1.Effect of long non-coding RNA F19 on secondary brain injury after traumatic brain injury in mice
Jianhua PENG ; Jinwei PANG ; Yue WU ; Yuke XIE ; Kecheng GUO ; Tianqi TU ; Qiancheng MU ; Yuyan LIAO ; Fang CAO ; Liang LIU ; Ligang CHEN ; Xiaochuan SUN ; Yong JIANG
Chinese Journal of Trauma 2019;35(3):267-273
Objective To investigate the effect of long non-coding RNA F19 (lncRNA F19) on secondary brain injury following traumatic brain injury (TBI) in mice. Methods (1) A total of 96 C57BL/6J male wild-type mice were divided into sham group, sham+control lentivirus group, sham+F19 lentivirus group, TBI group, TBI+control lentivirus group and TBI+F19 lentivirus group according to the random number table. Each group consisted of two subgroups of 1 day and 3 days after TBI, with eight mice per subgroup. The expression and silence efficiency of lncRNA F19 were detected. ( 2 ) A total of 96 C57BL/6J male wild-type mice were divided into sham group, TBI+control lentivirus group and TBI + F19 lentivirus group according to the random number table. Each group consisted of two subgroups of 1 day and 3 days after TBI, with 16 mice per subgroup. The effect of lncRNA F19 on neuronal apoptosis after TBI was recorded. The mice TBI model was established using the controlled cortical damage method (CCI). The lncRNA F19 lentivirus or control lentivirus were administrated by intracerebroventricular injection 5 days before injury. The expressions of lncRNA F19 ( 2 -ΔΔct ) were detected by real-time quantitative PCR ( qRT-PCR ) at 1 day and 3 days after injury. The Toll-like receptor 4 (TLR4), B lymphocyte tumor-2 (Bcl-2) and Bcl-2 related protein (Bax) expressions were detected by Western blot. The TUNEL was used to detect apoptosis around the traumatic lesions. Results From the first day after injury, both in the sham operation and TBI groups, the control lentivirus had no effect on the level of lncRAN F19 (P >0. 05). One day after injury, compared with sham +control lentivirus group, the levels of lncRNA F19 in sham + F19 lentivirus group were significantly decreased (0. 07 ± 0. 07:0. 93 ± 0. 17);compared with TBI+control lentivirus group, levels of lncRNA F19 in TBI+F19 lentivirus group were significantly decreased (2. 91 ± 1. 18:0. 52 ± 0. 32) (P<0. 05). There were significantly lower protein levels of TLR4 (0. 51 ± 0. 13:0. 66 ± 0. 15), Bax (0. 45 ± 0. 06:0. 67 ± 0. 16), lower TUNEL-positive neurons ratio [(23. 55 ± 6. 85)% : (31. 58 ± 7. 52)%], but higher protein levels of Bcl-2 (0. 76 ± 0. 16:0. 47 ± 0. 12) in TBI+F19 lentivirus group compared with the TBI+ control lentivirus group (P <0.05). Three days after injury, compared with sham + control lentivirus group, levels of lncRNA F19 in sham+F19 lentivirus group were significantly decreased (0. 11 ± 0. 09:0. 96 ± 0. 09); compared with TBI+control lentivirus group, levels of lncRNA F19 in TBI+F19 lentivirus group were significantly decreased (0. 54 ± 0. 24:3. 39 ± 0. 90) (P <0. 05). There were significantly lower protein levels of TLR4 (0. 60 ± 0. 20):(0. 85 ± 0. 09)], lower Bax (0. 60 ± 0. 12:0. 88 ±0. 21), lower TUNEL-positive neurons ratio [(29. 10 ± 7. 37)% :(39. 22 ± 10. 64)%], but higher protein levels of Bcl-2 (0. 66 ± 0. 12:0. 35 ± 0. 16) in TBI+F19 lentivirus group compared with the TBI+control lentivirus group (P<0. 05). Conclusion Inhibition of lncRNA F19 can significantly reduce the TLR4-induced neuronal apoptosis in cortex after TBI in mice and alleviate reduce the secondary brain injury.
