Effect of <italic style="font-style: italic">Jisuikang</italic>-containing Serum Combined with SOX-2 on Astrocyte Being Reprogrammed and Induced into Neurons

WANG Haotian; WANG Jianwei; WU Mao; WANG Guopeng; LI Shaoshuo; RONG Yi; SHAO Yang

doi:10.13288/j.11-2166/r.2023.03.013

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Effect of Jisuikang-containing Serum Combined with SOX-2 on Astrocyte Being Reprogrammed and Induced into Neurons

更新时间：2023-01-29

- Effect of Jisuikang-containing Serum Combined with SOX-2 on Astrocyte Being Reprogrammed and Induced into Neurons
- Journal of Traditional Chinese Medicine Vol. 64, Issue 3, Pages: 288-294(2023)
- 作者机构：
  
  1.南京中医药大学，江苏省南京市仙林大道138号，210023
  2.江苏省无锡市中医医院
- 作者简介：
- 基金信息：
- DOI：10.13288/j.11-2166/r.2023.03.013
  CLC：
- Received：08 August 2022，
  
  Revised：2022-11-21，
  
  Published：02 February 2023
- 稿件说明：
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王浩阗,王建伟,吴毛等.脊髓康含药血清联合SOX-2对星形胶质细胞重编程诱导为神经元细胞的影响[J].中医杂志,2023,64(03):288-294.

WANG Haotian,WANG Jianwei,WU Mao,et al.Effect of Jisuikang-containing Serum Combined with SOX-2 on Astrocyte Being Reprogrammed and Induced into Neurons[J].Journal of Traditional Chinese Medicine,2023,64(03):288-294.
王浩阗,王建伟,吴毛等.脊髓康含药血清联合SOX-2对星形胶质细胞重编程诱导为神经元细胞的影响[J].中医杂志,2023,64(03):288-294. DOI： 10.13288/j.11-2166/r.2023.03.013.

WANG Haotian,WANG Jianwei,WU Mao,et al.Effect of Jisuikang-containing Serum Combined with SOX-2 on Astrocyte Being Reprogrammed and Induced into Neurons[J].Journal of Traditional Chinese Medicine,2023,64(03):288-294. DOI： 10.13288/j.11-2166/r.2023.03.013.

摘要

目的

研究脊髓康联合SRY转录盒因子2（SOX-2）治疗脊髓损伤的作用机制。

方法

成年SD大鼠随机分为空白组和给药组各5只。给药组大鼠以生药量20 g/（kg·d）脊髓康灌胃，空白组给予16 ml/（kg·d）生理盐水灌胃，均连续灌胃3天后腹主动脉采血制备含药血清和空白血清。体外培养1~4日龄大鼠幼仔皮层脑组织三代星形胶质细胞，分为空白血清组、SOX-2组、SOX-2+脊髓康血清组。SOX-2组加入10 mmol/L SOX-2；SOX-2+脊髓康血清组加入2.5%脊髓康含药血清和10 mmol/L SOX-2，使培养基中血清终浓度为10%；空白血清组加入10%空白血清。诱导培养21天后，光镜下观察3组神经元样细胞形态改变，细胞免疫荧光检测神经元轴突标志物微管相关蛋白2（MAP2）的表达，Western-Blot检测细胞中下游代谢产物S腺苷甲硫氨酸（SAM）、乙酰辅酶A（acetyl-CoA）表达，PCR定量分析星形胶质细胞中糖酵解相关基因己糖激酶1（HK1）、己糖激酶2（HK2）、磷酸果糖激酶1（PFK1）、磷酸果糖激酶2（PFK2）、M2型丙酮酸激酶同工酶（PKM2）基因表达。细胞能量代谢分析仪比较SOX-2组、SOX-2+脊髓康血清组细胞外酸化率（EACR）和耗氧率（OCR）。

