| Background: Holism is the basic philosophy of traditional Chinese medicine(TCM),which emphasize the interaction between the whole body and the local pathology.In recent years,the research on the relationship between type 2 diabetes mellitus(T2DM)and degenerative lumbar spinal stenosis(DLSCS)has become the focus in modern medicine.TCM also reveal that Xiao Ke,a systemic disease,has an impact on local disorder of Yao Bi,and the mediator of the two may be the blood stasis.The concept of inflammatory response in modern medicine is an important part of blood stasis in TCM,which is also an important part of connective tissue fibrosis.Under the guidance of the theory of holistic view of TCM,it is essential to explore the specific mechanism of effect of systemic metabolic disease diabetes mellitus on lumbar spinal disease and the curcuma longa's potential value in the prevention and treatment.Objective: Retrospective analysis was performed under the guidance of TCM holistic view to explore the influence of Xiao Ke(such as T2DM)on the ligamentum flavum and other important spinal canal parameters from patients with Yao Bi(such as DLSCS).Studies in vivo and vitro were applicated to detect the content of macrophage migration inhibitory factor(MIF)and its related inflammatory mediators in the lumbar ligamentum flavum(LLF)and reveal the pathological changes of the LLF to clarify therelationship between inflammation and blood stasis.This study also aimed to explore the pro-inflammatory mechanism of MIF on the LLF effect cells and the inflammatory intervention effect of curcumin,the effective component of Curcuma longa,according to the theory of "treating from stasis".Methods:(1)A retrospective analysis was performed on patients with DLSCS from January 2016 to January 2019 in the PLA General Hospital of Central Theater Command.The differences were compared in the thickness of the LLF,distance between the inferior articular processes and sagittal diameter of the effective space of spinal canal between two groups with and without T2DM(DM+ and DM-).(2)Prospective design: According to the criteria of diagnosis,inclusion and exclusion,40 patients with degenerative lumbar spinal stenosis were enrolled in all spine surgery departments of Hubei 672 Orthopaedics Hospital of Integrated Chinese&Western from August 27,2018 to May 29,2019.Forty patients with degenerative lumbar spinal stenosis were divided into DM+ group complicated with type 2 diabetes mellitus and DM-group without type 2 diabetes mellitus.Among the 40 patients with type 2 diabetes mellitus(DM + group),there were 8 males and 12 females,with an average age of 61.55 ± 2.41 years,BMI of 25.62 ± 0.785.In this group,there were 14 cases with L4 / L5 level and 4 cases with L5 / S1 level,there were 2 cases of L3 / L4 level,20 cases of no diabetic patients(DM-group),including 11 males and 9 females,with an average age of 58.85 ± 3.78 years,BMI of 23.60 ± 0.643.In this group,there were 15 cases of L4 / L5 level,3 cases of L5 / S1 level and 2 cases of L3 / L4 level.The thickness of the lumbar ligamentum flavum was measured preoperatively on the horizontal view of preoperative MRI according to the Jong beom Park ligamentum flavum measurement method.During the lumbar surgery,LLFwere moved completely and divided into a and b parts according to the left and right sides of spinal canal(Part a of dry ice box was frozen after collection,part b was fixed with formaldehyde).Part a was measured by ELISA for the content of MIF and related inflammatory mediators.Part b was measured by Masson staining and immunohistochemistry(IHC).Image,biochemical and statistical methods were used to compare the differences between the two groups.(3)NIH3T3 cells were resuscitated,cultured and passaged by conventional methods(3-5 generations for experiment).1.Normal(5m M glucose)and high glucose(25m M glucose)were used to stimulate NIH3T3 cells for 48 hours.Macrophage migration inhibitory factor(MIF)expression was detected by Western Blot(WB),and cell proliferation was compared by microscope in those two groups.2.Exogenous MIF(recombinant mouse macrophage migration inhibitory factor,rm MIF)was introduced and set as N group with normal concentration rm MIF(2ng/ml),L group with low concentration rm MIF(4ng/ml),M group with medium concentration rm MIF(10ng/ml),H group with high concentration rm MIF(50ng/ml).NIH3T3 cells were cultured into those N,L,M,H groups to observe following items: 1)Proliferation of NIH3T3 cell by microscope at 48 h.2)CCK-8 kit was used to test NIH3T3 cells at 0,24,48 and 72 h respectively.A450 value from N,L,M,H groups were compared at 48 h.3)The S-phase of fibroblast proliferation was tested and compared at 48 h by flow cytometry.3.The m RNA and protein expression of cyclin D1 were detected by real time polymerase chain reaction(RT-PCR)and WB after 48 h stimulation of NIH3T3 cells with normal concentration of rm MIF(2ng/ml),high concentration of rm MIF(50ng/ml)and high concentration of rm MIF+Rho kinase specific inhibitor Y27632.