2.Effect of biglycan on neural apoptosis in mice with early brain injury after subarachnoid hemorrhage
Yuke XIE ; Kecheng GUO ; Jianhua PENG ; Jinwei PANG ; Jian ZHOU ; Long GU ; Lifang ZHANG ; Shigang YIN ; Ligang CHEN ; Yong JIANG
Chinese Journal of Neuromedicine 2020;19(2):138-145
Objective To investigate the effect ofbiglycan (BGN) on neural apoptosis in mice with early brain injury (EBI) after subarachnoid hemorrhage (SAH).Methods SAH models were induced by endovascular perforation in young male C57BL/6J mice.(1) Totally,48 mice were randomly divided into sham-operated group,SAH 6 h group,SAH 12 h group,SAH 24 h group,SAH 48 h group,and SAH 72 h group (n=8);the BGN protein and mRNA expressions were detected by Western blotting and real-time quantitative PCR (qRT-PCR).(2) Totally,16 mice were randomly divided into sham-operated group and SAH 48 h group (n=8);immunofluorescent double staining was conducted to explore the BGN expression in the neurons of brain tissues.(3) Totally,24 mice were randomly divided into sham-operated group,sham+control lentivirus group,and sham+BGN lentivirus group (n=8);BGN lentiviral vector and control lentivirus were administered intracerebroventricularly 7 d before sham-operation;qRT-PCR was performed to explore the BGN mRNA expression.(4) Totally,48 mice were randomly divided into sham-operated group,SAH+control lentivirus group,and SAH+BGN lentivirus group (n=16);BGN lentiviral vector and control lentivirus were administered intracerebroventricularly 7 d before SAH;neurological scores were detected by modified Garcia scale and beam balance tests;TUNEL was used to detect the neuronal apoptosis,and Western blotting was performed to explore the expressions of nuclear transcription factor kappa B (NF-κB) and phosphorylated-(p-) NF-κB.Results (1) Mice in the SAH 48 h group had the highest BGN protein and mRNA expressions,which showed statistical differences as compared with the sham-operated group (P<0.05).(2) A majority of BGN expressions were detected in the neurons 48 h after SAH.(3) The sham+BGN lentivirus group had statistically lower BGN mRNA expression than the sham+control lentivirus group (P<0.05).(4) As compared with those in the SAH+control lentivirus group,both scores of modified Garcia scale and beam balance tests were significantly higher in SAH+BGN lentivirus group (6.125±1.246 vs.13.000±1.309;1.125±1.126 vs.2.875±0.835),and neural apoptosis ratio and ratio of p-NF-κB/NF-κB were significantly lower in the SAH+BGN lentivirus group (51.950%±11.166% vs.31.938%±7.705%;1.161±0.156 vs.0.886±0.142,P<0.05).Conclusion Inhibition of BGN can effectively reduce neuronal apoptosis in mice with EBI after SAH,and attenuate neurological deficits.
3.Study on mechanism of berberine inhibiting tumor stem cells proliferation and its in vivo safety evaluation
Jinjin XIE ; Yan CHEN ; Xin DU ; Yuke LI ; Mengnan ZHAO ; Sanjun SHI
China Pharmacy 2024;35(12):1443-1450
OBJECTIVE To investigate the in vitro inhibitory mechanism of berberine on the proliferation of tumor stem cells and evaluate its in vivo safety. METHODS Flow cytometry was used to select tumor stem cells from mouse skin melanoma B16F10 cells; CD44, CD133, Nanog homologous box protein (NANOG) and octamer-binding transcription factor 4 (OCT4) were used as indicators to characterize tumor stem cells. Tumor stem cells were divided into control group, all-trans retinoic acid (ATRA) group, and berberine group, and the CCK-8 method was used to detect the effects of berberine on the viability of tumor stem cells; flow cytometry was adopted to detect cell apoptotic rate, the proportion of CD44+/CD133+ and the positive cell rate of sex determining region Y box protein 2 (SOX2); the morphological changes of tumor balls were recorded after treatment with berberine; the morphology of cell pyroptosis in each group was recorded, and the release rate of lactate dehydrogenase (LDH) was detected; Western blot assay was adopted to detect the expressions of pyroptosis-related protein gasdermin E (GSDME), GSDME- N, caspase-3 and cleaved caspase-3. Preliminary evaluation of in vivo safety of berberine was conducted by using zebrafish embryo toxicity experiments. RESULTS Compared with B16F10 cells, the proportion of CD44+/CD133+ cells in tumor stem cells and the fluorescence intensity of NANOG and OCT4 were significantly increased (P<0.000 1). The half-inhibitory concentration of berberine to tumor stem cells was 50.98 μmol/L. Compared with the control group, the apoptotic rate of cells in the berberine group was significantly increased, while the proportion of CD44+/CD133+ cells and the rate of SOX2 positive cells were reduced significantly (P<0.000 1); tumor stem cell spheroids were atrophied, with partial cell death. After treatment with berberine, tumor stem cells exhibited swelling in their outermost layer, the release rate of LDH of cells was significantly increased and the release rate of LDH increased with increasing dose; the protein expressions of GSDME-N and cleaved-caspase-3 of cells in berberine 20, 40 μmol/L groups were significantly increased, and the protein expressions of GSDME and caspase-3 were significantly reduced (except for berberine 20 μmol/L group, P<0.05). The embryonic development of zebrafish treated with berberine was almost unaffected, and the survival rate of embryo reached 100%, with no obvious abnormalities observed. CONCLUSIONS Berberine has good activity against the proliferation of tumor stem cells, and its mechanism of action may be related to activating GSDME and promoting cell pyroptosis; berberine has good in vivo safety.