巴戟天多糖通过P2X7R/NLRP3通路抑制大鼠骨质疏松

李志豪; 邓祖跃; 黄逸伦; 陈书恺; 蒋霞; 任艳云

doi:10.13748/j.cnki.issn1007-7693.20243275

巴戟天多糖通过P2X7R/NLRP3通路抑制大鼠骨质疏松

Morinda Officinalis Polysaccharides Inhibit Osteoporosis in Rats via the P2X7R/NLRP3 Pathway

摘要

摘要:
目的研究巴戟天多糖抑制骨质疏松的作用机制，尤其是其对P2X7R/NLRP3炎症小体通路的调节作用。
方法将100只雌性SD大鼠随机分成10组，包括假手术组，模型组，巴戟天多糖低、中、高剂量组(50、100、200 mg·kg⁻¹，口服)，己烯雌酚阳性组(0.1 mg·kg⁻¹，口服)，考马斯亮蓝G组(75 mg·kg⁻¹，腹腔注射给药)，考马斯亮蓝G联合给药组(巴戟天多糖100 mg·kg⁻¹+考马斯亮蓝G 75 mg·kg⁻¹联合给药)，氟甲基酮组(0.15 mg·kg⁻¹，静脉给药)，氟甲基酮联合给药组(巴戟天多糖 100 mg·kg⁻¹+氟甲基酮 0.15 mg·kg⁻¹联合给药)，每组各10只。通过双侧卵巢摘除法制备骨质疏松模型。各治疗组按照相应剂量给予药物，假手术组和模型组灌胃等量生理盐水。经过6周的连续给药后，处死各组大鼠。采用Micro-CT测定骨微结构参数(骨密度、骨小梁数量、骨小梁分离度、骨小梁厚度、骨体积分数)，利用ELISA试剂盒检测血清中骨代谢相关因子(BMP-2、BALP、OC、CTX-I)及炎症因子(TNF-α、IL-6、IL-1β、IL-18)的水平；通过Western blotting法检测P2X7R、NLRP3、Caspase-1、GSDMD-N、OPG、RANKL等蛋白的表达情况。最后，采用HE染色观察各组大鼠骨组织的病理变化。
结果给予巴戟天多糖治疗的大鼠骨质疏松愈合明显，骨微结构参数骨密度、骨小梁数量、骨小梁厚度和骨体积分数显著增加，骨小梁分离度降低；病理结果显示，骨小梁数量增加，平均骨小梁面积增加，骨小梁间隙显著降低，炎性细胞浸润减少；同时，血清中BMP-2、BALP、OC水平上升，CTX-I、TNF-α、IL-6、IL-1β、IL-18水平下降。此外，巴戟天多糖也上调了骨组织中OPG蛋白的表达，下调了P2X7R、RANKL、NLRP3、Caspase-1和GSDMD-N蛋白的表达。考马斯亮蓝G和氟甲基酮均可增强巴戟天多糖对骨质疏松愈合的促进作用。
结论巴戟天多糖可通过抑制P2X7R-NLRP3炎症小体通路调节骨质疏松状态下的炎症因子水平，从而发挥抗骨质疏松作用。

Abstract:
OBJECTIVE To investigate the mechanism of Morinda officinalis polysaccharides inhibiting osteoporosis, especially its regulatory effect on P2X7R/NLRP3 inflammasome pathway.
METHODS The 100 female SD rats were randomly divided into 10 groups, including sham group, model group, low, medium and high doses of Morinda officinalis polysaccharides groups(50, 100, 200 mg·kg⁻¹, oral), diethylstilbestrol positive group(0.1 mg·kg⁻¹, oral), Coomassie brilliant blue G group(75 mg·kg⁻¹, intraperitoneal injection), Coomassie brilliant blue G co-administration group(Morinda officinalis polysaccharides 100 mg·kg⁻¹+Coomassie brilliant blue G 75 mg·kg⁻¹ administered in combination), fluoromethyl ketone group(0.15 mg·kg⁻¹, intravenous), fluoromethyl ketone co-administration group(Morinda officinalis polysaccharides 100 mg·kg⁻¹+fluoromethyl ketone 0.15 mg·kg⁻¹ administered in combination), 10 rats in each group. Osteoporosis model was established by bilateral ovariectomy. Each treatment group was given drugs according to the corresponding dose, and the sham group and model group were given a gavage of an equal volume of saline. After 6 weeks of continuous administration, the rats of each group were sacrificed. Bone microstructural parameters(bone mineral density, trabecular number, trabecular separation, trabecular thickness, and bone volume/tissue volume) were measured by Micro-CT. Serum levels of bone metabolism related factors(BMP-2, BALP, OC, CTX-I) and inflammatory factors(TNF-α, IL-6, IL-1β, IL-18) were measured by ELISA kit. Expression of P2X7R, NLRP-3, Caspase-1, GSDMD-N, OPG, RANKL and other proteins were detected by Western blotting. Finally, HE staining was used to observe the pathological changes of bone tissue in each group.
RESULTS After the treatment of Morinda officinalis polysaccharides, osteoporosis healed obviously, bone mineral density, trabecular number, trabecular thickness and bone volume/tissue volume increased significantly, trabecular separation decreased significantly; pathological results showed that the number of trabecular bone increased, the average trabecular bone area increased, trabecular bone gap decreased significantly, inflammatory cell infiltration decreased; meanwhile, BMP-2, BALP, OC levels increased, CTX-I, TNF-α, IL-6, IL-1β, IL-18 levels decreased in serum. MOP also up-regulated OPG protein expression and down-regulated P2X7R, RANKL, NLRP3, Caspase-1 and GSDMD-N protein expression in bone tissue. Coomassie brilliant blue G and fluoromethyl ketone could enhance the promoting effect of Morinda officinalis polysaccharides on osteoporotic healing.
CONCLUSION Morinda officinalis polysaccharides can regulate the inflammatory factor level in osteoporosis by inhibiting P2X7R-NLRP3 inflammasome pathway, so as to play an anti-osteoporosis role.

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