| Objective:To investigate the involvement of the paravertebral lymphatic system and to reveal the influence of the structure and function of the paravertebral lymphatic vessel system in the nucleus pulposus herniation resorption.To explore the mechanism of BSTL formula regulating the lymphatic vascular system and promoting the resorption of nucleus pulposus herniation and to elucidate the modern scientific connotation of the pathogenesis of Traditional Chinese Medicine(TCM)stasis and the method of removing blood stasis.Methods:1.Lymphatic vessel expression in surgical specimens of patients with clinical lumbar disc herniation(LDH)The appearance of surgical specimens were observed,ABOG staining and tissue immunofluorescence staining were used to detect the pathological morphology and lymphatic vessel expression of the surgical specimens,and the distribution characteristics of lymphatic vessels in surgical specimens were described.2.Structural and functional changes of lymphatic vessel system in spontaneous LDH miceIn this study,Secreted protein acidic and rich in cysteine(SPARC)knockout mice were used as research objects.Magnetic resonance imaging(MRI)technology was used to detect the herniation of the intervertebral disc in the mice.Near infared-Indocyanine Green(NIR-ICG)paravertebral lymphatic imaging technique was used to evaluate paravertebral lymphatic draining function in mice.Draining lymph nodes were sampled and fixed to detect volume changes.ABOG staining was used to observe the pathological changes of intervertebral discs,and histoimmunofluorescence staining was used to assess the changes of lymphatic vessel expression in draining lymph nodes and intervertebral disc tissue.3.The regularity of lymphatic vessel system in the process of nucleus pulposus reabsorptionThe nucleus pulposus herniation model was used,which was induced by acupuncture of the annulus fibrosus in the intervertebral disc of the mouse tail.Ultrasound and vernier calipers were used to assess the swelling volume and diameter of the mouse modeling site before surgery,and 1 d,7 d,14 d,21 d and 28 d after surgery.The NIR-ICG imaging technique was used to observe the draining function of paravertebral lymphatic vessel system in mice at 1 W,2 W and 4 W after surgery.The expression of superficial lymphatic vessels in the mouse modeling site was detected by whole mount staining at 1 W,2 W and 4 W after surgery.Sciatic lymph nodes and iliac lymph nodes were collected and fixed to detect volume changes at 1 W,2 W and 4 W after surgery.ABOG staining was used to evaluate the pathological changes of intervertebral disc in mice at 1 W,2 W and 4 W after surgery.The expression of lymphatic vessels in the draining lymph nodes and intervertebral disc tissues of mice were detected by tissue immunofluorescence staining at 1 W,2 W and 4 W after surgery.4.The effect of inhibiting lymphatic draining function in the resorption of nucleus pulposus herniationIn this study,we selected conditional VEGFR3 knockout mice and induced nucleus pulposus herniation models in those mice.Ultrasound and vernier calipers were used to detect the swelling volume and diameter of the mouse modeling site before surgery and 1 d,7 d and 14 d after surgery.The NIR-ICG imaging technique was used to assess the draining function of paravertebral lymphatics in mice at 2 W after surgery.Whole mount staining was used to observe the expression of superficial lymphatic vessels in the mouse modeling site at 2 W after surgery.Sciatic lymph nodes were collected and fixed to measure volume changes at 2 W after surgery.ABOG staining was used to observe the pathological changes of intervertebral disc in mice at 2 W after surgery.The expression of lymphatic vessels in the draining lymph nodes and intervertebral discs of mice were evaluate by tissue immunofluorescence staining at 2 W after surgery.5.The mechanism of BSTL formula regulating the lymphatic vascular system and promoting the resorption of nucleus pulposus herniationLow,medium and high doses of BSTL were administered by gavage to mice with herniated nucleus pulposus.Ultrasound and vernier calipers were used to assess the swelling volume and diameter of the mouse modeling site before surgery and 1 day,7 days and 14 days after BSTL intervention.The NIR-ICG imaging technique was used to observe the lymphatic draining function of mice after 2 W gavage.Whole mount staining was used to evaluated the expression of superficial lymphatic vessels in the mouse modeling site after 2 W gavage.Sciatic lymph nodes were collected and fixed,and the volume changes were detected after 2 W gavage.ABOG