AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .

OBJECTIVE To investigate the molecular mechanisms of resveratrol( Res)in renal interstitial fibrosis(RlF)in rats with unilateral ureteral obstruction(UUO). METHODS Forty-eight Spra-gur-Dawley rats were randomly divided into UUO( normal saline,n = 16),UUO with Res treatment (Res,20 mg·kg-1 ,n=16),and sham-operation(sham,n=16)models. The kidneys were excised on the 7th and 14th day. The deposition of collagen fiber in the kidney was detected with HE and Masson staining. The levels of sonic hedgehog(SHH,an inducer of SHH pathway)in kidney tissues were deter-mined by ELlSA. lmmunohistochemical analysis was performed to evaluate the protein expression of SHH signaling-related molecules,including SHH,smoothened(Smo),patched-1(Ptch1),and Gli1, proliferating cell nuclear antigen(PCNA)and matrix component typeⅢ collagen. The mRNA expression levels of Smo,Ptch1 and Gli1 were detected by real-time RT-PCR. RESULTS The degree of RlF observed with HE and Masson staining was obviously increased in UUO kidneys,but decreased in Res-treated kidneys. Enhanced expression levels of typeⅢ collagen and PCNA in UUO rats were suppressed by Res treatment(P﹤0.05). Res administration decreased the expression levels of SHH,Smo,and Gli1 (P﹤0.05),but increased the expression of Ptch1(P﹤0.05),suggesting that Res inhibit the obstruction-induced activation of SHH signaling. CONCLUSION Res can attenuate RlF in UUO rats,and the possi-ble mechanism is that Res down-regulates the activity of SHH signaling and inhibits cellular proliferation, resulting in inhibition of matrix accumulation in renal interstitium of UUO rats.

Objective To investigate the expression of monocyte-macrophage-related factors and interstitial fibrosis in kidney tissues of rats with ureter obstruction and recanalization .Methods Forty-eight male Spragur-Dawley rats were divided randomly into the obstructive group:sham (n=6), unilateral ureteral obstruction(UUO)3 days (n=6), UUO 7 days (n=6), and UUO 14 days (n=6) and recanalization group:bilateral ureteral obstruction(RBUO)0 day (n=6), 3 days after RBUO (n=6), 7 days after RBUO (n=6), and 14 days after RBUO (n=6).The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fibers in kidney was detected with HE and Masson staining . Immunohistochemical analysis was performed to evaluate the protein expressions of monocyte chemoattractant protein -1 (MCP-1), macrophage colony-stimulating factor (M-CSF) and activated-macrophage marker CD68.Real-time PCR was used to detect the mRNA expressions of MCP-1 and M-CSF.TGF-β1 levels were determined by ELISA .Results Fibrosis observed with HE and Masson staining was obviously increased in kidney tissue of UUO rats , and aggravated as time prolonged, but alleviated in rats with recanalization .TGF-β1 levels were increased obviously in the UUO group , but decreased in rats with recanalization compared with those in BUO rats .In UUO rats, mRNA and protein expression levels of MCP-1 and M-CSF were increased .MCP-1 and M-CSF expression was gradually decreased in rats with recanalization compared with those in BUO rats .The dynamic change in expression of MCP-1 and M-CSF in both UUO rats and recanalization rats was consistent with the change in expression of CD 68. Conclusion Dynamic change in expression of MCP-1 and M-CSF in kidney tissues reflects change of activated and accumulated monocyte -macrophages , which may be one of the major mechanisms contributing to fibrosis induced by ureter obstruction .Renal fibrosis is alleviated by down-regulated expression of monocyte-macrophages factors with recanalization operation .

