To explore the effect of naringin (NG) on MC3T3-E1 cell viability under high glucose environment and its possible molecular mechanism.

Cultured in vitro, mouse MC3T3-E1 cells were were divided into 5 groups, including normal control group, high glucose group (25 mmol/L glucose) , 0.1 μmol/L NG+25 mmol/L glucose group, 1 μmol/L NG +25 mmol/L glucose group, 10 μmol/L NG +25 mmol/L glucose group. After drug intervention, the cell viability was measured by CCK-8 assay. The mRNA expression of IGF-1, Akt1 and Runx2 were detected by qPCR. The protein expression of IGF-1, Akt1 and ALP were determined by Western blot. Univariate analysis of variance was used for comparison among groups, and LSD-t test was used for pairwise comparison between groups.

The results of CCK8 assay showed that compared with the control group, the OD values of the high glucose group (12 h: 0.90±0.01 vs 0.80±0.01, 24 h: 1.00±0.05 vs 0.84±0.01, 48 h: 1.09±0.03 vs 0.90±0.01) were all reduced, and the difference was statistically significant (P < 0.01) . Compared with the high glucose group, the cell OD values were increased in 0.1 μmol/ L+glucose group (24 h: 0.93±0.05, 48 h: 0.99±0.01) , 1 μmol/L+ glucose group (12 h: 0.92±0.01, 24 h: 1.01±0.04, 48 h: 1.12±0.02) and 10 μmol/L+ glucose group (12 h: 1.01±0.32, 24 h: 1.16±0.03, 48 h: 1.20±0.02) , the difference was statistically significant (P < 0.05) . Compared with the control group, the high-glucose group decreased in Runx2, IGF-1, and Akt1 expression levels (24 h: 1.00 vs 0.34±0.02, 1.00 vs 0.34±0.01, 1.00 vs 0.15±0.02; 48 h: 1.00 vs 0.72±0.03, 1.00 vs 1.09±0.07, 1.00 vs 0.38±0.04) , and the differences were statistically significant. Compared with the high glucose group, 0.1 μmol/L+ glucose group had no significant difference in the gene expression. While the cell mRNA expression of Runx2, IGF-1 and Akt1 were increased in both 1 μmol/L+glucose group (24 h: 0.62±0.09, 0.77±0.03, 0.24±0.08; 48 h: 1.27±0.02, 2.44±0.19, 0.86±0.06) and 10 μmol/L+glucose group (24 h: 0.64±0.05, 1.02±0.07, 0.40±0.09; 48 h: 1.37±0.02, 2.73±0.04, 1.43±0.03) the differences were statistically significant. Compared with the control group, the cell expression levels of ALP, Akt1 and IGF-1 protein in the high glucose group were reduced (48 h: 1.00 vs 0.72±0.02, 1.00 vs 0.89±0.03, 1.00 vs 0.09±0.01) , and the differences were statistically significant (P < 0.05) . Compared with the high glucose group, after 48 hours of drug intervention, the expression of ALP, Akt1 and IGF-1 protein increased in 0.1 μmol/L+glucose group (1.92±0.02, 1.50±0.03, 1.75±0.01) , 1 μmol/L+glucose group (2.30±0.30, 1.43±0.04, 2.30±0.31) , 10 μmol/ L+GLUgroup (3.09±0.10, 1.40±0.13, 2.07±0.07) , the difference was statistically significant (P < 0.05) .

NG reversed the viability reduction of MC3T3-E1 cells induced by high glucose, while improved the inhibition of high glucose and promoted the expression of IGF-1, Akt-1, Runx2 mRNA and IGF-1, Akt-1, ALP protein in MC3T3-E1 cells.

To explore influence factors of steroid-resistant acute gastrointestinal graft versus host disease (aGVHD) in allogeneic hematopoietic stem cell transplantation.

A retrospective analysis was conducted on 20 patients with aGVHD who underwent allogeneic hematopoietic stem cell transplantation in our hospital from January 2012 to December 2019. Patients were divided into steroid-sensitive group (13 cases) and steroid-resistant group (20 cases) . Patients' gender, age, diagnosis as well as pre-transplant MRD, transplantation type, donor's age, donor-recipient relationship, ABO blood type, mononuclear cell number, CD34⁺ cell number, CD3⁺ cells in graft, neutrophil and platelet engraft time, CMV and EB infection, aGVHD onset time in gastrointestinal tract, of the above factors' relationship with steroid-resistant gastrointestinal aGVHD were analyzed by univariate logistics regression.