(4)NIH3T3 cells were cultured into group A(control group),group B with rm MIF(50ng/ml),group C with rm MIF(50ng/ml)+ ISO-1(20?M),group D rm MIF(50ng/ml)+ curcumin(20?M)for 48 h.1)A450 value was tested and compared by CCK-8 kit in the groups of A B D.2)RT-PCR and Western Blot were used to detect and compare the inflammatory mediators(TGF-?1,MMP-13,IL-1?,IL-6,TNF-?)and collagen(COL-1,COL-3)in four groups.Results:(1)The thickness of LLF in DM+ group and DM-group were 5.23±0.71 mm VS 4.42±0.60 mm.The distances between inferior articular processes were11.32±1.23 mm VS 12.09±1.57 mm.The sagittal diameter of the effective lumen of the vertebral canal were 7.57±1.87 mm VS 8.24±1.74 mm.There were statistical differences in these three parameters between those two groups(P<0.05).(2)The thickness of LLF in DM+ group and DM-group were 5.567±1.090 mm VS 4.828±0.552.The content of MIF were(312.105±80.820)vs 210.363±62.182)pg/mg protein.TGF-?1were(2728.121± 890.373)vs(2170.131 ±689.459)pg/mg protein.MMP-13 were(564.167±189.391)vs(379.841±101.363)pg/mg protein.IL-1?were(15.585±5.797)vs(10.785±2.787)pg/mg protein.IL 6 were(17.238±4.449)vs(12.523±2.842)pg/mg protein.TNF-?were(13.947±4.688)vs(9.829±2.616)pg/mg protein.Masson staining showed the volume fraction of collagen were(50.354 ± 9.762)vs(41.803 ± 5.873).The IOD of MIF positive staining of IHC were(2808.882 ± 699.779)vs(1615.892 ± 408.609).Statistically significant differences from those items were found between DM+ and DM-groups(P< 0.05).The MIF content in LLF revealed a positive correlation with its thickness(r=0.768,P=0.000).(3)1.Compared with normal glucose group,NIH3T3 cells cultured in high glucose group showed higher expression of MIF protein,and NIH3T3 cells proliferated significantly in high glucose group.2.1)NIH3T3 cells'morphology were different in four concentration of rm MIF at the same time point(48h).Cell density was increased with higher concentration of rm MIF.2)CCK-8 kit at the 48 h showed that there was a significant difference in A450 between the normal,low,medium and high rm MIF concentration groups(P<0.05).Compared with the N group,the proliferation activity of the L,M and H group were gradual higher.The significant statistically differences were found in every two groups(P<0.05).3)Flow cytometry showed that the percentage of S phase cells increased with the increase of rm MIF concentration.3.The m RNA and protein of cyclin-D1 in NIH3T3 cells increased under rm MIF stimulation for 48 hours and decreased by Rho pathway inhibitor block.(4)CCK-8 kit showed that the A450 value of group B was higher than that of group A(P<0.05).The A450 value of group D was lower than that of group B(P<0.05).No statistical difference was found between the group A and group D(P>0.05).RT-PCR and WB showed that the m RNA and protein expression of inflammatory mediators and?,? type collagen in group B were higher than that in group A(P<0.05).The m RNA and protein expression of those in group D were lower than that in group B(P<0.05).There were no significant difference between group D and group C(P>0.05)Conclusion: 1.Retrospective analysis show that the severity of hypertrophy of LLF and other two parameters in the DM+ group are significantly higher than that in the DM-group,which confirm that T2 DM is a susceptible factor for lumbar stenosis especially in hypertrophy of LLF.It is consistent with the theory of holistic view of traditional Chinese medicine to reveal that systemic metabolic factors could affect the local lumbar spine.2.Study in vivo and vitro show that MIF and its related inflammatory mediators in the LLF of DM+ group are higher than that of DM-group.MIF reveal positive correlation with its thickness.In the patients with type 2 diabetesmellitus,there are more obvious degradation of elastic fibers and proliferation of collagen fibers in the samples of ligamentum flavum,and the MIF content in this group are higher and more widely distributed in the ligamentum flavum in this group.3.The high glucose condition could stimulate the high expression of MIF in fibroblasts to promote the proliferation and the expression of inflammatory mediators.The inflammatory response of MIF may be the main reason of mediating LLF hypertrophy.Among them,the proliferation of fibroblasts promoted by MIF may be an important pathological part of scar reshaping in LLF.Inflammatory injury and repair are important parts of inflammatory response,which is one of the main connotations of blood stasis theory in traditional Chinese medicine.4.study in vitro show that curcumin could effectively block the inflammatory effect of MIF on fibroblasts,which indicate that curcumin may be one of the effective medicine to prevent and treat T2 DM complicated with LLF hypertrophy.The above study have illustrated the important guiding role of TCM holistic view in analysis of the mechanism of LLF hypertrophy in patients with T2 DM.Meanwhile,it has proved that the theory of "treating from stasis" in TCM may be one of the important direction in effective prevent and treatment for this systemic chronic disease complicated with LLF hypertrophy. |
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