OBJECTIVE To investigate the effect ofγ-secretase inhibitor N-[N-(3,5-difluorophen?acetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on phenotypic transformation and matrix accu?mulation induced by aristolochic acid(AA) in renal tubular epithelial cells(NRK-52E)and explore the mechanism. METHODS NRK-52E cells were divided stochastically into normal cell control group,AA 10 mg·L-1 group and AA 10 mg·L-1+DAPT 1 and 10μmol·L-1 group. After 24 h,the mRNA expressions of Notch1,Jagged1,Numb,E-cadherin,transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),bone morphogenic protein 7 (Bmp7),typeⅠ a1 (Col1a1) and Ⅲ collagens a1 (Col3a1)were quantified by quantitative real-time RT-PCR. The protein expressions of Notch1,Jagged1,α-SMA,and Col3a1 in NRK-52E cel s were detected by immunofluorescence staining. RESULTS In NRK-52E cells,AA enhanced the expression of TGF-β1,α-SMA and Col3a1 mRNA(P<0.05),reduced the expression of E-cadherin mRNA(P<0.05),up-regulated the mRNA expression of Notch1 mRNA(P<0.01)and Jagged1(P<0.05),and down-regulated the mRNA expression of Numb mRNA(P<0.05) compared with normal cell control group,indicating that phenotypic transformation and matrix accumu?lation occurred in AA-treated NRK-52E cells,accompanied by activated Notch signaling. Treatment with DAPT inhibited Notch signaling by decreasing the expression of Notch1 and Jagged1 (P<0.05),and increasing the expression of Numb mRNA(P<0.05). Furthermore, DAPT also down-regulated the expression levels of TGF-β1,α-SMA,Col1a1 and Col3a1 mRNA(P<0.05), and up-regulated the expression level of Bmp7 and E-cadherin mRNA(P<0.05) compared with AA group,suggesting that DAPT inhibited phenotypic transformation and matrix accumulation in AA-treated NRK-52E cells. CONCLUSION AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells,which is inhibited by DAPT treatment. The possible mechanism is that DAPT suppresses the activation of Notch signaling,resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.

Objective To analyze clinical and laboratory features, viral load and viral shedding period of patients with mild or severe H1N1 influenza A infection. Methods Seventy mild cases and 16 severe cases with concurrent pneumonia were included from Shcnzhen area for analysis.Nasopharyngeal-swab specimens of patients were collected and viral load was detected by real-time quantitative polymerase chain reaction (PCR) assay during their hospitalization. The viral load and viral shedding period were compared between patients over 14 years old and less than 14 years old, and between 70 mild cases without pneumonia and 16 severe cases with pneumonia. The statistic analysis was performed using t test and chi square test. Results The most common symptoms and signs of the patients were fever, cough and enlargement of tonsils. However, the severe cases suffered more frequently from cough, dyspnea and high fever compared with the mild cases (x2 = 10. 9 and 14.3, respectively, t=3.65; both P＜0.01 ). The levels of white blood cell (WBC) count and alanine arninotransferase (ALT) of severe patients were both significantly higher than those of mild patients(t= 3.2, 2.4,respectively; both P＜0.05). The chest radiology of the severe cases showed interstitial pneumonia,mostly with ground glass image. The viral load of patients under 14 years was significantly higher than those over 14 years [(4.86± 1.23) lg vs (4. 17±0.89) lg; t=2.3, P＜0.05], and the viral shedding period of patients under 14 years was significantly longer than those over 14 years [(5.33±0. 49) d vs(3. 63±0.28) d; t=3.4, P＜0.01]. The severe patients also displayed significantly higher viral load and prolonged viral shedding period than the mild patients [(6. 36±1. 44) lg vs (4. 35±0.99) lg, t=6.1,P＜0.01; (5.75±1.77) d vs (4. 24±1. 96) d, t=3.2, P＜0.01]. Conclusion Age anddisease severity of patients with H1N1 influenza A infection are significantly associated with viral load and viral shedding period.

Objective To assess the effects of the risk factors of grafts from donors after cardiac death (DCD) on the prognosis of liver transplantation (LT).Methods In this retrospectively study,215 cases of LT using DCD donor grafts were performed at our institution from September 2013 to January 2017.Due to the loss to follow-up in 4 cases,211 cases were enrolled in the study.The following DCD donor data were collected:gender,age,primary disease,ABO blood type,body mass index (BMI),medical history (fatty liver,hypertension),ICU hospitalization time,mechanical ventilation time,warm ischemia time,cold ischemia time,and indexes of routine laboratory test before donation.Statistical analyses using the Kaplan-Meier method,log-rank test,multivariate step-wise Cox regression were performed.Results Of the 211 donors,univariate analysis showed that the overall 6-month,1-,and 3-year survival rate after DCD LT was 88％,84％,and 82％,respectively.Univariate analysis showed that donor serum sodium level ＜136 mmol/L (P =0.018) and cold ischemia time ＞9 h (P =0.013) were all significant risk factors affecting overall survival after DCD LT.Additionally,donor BMI ＞30 kg/m2 (P =0.011) and donor age ＞60 years (P =0.025) were significantly associated with postoperative complications.Multivariate analysis showed that donor serum sodium level (P=0.025) was an independent risk factor of survival after DCD LT.Conclusion To select suitable DCD liver allografts and control risk factors of donor can help to improve outcomes of recipients.