staining was used to observe the pathological changes of intervertebral disc in mice after 2 W gavage.The expression of lymphatic vessels in the draining lymph nodes and intervertebral discs of mice were observed by tissue immunofluorescence staining after 2 W gavage.Results:1.The appearance of the control specimens were white,and the texture of the control specimens was soft;however,the herniated specimens were grey,yellow,and the texture of the herniated specimens was hard.ABOG staining showed that the cells in the control specimens were scattered and vacuumed,and the tissue blue staining was obvious,indicating that the extracellular matrix was abundant;however,the disappearance of blue staining in the herniated specimens indicated a significant loss of extracellular matrix components and infiltration of inflammatory cells in the tissue.Tissue immunofluorescence showed that the herniated specimens expressed lymphatic vessels,mainly capillary lymphatic vessels(P<0.05).2.The wild type(WT)mice in the littermate control group had a normal body shape with slightly curved spines,while the SPARC knockout mice had abnormally curved spines and curled up tails.In body weight,the SPARC knockout group decreased compared with the WT control group(P<0.05).MRI imaging results showed that the physiological curvature of the spine existed in the WT control group,the intervertebral discs were not herniated,and the signal brightness of the lumbar intervertebral disc was slightly reduced;however,the spine was abnormally curved,the signal brightness of the intervertebral disc was significantly reduced,and the intervertebral disc was herniated in the SPARC knockout group.The results of ICG clearance rate showed that compared with the WT control group,the ICG clearance rate of the SPARC knockout group significantly increased(P<0.05),indicating that the lymphatic drainage function was significantly enhanced.The draining lymph nodes in the WT control group were white and oval or round,while the draining lymph nodes in the SPARC knockout group were enlarged,slightly gray-brown,and round or oval.The volume of draining lymph nodes in SPARC knockout mice increased when compared with the WT group(P<0.05).The expression of lymphatic vessels in the draining lymph nodes showed that compared with the WT control group,the lymphatic vessels in the draining lymph nodes of the SPARC knockout group were significantly expanded(P<0.05).The results of ABOG staining showed that the lumbar intervertebral disc in the WT control group had no herniation,the nucleus pulposus was centrally located in the intervertebral disc and was dark blue,and the outer annulus fibrosus had a slight bulge;while the SPARC knockout group showed complete rupture of the annulus fibrosus,the nucleus pulposus was herniated,the extracellular matrix components significantly reduced,and the inflammatory infiltration obviously.The results of immunofluorescence staining showed that compared with the WT control group,the expression of lymphatic vessels in the draining lymph nodes and intervertebral disc tissues of SPARC knockout mice increased(P<0.05).3.There was no difference in each group before surgery and the end of the experiment in body weight(all P>0.05),indicating that modeling had no significant effect on the body weight of mice.The results of the swelling volume and diameter of the modeling site between the 4 W sham group and the 4 W model group showed that there was no significant difference in the swelling volume and diameter of the modeling site between the two groups before surgery(P>0.05);the swollen volume and diameter of the modeling site increased at 1 d,7 d,14 d,21 d and 28 d after surgery(P<0.05).The results of lymphatic draining function showed that there was no significant difference in the clearance rate of ICG in each group of mice before surgery(P>0.05);Compared with the 1 W sham group,the ICG clearance rate of the 1 W model group was significantly increased(P<0.05);Compared with the 2 W sham group,the ICG clearance rate of the 2 W model group was significantly increased(P<0.05);Compared with the 4 W sham group,ICG clearance rate of the 4 W model group was significantly decreased(P<0.05).The results of whole mount staining showed that there were very few lymphatic vessels in the skin of the modeling site in the sham group,while the number of lymphatic vessels in the model group increased significantly.Lymphangiogenesis at the modeling site increased at 1W,2W,and 4W after surgery,compared with the sham group(P<0.05).The smooth muscle of collecting lymphatic vessels in the 1 W,2 W and 4 W sham groups were closely