Among 20 patients with gastrointestinal aGVHD, 7 patients were steroid-resistant. When acute GVHD occurs less than 1 month , the risk of steroid resistance was increased (OR = 13.500, 95 ﹪CI = 1.197 ~ 152.211, P = 0.035) . Gender, age, diagnosis, pre-transplant MRD, transplant type, donor age, donor-recipient relationship, donor-recipient ABO blood group, transfused monocytes, CD34⁺ cells, CD3⁺ cells, neutrophils and platelet implantation time, CMV and EB virus infection have no effect in steroid resistance (P > 0.05) . Logistic regression analysis showed that aGVHD onset time in gastrointestinal tract was negatively correlated with steroid-resistant. 7 patients with steroid-resistant gastrointestinal aGVHD were treated with CD25 monoclonal antibody while 2 patients died after ineffective treatment. One year overall survival and progression free survival rates of the steroid-sensitive patients were 64﹪and 28﹪, and one year overall survival and progression free survival rates of the steroid-resistant patients were 52﹪ and 32﹪. There was no significant difference of 1-years OS and PFS between two groups (P > 0.05) .

Steroid-resistant gastrointestinal aGVHD was related to its onset time after allogeneic hematopoietic stem cell transplantation. The earlier it occurs, the more likely it is to be steroid resistance.

To investigate the effect and mechanism of interfering FSCN1 gene expression on apoptosis and reactive oxygen species (ROS) level in prostate cancer cells.

The normal prostate epithelial cell RWPE-1 was used as the control, and the expression of FSCN1 mRNA and protein in the LNCaP, DU145 and PC-3 cells of prostate cancer were detected by RT-PCR and Western blot, respectively. Lipofectamine^TM 2000 was used as the carrier, DU145 cells were divided into si-FSCN1 group (small interfering RNAs targeting FSCN1 transfected DU145 cells) , negative control group (negative control siRNA transfected DU145 cells) and blank control group (untransfected cells) , siRNA was transfected for 48 h, and Western blot was used to detect the protein expression of FSCN1, PCNA, Cleaved caspase3, NF-κB p65, p-NF-κB p65, IKK and p-IKKα. Cell viability was detected by CCK8, and apoptosis rate and ROS level were detected by flow cytometry. One-way analysis of variance was used for comparison between groups, and SNK-q test was used for pairwise comparisons between groups.

Compared with RWPE-1 cells, the expressions of FSCN1 mRNA in LNCaP, DU145 and PC-3cells (1 vs 2.561±0.189, 7.183±0.882, 4.796±0.567, 4.796±0.567) and protein (0.053±0.007 vs 0.217±0.013, 0.654±0.058, 0.316±0.035) were increased (all P< 0.05) . Compared with the negative control group, the expression of FSCN1 in the si-FSCN1 group were decreased (0.473±0.052 vs 0.086±0.010, P< 0.05) . Compared with the si-FSCN1 group, the cell viability of the blank control group and the negative control group (0.302±0.033 vs 0.787±0.069, 0.764±0.063) were increased, while the apoptotic rate (24.54±1.47﹪vs 3.04﹪±0.36﹪, 3.28﹪±0.40﹪) and ROS relative fluorescence intensity (90.04±5.73 vs 47.88±3.62, 49.62±4.11) were decreased (all P< 0.05) . Compared with si-FSCN1 group, the expression of PCNA (0.255±0.032 vs 0.654±0.062, 0.631± 0.058) , NF-κB p65 (0.092±0.011 vs 0.296±0.032, 0.318±0.037) , p-NF-κB p65 (0.042±0.008 vs 0.155±0.018, 0.151±0.016) , IKKα (0.112±0.01 vs 0.172±0.020, 0.192±0.023) and p-IKKα protein (0.051±0.005 vs 0.102±0.011, 0.091±0.009) were increased in blank control group and negative control group, while the expression of Cleaved caspase3 protein (0.206±0.018 vs 0.074±0.009, 0.085±0.010) decreased (all P< 0.05) . There was no significant difference in cell viability, apoptosis rate, ROS levels, and protein expression of FSCN1, PCNA, Cleaved caspase3, NF-κB p65, p-NF-κB p65, IKKα, and p-IKKα in the negative control group and the blank control group (P> 0.05) .

Interfering with FSCN1 gene expression could reduce the activity and induce apoptosis of prostate cancer cells. The mechanism may be related to the increase of ROS level and the decrease of NF- κB signal.

To investigate the effect of miR-652-3p targeting autonomous heterologous nuclear gene 1 (PRRX1) in the regulation of angiotensinⅡ (AngII) -induced cardiomyocyte apoptosis.

