Effects of Puerarin on Th17/Treg Cell Immune Homeostasis and the Expression of Related Transcription Factors in Rats with Periodontitis
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摘要:
目的 探究葛根素对牙周炎大鼠辅助性 T 细胞 17( T helper cell 17,Th17) /调节性 T 细胞 (Regulatory T cell, Treg) 免疫平衡及相关转录因子的影响。 方法 采用分离牙龈、丝线结扎配合龈下注射大肠杆菌内毒素(E-LPS)法建立大鼠牙周炎模型。将大鼠随机分为正常对照组(A组)、牙周炎模型组(B组)、200 mg/(kg·d)葛根素组(C组)。HE 染色观察牙周组织病理形态学变化。通过Micro-CT对骨生物学参数定量分析牙槽骨吸收情况。流式细胞术、免疫印迹法(western blot,WB) 分别检测牙周组织中 Th17、Treg 细胞比例、白介素-17( IL-17) 、视黄酸相关孤儿受体( RORγt) 、IL-10、叉头状转录因子-3( Foxp3) 蛋白表达。 结果 HE染色中葛根素组大鼠牙根部可见明显新生骨细胞增生,牙周膜可见少量炎症细胞聚集,牙槽骨结构较整齐。Micro-CT显示治疗组大鼠牙槽骨较模型组骨量略有下降,骨质量及强度有所上升。流式细胞术检测全血细胞发现葛根素组的TH17、Treg细胞比例均下降,且TH17/Treg的细胞比例也下降。 IL-10、Foxp3在葛根素组蛋白表达量上调,而IL-17、RORγt在葛根素组蛋白表达量下调,差异有统计学意义(P < 0.05)。 结论 葛根素可缓解牙周炎大鼠牙周组织炎性反应、牙槽骨吸收,通过调节Th17 /Treg 细胞及相关转录因子的表达,使Th17 /Treg 细胞免疫平衡轴往有利于牙周组织愈合方向发展,其作用机制可能与葛根素有效调节Th17 /Treg细胞免疫平衡轴有关。 Abstract:Objective To study the effects of puerarin on the immune homeostasis of T helper 17 (Th17)/regulatory T (Treg) cells and the expression of related transcription factors in rats with periodontitis. Methods A rat periodontitis model was established using gingival isolation, silk ligature application and subgingival injection of Escherichia coli endotoxin (E-LPS). The rats were randomly divided into a normal control group (group A), a periodontitis model group (group B), and a 200 mg/(kg-d) puerarin group (group C). HE staining was used to observe the changes of periodontal tissue. Micro-ct correlated bone indexes were also analyzed to quantify the resorption of alveolar bone. The percentages of Th17 and Treg cells and the expression of interleukin (IL)-17, retinoic acid-related orphan receptor gamma t (RORγt), IL-10 and forkhead transcription factor-3 (Foxp3)in periodontal tissues were monitored by flow Cytometry and Western blot (WB), respectively. Results In the study, the roots of the teeth in the puerarin group were significantly proliferated with new osteoblasts. Flow cytometry showed that the proportion of TH17 and Treg cells in the puerarin group decreased, and the proportion of TH17/Treg cells also decreased . The protein