distributed.The coverage area of the smooth muscle of the collecting lymphatic vessels did not change in the 1 W and 2 W model groups(P>0.05),but it was significantly decreased in the 4 W model group(P<0.05).The results of the drainage lymph node volume showed that compared with the sham group,the volume of the sciatic lymph nodes in the 1W,2W and 4 W models were significantly increased(P<0.05);compared with the sham group,the iliac lymph node volume were not significantly changes in the 1W,2W and 4W models(P>0.05).The expression results of lymphatic vessels in the draining lymph nodes showed that compared with the sham group,the lymphatic vessels in the lymph nodes of the 1 W,2 W,and 4 W model groups were significantly expanded(P<0.05).ABOG staining results showed that the intervertebral disc in the sham group was complete in shape,the annulus fibrosus was neatly arranged,the nucleus pulposus was centered,and the content was abundant;while the intervertebral discs showed disorder of the annulus fibrosus,the boundary between the annulus fibrosus and the nucleus pulposus was unclear,the nucleus pulposus herniated to both sides,and the staining of glycosaminoglycans significantly reduced or disappeared in the 1,2,and 4 W after surgery.The results of inflammatory infiltration of the intervertebral disc showed that compared with the sham group,1,2,and 4 W model groups had obvious inflammatory cell infiltration(P<0.05).The results of immunofluorescence staining showed that there were no lymphatic vessels and blood vessels in the intervertebral disc of the sham group,and a few lymphatic vessels and blood vessels were distributed in the paravertebral tissue.The lymphatic vessels in each group were mostly capillary lymphatic vessels,and there was no obvious distribution of collecting lymphatic vessels.Compared with the 1 W sham group,the 1W model group had lymphatic vessels and blood vessels ingrowth in the intervertebral disc(both P<0.05);compared with the 2 W sham group,the 2 W model group had more intervertebral disc lymphatic vessels and blood vessels(both P<0.05);compared with the 4 W sham group,the expression of intervertebral disc lymphatic vessels and blood vessels in the 4 W model group increased(P<0.05).4.In order to clarify the body weight changes of VEGFR3 knockout mice,body weight of mice in each group were detected before induction,after induction and before euthanasia.The results showed that the baseline body weight of VEGFR3flox/flox mice and VEGFR3 knockout mice were consistent with the pre-induction weights(both P>0.05),there was no significant about the body weight of the mice in each group after induction and before euthanasia(all P>0.05).The results of swelling volume and diameter showed that there was no significant difference in each group before modeling(all P>0.05);compared with the VEGFR3flox/flox sham group,the swelling volume and diameter of the modeling site in the VEGFR3flox/flox model group increased(both P<0.05),while the VEGFR3 knockout sham group had basically no obvious swelling(both P>0.05)at 1 d,7 d and 14 d after model was induced;compared with the VEGFR3 knockout sham group,the swelling volume and diameter of the VEGFR3 knockout model group increased(both P<0.05)at 1,7 and 14 days after model was induced;compared with the VEGFR3flox/flox model group,the swelling volume and diameter of the VEGFR3 knockout model group did not increase significantly at 1 and 7 days after modeling(both P>0.05).but significantly increased at 14 days after surgery(all P<0.05).The baseline ICG clearance rates of VEGFR3flox/flox control group and VEGFR3 knockout group were consistent before induction(P>0.05);compared with the VEGFR3flox/flox group.the ICG clearance rate of the VEGFR3 knockout group significantly decreased after induction(P<0.05).The results of ICG clearance rate in each group showed that compared with the VEGFR3flox/flox sham group,the VEGFR3flox/flox model group increased(P<0.05),and the VEGFR3 knockout sham group decreased(P<0.05);compared with the VEGFR3 knockout sham group,the ICG clearance rate of the VEGFR3 knockout model group did not change significantly(P>0.05);compared with the VEGFR3flox/flox model group,the ICG clearance rate of the VEGFR3 knockout model group decreased significantly(P<0.05)at the end of the experiment.The results of whole mount staining showed that the VEGFR3flox/flox sham group had fewer lymphatic capillaries,compared with the VEGFR3flox/flox sham group,the VEGFR3flox/flox model group significantly increased(P<0.05).There was almost no lymphatic capillary expression in the VEGFR3 knockout sham group,the