Rat cardiac cell H9c2 cultured under normal conditions were used as the control group, while AngⅡgroup were established in medium containing 1 μmol/L AngⅡ. These cells were transfected with miR-652-3p+Vctor and miR-652-3p mimics respectively. In addition AngⅡ-H9c2 cells transfected with miR-652-3p mimics were transfected with pc-PRRX1 and overexpressed-PRRX1. The expression of miR-652-3p in H9c2 was determined by quantitative real-time PCR (qRT-qPCR) . Detection of cell apoptosis was evaluated by using flow cytometry. In addition the protein expression levels of Bax and Bcl-2 were assessed by Western blot analysis. Gene regulatory relations between miR-652-3p and PRRX1 were confirmed by Dual-Luciferase reporter gene system. The t-test was applied for comparison between two groups and single factor variance analysis (one-way ANOVA) was used for comparison among several groups. The SNK-q test method was used to compare between groups.

Compared with control group, the level of miR-652-3p (1.00±0.08 vs 0.21±0.05) and Bcl-2 protein (0.83±0.08 vs 0.40±0.04) in H9c2 cells were lower in AngⅡgroup, while PRRX1 protein level (0.06±0.01 vs 0.41±0.04) , apoptosis rate (5.02﹪±1.41﹪vs 25.33﹪±3.75﹪) , Bax protein level (0.46±0.05 vs 0.96±0.10) were higher (all P< 0.05) . Compared with AngⅡ+NC group, the expression level of miR-652-3p (0.24±0.06 vs 0.98±0.07) and Bcl-2 protein level in the AngⅡ+miR-652-3p group (0.38±0.04 vs 0.72±0.07) were higher while the PRRX1 protein level (0.39±0.04 vs 0.13±0.01) , and apoptosis rate (27.02﹪±4.11﹪ vs 12.19﹪±1.63﹪) , Bax protein level (0.95±0.09 vs 0.53±0.05) were lower (all P< 0.05) . Compared with the AngⅡ+miR-652-3p+Vctor group, the apoptosis rate of H9c2 cells (12.88﹪±1.84﹪vs 25.45﹪±3.58﹪) as well as expression of PRRX1 protein (0.13±0.01 vs 0.35±0.04) and Bax protein (0.54±0.05 vs 0.82±0.08) in the AngⅡ+ miR-652-3p+PRRX1 group were higher (all P< 0.05) , whereas Bcl-2 protein level (0.72±0.07 vs 0.46±0.05) was lower (all P< 0.05) .

AngⅡcould down-regulatethe expression of miR-652-3p in cardiomyocytes. Additionally, Up-regulation of miR-652-3p could reduce the apoptosis of H9c2 cells induced by AngⅡ by targeting the inhibition of PRRX1 expression.

To investigate the anti-tumor effect and mechanism of shikonin on colorectal cancer cell LoVo.

The colorectal cancer cell LoVo was treated with different concentration gradients (0, 2, 4, 6 μmol/L) for 24 hours. Besides, the other colorectal cancer cell LoVo was treated with 4μmol/L of shikonin according to different time gradients (0, 12, 24, 48 h) . The apoptosis rate was determined by flow cytometry combined with Annexin V-FITC/PI double staining. The expression and cleavage of caspase-9 protein were detected by Western blot. ANOVA and LSD-t tests were conducted to compare the average values between the control and experimental groups.

Compared with the colorectal cancer cell LoVo treated with 0 μmol/L for 24 hours, the apoptosis rate of colorectal cancer cell LoVo treated with 2, 4, 6 μmol/L for 24 hours were increased [ (6.94±1.02) ﹪ vs (10.61±1.12) ﹪, (15.55±1.35) ﹪ and (36.51±1.46) ﹪]. Compared with the colorectal cancer cell LoVo treated with 4 μmol/L for 0 hours, the apoptosis rate of colorectal cancer cell LoVo treated with 4 μmol/L for 12 hours, 24 hours and 48 hours were increased [ (1.33±0.59) ﹪vs (19.23±1.24) ﹪, (22.24±1.41) ﹪ and (28.41±1.52) ﹪], differences were statistically significant (P< 0.001) . In the mean time, caspase-9 protein was up-regulated and activated when shikonin dose was≥2 μmol/L and treatment time was≥12 h, while caspase inhibitor (Z-LEHD-FMK) pretreatment could reduced the apoptosis rate significantly (P< 0.001) , by approximately 38.7﹪ (P< 0.05) .

Shikonin could induce apoptosis of colorectal cancer cells by regulating the expression of caspase-9 protein and its cleavage activity.

To investigate the effects and mechanisms of dichloroacetic acid (DCA) on the invasion and migration of the renal cell carcinoma cell line A498.

