosis degree at 28th of rats' lung reduced on sodium aescinate group. The expression of hydroxyproline in rats' lung of 7th day[ (1.246±0.018) µg/mg], 14th day [ (1.269±0.034) µg/mg] and 28th[ (1.283±0.028) µg/mg] of sodium aescinate group were lower than the corresponding period of PQ groups', and the difference had statistical significance (P<0.05) . Conclusion: sodium aescinate could reduce the pulmonary inflammatory injury and hydroxyproline value of PQ poisoning rats, so sodium aescinate could ameliorate lung injury induced by PQ.

[Role of green tea polyphenols in noise-induced hearing loss].

Objective: To investigate the role and mechanism of action of green tea polyphenols in noise-induced hearing loss. Methods: Male specific pathogen-free guinea pigs were randomly divided into normal control group with 9 guinea pigs, noise exposure group with 36 guinea pigs, and green tea polyphenol intervention group with 36 guinea pigs. Auditory brainstem response (ABR) threshold shift was examined before noise exposure and at 1, 3, 7, and 14 days of noise exposure. The surface preparation of cochlear basilar membrane was used for hair cell count and the morphology of hair cells was also observed. Western blot was used to observe the expression of cysteinyl aspartate-specific protease-9 (caspase-9) and cysteinyl aspartate-specific protease-3 (caspase-3) in cochlear tissue. Results: Both the noise exposure group and the green tea polyphenol intervention group had an increase in ABR threshold after noise exposure, and the green tea polyphenol intervention group had a significantly lower ABR threshold shift than the noise exposure group at all time points (P<0.05). Both groups had enlargement, atrophy, or loss of hair cells after noise exposure, and at 7 and 14 days of noise exposure, the noise exposure group had a significantly higher rate of abnormal hair cells than the green tea polyphenol intervention group (P<0.05). Both groups had an increase in the expression of caspase-9 and caspase-3 after noise exposure, and the noise exposure group had a significantly greater increase than the green tea polyphenol intervention group (P<0.05). Conclusion: Green tea polyphenols can reduce noise-induced hearing loss and hair cell injury, possibly by regulating the expression of caspase-9 and caspase-3.

[Changes of integrin-linked kinase expression on rats' pulmonary fibrosis induced by paraquat].

Objective: To observe the expression of integrin-linked kinase on pulmonary fibrosis of paraquat (PQ) poisoning rats, and to discuss the relationship between ILK with pulmonary fibrosis induced by paraquat. Methods: Forty male Sprague-Dawley (SD) rats were randomly divided into control group and paraquat group, 20 in each group; the PQ group rats were intraperitoneally injected PQ liquid (18 mg/kg) , and the control group rats were injected the same volume of saline; 5 rats of these two groups were respectively sacrificed after 7, 14, 28, 56 days of PQ injection; according to the results of lung biopsy HE staining and Masson staining to observe the lung pathologic changes, measure the value of lung hydroxyproline and the expression of ILK. Results: HE and Masson staining of lung pathological biopsy showed, the 7th day after PQ exposure lung tissue mostly had congestion, edema, inflammatory cells infiltration; the 14th inflammation reduced, fibrosis change appeared gradually; the 28th and 56th showed the lung tissue structure disorder and occurred apparent hydroproline with blue dye in pulmonary interstitium. Compared with control group in the same experiment period, the value of lung hydroxyproline in each experiment period of PQ group increased (P<0.05) , and the value of lung hydroxyproline of PQ group rose with the increasing of the time of PQ poisoning. The expression of ILK mRNA and protein in each experiment period of PQ group was higher than the control group in the same experiment period (P<0.05) ; ILK mRNA and protein of PQ group began to increase on 7 day phase, reached the highest on 28 day phase, and decreased on 56 day phase. Conclusion: The expression of ILK mRNA and protein increased with the lung fibrosis progression of PQ poisoning rats, so ILK could be the key molecule target which induced pulmonary fibrosis of PQ poisoning.

Anti-inflammatory effects of Lactobacillus brevis K65 on RAW 264.7 cells and in mice with dextran sulphate sodium-induced ulcerative colitis.

Lactic acid bacteria (LAB) with anti-inflammatory effects may be beneficial to the prevention or treatment for inflammation-related diseases, such as inflammatory bowel diseases. In an in vitro assay, heat-killed Lactobacillus brevis K65 (K65) reduced lipopolysaccharide-induced production of nitric oxide, tumour necrosis factor (TNF)-α and prostaglandin E2 in RAW 264.7 cells. In RAW 264.7 cells stably expressing an ind=ucible nitric oxide synthase (iNOS) reporter, viable K65 showed greater inhibition of iNOS production than its heat-killed form. In order to further examine the in vivo anti-inflammatory effect of K65, viable K65 was orally administered to BALB/c mice before and during the period of dextran sulphate sodium (DSS)-induced ulcerative colitis (UC). K65 improved UC symptoms, including reduced the levels of the pro-inflammatory cytokines, TNF-α, interleukin (IL)-6 and IL-1ß, and lowered the activity of myeloperoxidase. Furthermore, K65 inhibited TNF-α, cyclo-oxygenase 2, forkhead box P3, and Toll-like receptor 4 mRNA expression in the colonic tissue of DSS-induced UC mice. Taken together, K65, a LAB with in vitro anti-inflammatory activity showed preventive effects on mice with DSS-induced UC by lowering the expression of inflammatory molecules.

Effect of small interfering RNA on the expression of connective tissue growth factor and type I and III collagen in skin fibroblasts of patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is characterized by an excessive production of extracellular matrix. It is widely accepted that fibrosis is induced by transforming growth factor (TGF)-beta in the early stage and is subsequently maintained by connective tissue growth factor (CTGF). CTGF is a cysteine-rich mitogenic peptide that has been involved in various fibrotic disorders and can be induced in fibroblasts by activation with TGF-beta. OBJECTIVES: To evaluate the effect of small interfering RNA (siRNA) targeting CTGF on the expression of CTGF and type I and type III collagen in SSc. METHODS: Skin fibroblasts from patients with SSc were cultured in vitro and later transfected using four CTGF-specific siRNAs and one nonspecific siRNA. The effect of CTGF-specific siRNAs on the expression of CTGF and type I and type III collagen was examined and quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry. RESULTS: Semiquantitative RT-PCR analysis showed that the four CTGF-specific siRNAs significantly reduced CTGF mRNA expression (P < 0.001), of which siRNA742 showed the strongest inhibitory effect with an inhibitory rate of 73%. Three of the four siRNAs could also depress the transcriptional levels of type I and type III collagen mRNA (P < 0.001), of which siRNA742 showed the strongest inhibitory effect with an inhibitory rate of 37% and 29% for type I and type III collagen, respectively. Western blot analysis further demonstrated that three CTGF-specific siRNAs could significantly decrease CTGF protein level (P < 0.001). In addition, immunocytochemical analysis showed that the expression of type I collagen was significantly decreased in fibroblasts after transfection with siRNA742, whereas inhibition of expression of type III collagen was modest. CONCLUSIONS: Our data for the first time showed that CTGF RNA interference could inhibit expression of CTGF and type I and III collagen in SSc fibroblasts and indicated that CTGF might be an upstream factor regulating type I and type III collagen synthesis, particularly type I collagen. Our findings suggest that silencing CTGF expression might facilitate a potential therapeutic approach for SSc.