expressions of IL-10 and Foxp3 were up-regulated in puerarin, while the histone expressions of IL-17 and RORγt were down-regulated in puerarin (P < 0.05). Conclusions Puerarin can alleviate the periodontal tissue inflammatory response and alveolar bone resorption in rats with periodontitis and favor periodontal tissue healing by regulating Th17/Treg cell immune homeostasis and the expression of related transcription factors. The mechanism of action may be related to effective regulation of Th17/Treg cell immune homeostasis by puerarin. -
Key words:
- Puerarin /
- Periodontitis /
- Rats /
- Helper T cells /
- Regulatory T cells
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第5跖骨近端骨折在临床上是足部最常见的损伤,占足部骨折的61%~78%[1]。其治疗主要取决于骨折的解剖位置,而对于第5跖骨近端的分类主要包括Stewart及Lawrence and Botte分类,前者将其分为粗隆骨折与琼斯骨折,而后者主要将第5跖骨近端骨折分为三区[2],2种分类中琼斯骨折与二区、三区骨折重叠[3],但二区骨折通常指的就是琼斯骨折。粗隆部骨折松质结节有丰富的血液供应,非手术治疗可取得良好愈合,但相对无血管的琼斯骨折最好的治疗方式是手术固定[4]。累及二区且有移位的,以及运动员或需求高的非移位骨折患者,手术目的是减少愈合时间和加速康复,三区骨折最好选择手术治疗,因为这些骨折容易发生延迟愈合和骨不连。而对于二、三区的内固定选择,有关文献证实了髓内钉是首选之一[5-6]。
1. 资料与方法
1.1 一般资料
选择2021年1月至2022年3月在云南中医药大学第一附属医院骨科就诊行足部正斜位片的患者,且选择既往无手术或第5跖骨损伤,无足部骨折、脱位或畸形的患者,拍摄的120例患者中男60例、女60例符合纳入标准[7]。本研究已通过医院伦理委员会审批。
1.2 测量方法
选择标准足部正斜位作为测量图像,见图1(正位)中第5跖骨髓腔直径内侧线以第5跖骨基底部与骰骨组成的跖跗关节缘尖端为起点,做一条至第5跖骨干内侧髓腔皮质弯曲部的直线A,再从第5跖骨干外侧髓腔皮质弯曲点为起点,引出一条平行A至第5跖骨基底部的直线B,测量2条直线间的垂直距离作为第5跖骨髓腔直径(a)。在正位片上从第5跖骨干弯曲点外侧皮质做一平行于B至第5跖骨基底部的直线C,此条长度为髓内钉长度(c)。再从第5跖骨基底部外侧做一条平行于c至第5跖骨头关节面的直线D,此长度为第5跖骨长度(d)。图2(斜位)中第5跖骨髓腔直径内侧线以5跖骨基底部内侧尖端为起点,同图1做两平行线E、F,测量2条直线间的垂直距离为斜位髓腔直径(b),上述具体字母标识见图1、图2。
1.3 统计学处理
采用SPSS26.0统计学软件包进行统计学分析,正态分布计量资料采用均数据±标准差、平均值、中位数、最大值、最小值、标准差表示,正态分布计量资料2组组间比较采用两独立样本t检验,相关性分析采用Pearson相关性分析,P < 0.05表示差异有统计学意义。
2. 结果
2.1 数据统计描述
正位上髓腔直径(a)平均值为4.15 mm,范围(2.24~5.64 mm),斜位上第5跖骨髓腔直径(b)平均值为4.31 mm,范围(3.18~5.81 mm),髓内钉长度(c)平均值为39.74 mm,范围(28.07~53.46 mm),第5跖骨长度(d)平均值71.64 mm,范围(59.73~85.44 mm),髓内钉长度与第5跖骨长度比值(e)平均值为55.48%,范围(41.12%~68.05%),见表1。
表 1 基础数据描述Table 1. Description of basic data指标 平均值 中位数 最大值 最小值 标准差 年龄 43.79 40.00 79 19 16.40 a(mm) 4.15 4.10 5.64 2.24 0.58 b(mm) 4.31 4.26 5.81 3.18 0.69 c(mm) 39.74 39.58 53.46 28.07 4.80 d(mm) 71.64 71.42 85.44 59.73 5.78 e(%) 55.48 55.07 68.05 41.12 5.10 a:正位第5跖骨髓腔直径;b:斜位第5跖骨髓腔直径;c:髓内钉长度;d:第5跖骨长度;e:髓内钉长度与第5跖骨长度比值。 2.2 指标与性别的差异