Objective: To explore the effect of microRNA(miR)-222 on cell proliferation and apoptosis of fibroblasts in hypertrophic scar (HS) and the underlying mechanisms. Methods: The expression of miR-222 in the HS and the normal skin tissues was detected by real-time RT-PCR. The HS fibroblasts were transfected with miR-222 mimic and miR-222 inhibitor respectively. The cell viability was tested with MTT assay, cell cycle distribution and apoptosis were detected with flow cytometry and the expression levels of proliferation, apoptosis and cell cycle related proteins were determined with Western blot. Direct target of miR-222 was evaluated by dual-luciferase reporter assay. Results: miR-222 was significantly up-regulated in HS tissues compared with normal skin tissues(P<0.05). Overexpression of miR-222 enhanced the cell viability of HS fibroblasts; increased mRNA and protein expressions of proliferating cell nuclear antigen (PCNA), collagen alpha-1 (Ⅰ) chain (Col1A1) and collagen alpha-1 (Ⅲ) chain (Col3A1); increased cell population in S phase and protein expressions of cyclin D1, cyclin E1 and cyclin-dependent kinases 1 (CDK1); inhibited cell apoptosis and reduced protein expressions of caspase-3/9. Overexpression of MMP1 attenuated the effects of miR-222 on the cell viability and apoptosis in fibroblasts, reduced expression levels of PCNA, cyclin D1 and the expression of caspase-3 was increased. Conclusion: miR-222 enhances cell proliferation and inhibits cell apoptosis of HS fibroblasts through negative regulation of MMP1, which suggests that miR-222 and MMP1 might be used as novel biomarkers and targets in diagnostic and therapeutic approaches for HS.
Apoptosis ; genetics ; Cell Proliferation ; genetics ; Cicatrix, Hypertrophic ; Fibroblasts ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; MicroRNAs ; metabolism

ObjectiveBased on the network pharmacology system and quantitative spectroscopy of traditional Chinese medicine（TCM） compounds， a topological network analysis method with equilibrium constant as the core was established to further explore the interaction between allergenic components and their network targets in Shuanghuanglian injection（SHLI）， in order to provide new ideas and experimental basis for identifying and screening potential allergens of SHLI. MethodAfter one week of adaptive feeding， 72 SPF-grade SD male rats were randomly divided into blank group， SHLI standard group， Lonicerae Japonicae Flos（LJF） group， Scutellariae Radix（SR） group， Forsythiae Fructus（FF） group， and 7 groups of SHLI matching groups（groups 1-7）， with 6 rats in each group. Rats in each group were administered the drug intravenously and blood samples were taken after steady state， high performance liquid chromatography（HPLC） characterization profiles of the testing drugs and plasma components in each group were established， and the peak area changes of the drugs and plasma components in each group were calculated after the component groups were classified. Enzyme-linked immunosorbent assay（ELISA） was used to determine the changes of immunoglobulin E（IgE）， histamine（HIS）， tryptase（TPS）， total complement（CH50） and terminal complement complex（C5b-9） in animal blood samples. MATLAB R2020b v9.9.0 software was used to calculate the network balance constants of the component groups with the targets， and the eigenvalues of the matrices composed of network equilibrium constants were calculated and ranked according to their values. ResultELISA results showed that， compared with the blank group， groups 1-3 could significantly increase the IgE level， groups 1-2， groups 4-6 and SHLI standard group could significantly increase the HIS level， group 4 could significantly increase the CH50 level， groups 1， 3-4， LJF group and FF group could significantly increase the TPS level， SR group could significantly increase the C5b-9 level， and the differences were all statistically significant（P<0.05）. According to the retention time of chromatographic peaks， it was classified into 6 component groups from C1 to C6 by HPLC. The order of the network balance constants of each component group was C6>C4>C1>C5>C3>C2， indicating that C6 had the greatest effect on the allergic reaction， and was most likely to be the allergen. The sequence of eigenvalues was C2>C5b-9>C3>C1>CH50>C6>C5>IgE>TPS>C4>HIS， indicating that component group C2 had the greatest contribution to the whole network. ConclusionBased on the correlation analysis of SHLI component group and allergy-like target network， this study clarified that component group C6 may be a potential allergen in SHLI， and the component group C2 may be a key node in the mechanism of drug action， which can provide new strategies and methods for the screening of allergens in TCM injections.