Human renal cell carcinoma cell line A498 were cultured and divided into negative a control (NC) group, a low dose DCA group, a medium dose DCA group and a high dose DCA group. The 3 dose groups of DCA were treated with 5, 10, 20 mmol/L DCA, and the NC group was administered isovolumetric sterile saline for 48 hours. Transwell chamber test was used to detect invasive ability and the cell scratch test to detect migration ability. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of c-Jun amino-terminal kinase (JNK) , E-cadherin and N-cadherin genes. Western blot was used to detect the expressions of the above proteins and phosphorylated JNK (p-JNK) . The differences in the data of these groups were analyzed by one-way ANOVA in SPSS Statistics 25.0, and SNK-q test was used for comparison between each two groups.

Comparing with high dose DCA group, the invasive activity decreased in the middle dose DCA group, the low dose DCA group and the negative control group (75.33±10.09 vs 167.89±47.12, 372.74±50.06 and 530.26±81.22/field) , and the relative expression of E-cadherin mRNA decreased (1.52±0.12 vs 1.35±0.11, 1.02±0.11 and 0.85±0.12, respectively) , as well as the relative expression of E-cadherin protein decreased (1.32±0.19 vs 0.89±0.14, 0.37±0.08 and 0.13±0.03, respectively) (all P< 0.001) , while the mobility[ (10.05±2.31) ﹪vs (23.95±4.20) ﹪, (36.26±5.01) ﹪ and (53.59±6.71) ﹪, respectively], the relative expression of N-cadherin mRNA (0.42±0.06 vs 0.58±0.07, 0.80±0.10 and 0.95±0.11, respectively) , the relative expression of N-cadherin protein (0.15±0.03 vs 0.25±0.04, 0.74±0.07 and 0.95±0.11, respectively) , and level of p-JNK (0.14±0.03 vs 0.29±0.05, 0.68±0.07 and 0.95±0.10, respectively) were increased (all P< 0.001) . There was no significant difference in JNK mRNA and protein expression among four groups (P> 0.05) .

DCA mayinhibit the invasion and migration of human renal cell carcinoma cell line A498 in a dose-dependent manner within a certain range, which is presumed to be related to the expressions of genes and proteins related to invasion and migration.

To investigate the effect of argatroban on neuronal apoptosis, and the expression of caspase-3, Bcl-2 as well as Bax in rats with acute cerebral infarction.

The rat model of focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) . SD rats were divided into Sham group, MCAO model group and Argatroban treatment group, and 3 time windows were set (0, 6, 12 h) . The samples of 3 time windows at 0, 6, 12 h were selected for detection, respectively. TUNEL apoptosis detection kit was used to detect neuronal apoptosis in the rat brain; Western blot and ELISA were used to detect the expression of caspase-3, Bcl-2 and Bax in the rat brain; ANOVA was used for comparison among groups, and LSD-t test was used for comparison of two groups.

The results of TUNEL showed that the apoptosis index of the sham group and Argatroban group all decreased at 0, 6, 12 h when compared with MCAO group [0 h (14.91±4.11) ﹪ vs (2.13±0.58) ﹪, (4.22±1.01) ﹪; 6 h (24.75±3.88) ﹪ vs (3.19±0.28) ﹪, (12.14±1.90) ﹪; 12 h (48.22±2.69) ﹪ vs (5.75±1.21) ﹪, (28.45±7.84) ﹪, P< 0.05]. The results of ELISA showed that the expression of caspase-3 and Bax in rat brain neuron in MCAO group increased at 0, 6 and 12 h compared with that in the sham group, while the expression of Bcl-2 increased at 6 and 12 hours (P< 0.05) . And compared with MCAO group, the expression of caspase-3 and Bax in rat brain neuron in Argatroban group decreased at 0, 6 and 12 h, while the expression of Bcl-2 increased at 6 and 12 hours [the expression of caspase-3: 0 h (2.32±0.12) vs (1.25±0.06) 、6 h (5.91±0.60) vs (3.26±0.22) 、12 h (7.31±0.27) vs (6.42±0.18) ng/ml; the expression of Bax: 0 h (0.82±0.08) vs (0.45±0.03) 、6 h (1.00±0.05) vs (0.66±0.04) 、12 h (1.56± 0.11) vs (1.09±0. 07) ng/ml; the expression of Bcl-2: 6 h (1.07±0.08) vs (1.56±0.05) ng/ ml, 12 h (0.67±0.08) vs (1.45±0.06) ng/ml, P< 0.01]. The protein expression trends of caspase-3 and Bax in rat brain neuron among 3 groups were consistent with those of ELISA, while the expression of Bcl-2 protein in Argatroban group was higher than that in sham group and MCAO group (P< 0.01) .

Argatroban exerts neuroprotective effects by reducing neuronal apoptosis induced by acute cerebral infarction, inhibiting the expression of caspase-3 and Bax and further enhancing Bcl-2 levels.