经t检验证实男性与女性年龄差异无统计学意义(P > 0.05),男性和女性正斜位髓腔直径、第5跖骨长度、髓内钉长度,差异有统计学意义(P < 0.05),髓内钉长度与第5跖骨长度比值(e),差异无统计学意义(P > 0.05)。Pearson相关性分析:男性和女性d与e均无相关性(P > 0.05),见表2、表3。
表 2 男性和女性各资料比较($ \bar x \pm s $ )Table 2. Comparison of male and female data ($ \bar x \pm s $ )指标 男性组 女性组 t P 年龄 46.03 ± 17.92 41.55 ± 14.53 1.505 0.135 a(mm) 4.31 ± 0.56 3.99 ± 0.56 3.082 0.003* b(mm) 4.46 ± 0.64 4.17 ± 0.50 2.717 0.008 c(mm) 41.25 ± 4.64 38.23 ± 4.51 3.609 < 0.001* d(mm) 75.04 ± 4.68 68.23 ± 4.68 7.977 < 0.001* e(%) 54.95 ± 4.99 56.01 ± 5.30 −1.139 0.257 a:正位第5跖骨髓腔直径;b:斜位第5跖骨髓腔直径;c:髓内钉长度;d:第5跖骨长度;e:髓内钉长度与第5跖骨长度比值。*P < 0.05。 表 3 男性和女性d与e相关性分析Table 3. Correlation analysis of d and e between men and women性别 r P 男 0.03 0.799 女 0.05 0.694 3. 讨论
足外侧纵弓和横弓,是第5跖骨近端的重要组成部分,在足部生物力学中有重要作用,如足部力量的传导,及负重[8],而且腓骨短肌止点位于第5跖骨粗隆部,当足部重复性应力及外伤时容易造成骨折,若骨折累计二区、三区,因影响血液供应,容易造成骨折不愈合或延迟愈合[9]。因此选择良好内固定物与术式,对骨折愈合有巨大作用。Ryan等[10]在一项研究琼斯骨折外科技术中提到髓内螺钉固定仍是治疗选择的金标准,但有关金标准的提法并未取得共识。而且髓内螺钉固定也存在骨不连、再骨折等风险[11]。也有其更好的作用,经皮髓内螺钉对跖骨血供破坏较少,利于跖骨骨折愈合,而且切口在跖骨近端粗隆部,减少皮肤坏死等并发症,植入扩髓的腔隙增加了其稳定性,防止内固定的松动[12]。
此次研究根据120例患者的X片检测确定了第5跖骨的简要解剖结构,在笔者的研究人群中,第5跖骨的平均长度为71.64 mm,髓内钉长度的平均值为39.74 mm,髓内钉长度约占第5跖骨总长度的55.48%。髓腔平均值大小在正位上比斜位要小。Ochenjele等[7]在CT测量研究中发现40 mm螺钉长度已接近第5跖骨弯曲处,而在本次X片测量中髓内钉平均长度为39.74 mm,与本文献相映。因此在临床置钉中应该注意髓内钉的长度,钉道长度应该小于跖骨长度的55.48%,从而避免致内外侧皮质骨折。但是为了让骨折有良好的固定及稳定性,可以考虑选择更大的髓内钉直径。
不足之处在于利用X线拍摄足部正斜位片,只能得到部分信息,若考虑存在跖跗关节损伤时,需拍摄跖跗关节正斜位[13]。本研究样本量相对较少,可能数据存在偏倚,且髓腔大小随着跖骨解剖而变化,利用计算机断层扫描和尸体标本测量髓腔大小可能会更接近真实值,尽管对大量就诊患者第5跖骨的解剖进行了详细的研究和分析,但每个患者都是独立个体,髓内钉的直径及长度应据个体的术前影像学检查确定[14]。另外所有测量的起点都在第5跖骨基底部尖端进行的,可能不能提供一致的髓内钉轨迹。而且这是一项放射学研究,测量结果需大量的临床实践进行验证,才能确定髓内钉的良好固定、稳定性,及减少并发症。总之,此研究结果有助于指导临床髓内钉直径和长度的选择。
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表 1 各组Micro-CT扫描分析后骨体积分数、骨矿物质密度值(n = 12,
$ \bar x \pm s $ )Table 1. BV/TV and BMD values of each group obtained by micro-CT scan analysis (n = 12,
$ \bar x \pm s $ )组别 BV/TV(%) BMD(g/cm3) 对照组 0.720 ± 0.049 1004.269 ± 54.644△ 模型组 0.387 ± 0.0376* 894.119 ± 53.789* 葛根素治疗组 0.337 ± 0.036△ 1046.297 ± 54.565△ 与对照组比较,*P < 0.05;与模型组比较,△P < 0.05。 -
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