结果

光镜下见SOX-2+脊髓康血清组大量具有两极且长轴突的神经元样细胞，较SOX-2组明显增多；空白血清组见星形胶质细胞，未见神经元样细胞。与空白血清组和SOX-2组比较，SOX-2+脊髓康血清组MAP2、SAM、acetyl-CoA表达均升高，HK1、HK2、PFK1、PFK2、PKM2基因表达亦显著升高（

＜0.05或

＜0.01）。SOX-2+脊髓康血清组ECAR和OCR亦高于SOX-2组。空白血清组和SOX-2组MAP2、SAM、acetyl-CoA表达差异无统计学意义（

＞0.05）。

结论

脊髓康含药血清联合SOX-2可以将星形胶质细胞重编程诱导为神经元细胞，其机制可能是通过提高糖酵解能力从而提升星形胶质细胞重编程。

Abstract

Objective

To study the effect and machanism of

Jisuikang

（脊髓康） combined with SRY transcription box factor 2 （SOX-2） on spinal cordinjury （SCI）.

Methods

Adult SD rats were randomly divided into blank group and medication group with five rats in each. Rats in the medication group were given 20 g/（kg·d） of

Jisuikang

by gavage， while those in the control group received 18 ml/（kg·d） of normal saline by gavage， both for three days. After that， blood was collected from the abdominal aorta to prepare

Jisuikang

-containing serum and blank serum. Three generations of astrocytes were cultured in vitro in the cerebral cortex of 1~4 day newborn rats， and were divided into three groups including blank serum group， SOX-2 group， and combination of

Jisuikang

-containing serum plus SOX-2 group （SOX-2+JSK group）. Add 10 mmol/L of SOX-2 to the SOX-2 group， and 2.5%

Jisuikang

-containing serum plus 10 mmol/L of SOX-2 to the SOX-2 + JSK group， making the final serum concentration in the medium to 10%； blank serum group was added with 10% of blank serum. After 21 days of induction and culture， the morphology of neuron-like cells in the three groups were observed under light microscope. The expression of neuronal axon marker microtubule-associated protein 2 （MAP2） was detected by cellular immunofluorescence， and the expression of downstream metabolites S-adenosylmethionine （SAM） and acetyl-CoA （acetyl-CoA） were detected by Western Blot. Quantitative PCR analysis was performed for the glycolysis-related genes expression including hexokinase 1 （HK1）， hexokinase 2 （HK2）， phosphofructokinase 1 （PFK1）， phosphofructokinase 2 （PFK2）， and M2-type pyruvate kinase isozyme （PKM2）. The cell energy metabolism analyzer was used to compare the extracellular acidification rate （EACR） and oxygen consumption rate （OCR） of the SOX-2 group and the SOX-2+JSK serum group.

Results

Under the light microscope， a large number of neuron-like cells with bipolar and long axons were found in the SOX-2+JSK group， which was significantly more than that in the SOX-2 group； in the blank serum group， astrocytes were found， but with no neuron-like cells. The expression of MAP2， SAM and acetyl-CoA significantly increased in the SOX-2+JSK group compared to those in the blank serum group and the SOX-2 group， while the gene expressions of HK1， HK2， PFK1， PFK2， and PKM2 significantly increased （

＜0.05 or

＜0.01）. Higher ECAR and OCR were found in the SOX-2+JSK group than the SOX-2 group. There was no significant difference in the expressions of MAP2， SAM， and acetyl-CoA between the blank serum group and the SOX-2 group （

＞0.05）.

Conclusion

Iisuikang

-containing serum combined with SOX-2 can induce astrocyte reprogramming into neurons， and the machnism of astrocyte reprogramming may be improved through glycolysis.

关键词

Keywords

references

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Effect of Jisuikang-containing Serum Combined with SOX-2 on Astrocyte Being Reprogrammed and Induced into Neurons

DOI：10.13288/j.11-2166/r.2023.03.013

摘要

Abstract

关键词

Keywords

references