VEGFR3flox/flox sham group had the same trend(P>0.05);compared with the VEGFR3 knockout sham group,there was no increase in the VEGFR3 knockout model group(P>0.05);compared with the VEGFR3flox/flox model group,the VEGFR3 knockout model group decreased significantly(P<0.05).The results of draining lymph node volume showed that compared with the VEGFR3flox/flox sham group,the volume of the sciatic lymph nodes in the VEGFR3flox/flox model group increased significantly(P<0.05),while the VEGFR3 knockout sham group showed no significant change(P>0.05);compared with the VEGFR3 knockout sham group.the sciatic lymph nodes in the VEGFR3 knockout model group increased(P<0.05);compared with the VEGFR3flox/flox model group.there was no significant difference in the volume of sciatic lymph nodes between the VEGFR3 knockout model group(P>0.05).The results of lymphatic vessel changes in the draining lymph nodes showed that compared with the VEGFR3flox/flox sham group,the expression of lymphatic vessels in the lymph nodes of the VEGFR3flox/flox model group was significantly increased(P<0.05),and the area ratio of lymphatic vessels in the lymph nodes of the VEGFR3 knockout sham group was no significant changes(P>0.05);compared with the VEGFR3 knockout sham group,the lymphatic vessels in the VEGFR3 knockout model group had no significant changes(P>0.05);Compared with the VEGFR3flox/flox model group,the expression of lymphatic vessels in the lymph nodes of the VEGFR3 knockout model group was significantly decreased(P<0.05).The results of ABOG staining of the intervertebral disc showed that the shape of the intervertebral disc were complete in the two sham groups,while the annulus fibrosus in the two model groups were disordered,the boundary between the annulus fibrosus and the nucleus pulposus disappeared,and the nucleus pulposus herniated to both sides.The results of inflammatory infiltration area showed that compared with the VEGFR3flox/flox sham group,the area of inflammatory infiltration in the VEGFR3flox/flox model group was increased(P<0.05),and there was no inflammatory infiltration in the intervertebral disc and surrounding tissues in the VEGFR3 knockout sham group(P>0.05);compared with the VEGFR3 knockout sham group,the VEGFR3 knockout model group had obvious inflammatory infiltration(P<0.05).The results of changes in the capillary lymphatic vessels of the intervertebral disc showed that the VEGFR3flox/flox sham group had no lymphatic distribution in the intervertebral disc,and only a few lymphatic expression in the surrounding tissue;compared with the VEGFR3flox/flox sham group,the VEGFR3flox/flox model group significantly increased(P<0.05),the VEGFR3 knockout sham group no statistical difference in the distribution of lymphatic capillaries(P>0.05);compared with the VEGFR3 knockout sham group,the VEGFR3 knockout model group increased the expression of capillary lymphatic vessels(P<0.05);compared with the VEGFR3flox/flox model group,the expression of lymphatic capillaries in the VEGFR3 knockout model group significantly decreased(P>0.05).The results of blood vascular expression in the intervertebral disc of each group showed that there was no blood vascular distribution in the intervertebral disc of the VEGFR3flox/flox sham group,and only blood vascular expression was found in the outer and surrounding tissues of the annulus fibrosus;compared with the VEGFR3flox/flox sham group,the number of intervertebral disc blood vessels increased in the VEGFRflox/flox model group(P<0.05),while the blood vessel distribution in the VEGFR3 knockout sham group did not change(P>0.05);compared with the VEGFR3 knockout sham group,the number of blood vessels in the VEGFR3 knockout model group increased(P<0.05);compared with the VEGFR3flox/flox model group,there was no significant difference in the number of blood vessels in the VEGFR3 knockout model group(P>0.05).5.BSTL intervened in the model mice of nucleus pulposus herniation.Whatever before surgery or euthanasia,the body weight of the mice in each group had no difference(all P>0.05).The results of swelling volume and diameter showed that there was no difference in the mice in each group before surgery(all P>0.05);compared with the sham group,the swelling volume and diameter of the model group increased(P<0.05).compared with the model group,the swelling of the modeling site in the BSTL low-dose,medium-dose and high-dose groups did not change(all P>0.05)at 1 day after surgery;compared with the sham group,the swelling volume and diameter of the model group increased(P<0.05),compared with the model group,the BSTL low-dose did not change(P>0.05).while BSTL medium-dose and high-dose groups increased at 7 days and 14 days after surgery(all P<0.05).The results of ICG clearance rate showed that compared with the sham group,the ICG clearance rate of the mice in the model group increased(P<0.05);compared with the model group,the ICG clearance rate at the modeling site of the BSTL low and medium dose groups was not statistically significant(P>0.05),the ICG clearance rate significantly enhanced in the high-dose BSTL group(P<0.05).The results of whole mount staining showed that compared with the sham group,the lymphatic vessels in the superficial tissue of the mice in the model group increased significantly(P<0.05);compared with model group,the number of lymphatic capillaries at the modeling site in the low-dose BSTL group did not change significantly(P>0.05),while the lymphatic vessels was increased in the medium-dose and high-dose BSTL groups(P<0.05).The results of the volume of sciatic lymph nodes showed that compared with the sham group,the sciatic lymph nodes in the model group were significantly enlarged(both P<0.05);compared with the model group,the sciatic lymph nodes in the low-dose BSTL group had no significant changes(both P>0.05),but the volumes of the sciatic lymph nodes in the BSTL medium-dose and high-dose groups were reduced(all P<0.05).The results of lymphatic changes in lymph nodes showed that compared with the sham group,the expression of lymphatic vessels in the lymph nodes of the model group was significantly increased(P<0.05);compared with the model group,the area ratio of lymphatic vessels in the lymph nodes of the BSTL low-dose group did not change significantly(P>0.05),while the expression of lymph vessels in the lymph nodes of the BSTL medium-dose and high-dose groups decreased(both P<0.05).ABOG staining results showed that in the sham group,the shape of the intervertebral disc was complete,the inner and outer annulus fibrosus were neatly arranged,the nucleus pulposus was located in the center of the intervertebral disc,and the extracellular matrix abundantly expressed and filled in the nucleus pulposus tissue.In the model group,the annulus fibrosus of the intervertebral disc were ruptured,the boundary between the annulus fibrosus and the nucleus pulposus disappeared,the nucleus pulposus tissue was prominent,and the central glycosaminoglycan staining of the intervertebral disc were significantly reduced or disappeared.When mice were treated with BSTL,the structure of the intervertebral disc were partially restored.The results of inflammatory infiltration in the intervertebral disc tissue showed that compared with the sham group,the intervertebral disc and surrounding tissue in the model group had obvious inflammatory cell infiltration(P<0.05);compared with the model group,the inflammatory infiltration area of the intervertebral disc and surrounding tissue in the low-dose BSTL group did not decrease significantly(P>0.05),but in the BSTL medium-dose and high-dose groups decreased(P<0.05).Conclusion:1.Lymphatic vessels are distributed in the surgical specimens of 16 clinical LDH patients,and mainly capillary lymphatic vessels,indicated that the lymphatic system may be involved in the pathological mechanism of LDH.2.The intervertebral disc of 14-month-old SPACR knockout mice developed spontaneous nucleus pulposus herniation,the expression of lymphatic capillaries in the herniated nucleus pulposus increased,and the function of paravertebral lymphatic drainage enhanced in mice,suggested that paravertebral lymphatic system may be associated with the resorption of nucleus pulposus herniation.3.The paravertebral lymphatic system is involved in the process of reabsorption of nucleus pulposus herniation,the paravertebral lymphatic drainage function enhanced in the early stage of nucleus pulposus herniation and damaged in the later stage,suggested that the establishment of paravertebral lymphangiogenesis and draining function may be a concrete manifestation of TCM for removing blood stasis.4.It hindered the resorption of nucleus pulposus herniation and aggravate the pathological process of the intervertebral disc via inhibiting paravertebral lymphatic draining function.5.BSTL formula promotes paravertebral capillary lymphangiogenesis,enhances lymphatic draining function,accelerates the resorption of nucleus pulposus herniation,reduces the inflammatory response,and then delays the pathological process of intervertebral disc,suggested that TCM treatment of removing blood stasis may be achieved by promoting the lymphatic drainging function. |
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