Effects Of Adipose-derived Stem Cells Comb Ined With Morinda Officinalis How On Renal Injury And Immunoinflammatory Factors In B6.MRL-Faslpr/Nju Lupus Mice

Purpose and backgroundSystemic lupus erythematosus is an autoimmune disease characterized by secretion of autoantibodies,formation of a large number of immune complexes,and chronic inflammation and terminal organ damage.In recent years,the incidence of SLE in the world is on the rise,and it is more common in women aged 15 to 45 years.SLE is mainly treated with glucocorticoids and immunosuppressive agents,which leads to adverse reactions in patients.With the development of medical technology,a new treatment method,stem cell therapy,has emerged.Stem cells are a very different group of cells,called"universal cells”in the medical world.It can treat a variety of diseases,has the potential to differentiate multiple cells and has a strong ability to self-renew.In recent years,allogeneic mesenchymal stem cell transplantation has been used to treat patients with refractory and severe SLE.Adipose-derived mesenchymal stem cells(ADSCs)are one of mesenchymal stem cells,which have the characteristics of mesenchymal stem cells and can differentiate into a variety of cells and have immunomodulatory ability.Compared with other mesenchymal stem cells,ADSCs have the advantages of abundant source,safe application,low price and few ethical restrictions.Previous studies have reported that adipose-derived mesenchymal stem cells have a certain regulatory effect on immune system damage caused by systemic lupus erythematosus.Morinda officinalis How(MOH)has the effect of tonifying kidney,strengthening bones and strengthening rheumatism,and is called "the medicine for tonifying kidney and yang".Studies have shown that kidney-tonifying Chinese medicine can promote the proliferation of mesenchymal stem cells to treat lupus nephritis.Therefore,this study explored the treatment of B6.MRL-Faslpr/Nju lupus mice by ADSCs combined with Morinda officinalis How,and observed whether Morinda officinalis How can promote the treatment of ADSCs proliferation in kidney injury of lupus mice,and whether it affects SLE-related immunoinflammatory factors,and explore new uses of Morinda officinalis How.Method1.Separation and cultivation of ADSCsTake 10 adult C57BL/6J mice with adipose tissue in the groin,abdomen and axilla.The adipose tissue was washed with PBS and then added with 0.25%type I collagenase,and placed at 37℃and shaken for 40 min;Obtained chyle-like adipose tissue and adipose-derived mesenchymal stem cells after centrifugation.The chyle-like adipose tissue and the adipose-derived mesenchymal stem cells were cultured in an incubator,and passage was performed when the cell fusion reached 80 to 90%.2.ADCSs induced differentiation and identificationThe third generation logarithmic growth phase of ADSCs cells was inoculated into 12-well plates for scaffolding.When cell fusion reached 50-60%,the expression of CD29,CD34,CD45 and CD105 on ADSCs was identified by immunofluorescence.The third generation logarithmic growth phase of ADSCs cells was inoculated into a 6-well plate,and when the cell fusion reached 100%,the adipogenic induction solution was added for adipogenic differentiation,and the oil red "0" staining was performed after the adipogenic induction.The third generation logarithmic growth phase of ADSCs was seeded in a 6-well plate.when the cell fusion reached 60%,the osteogenic induction solution was added for osteogenic differentiation,and the endogenous induction was followed by alizarin red staining.3.Growth cycle of ADSCs and effect of Morinda officinalis How on ADSCs activityADSCs were inoculated into 96-well plates,with 7 groups of 5 replicate wells per group.CCK-8 was tested daily for 7 days.Calculate the cell survival rate based on the OD value and plot the growth curve.ADSCs were inoculated into 96-well plates.After 24 h,cells were treated with different concentrations of MOH for 48 h.The normal control group was set up with 5 replicate wells in each group,and the cell survival rate was measured by CCK-8.4.Effects of ADSCs combined with MOH on renal injury and immunoinflammatory factors in lupus mice.10 C57 mice were set as normal group,and 40 B6.MRL-Faslpr/Nju mice were randomly divided into model group,ADSCs group,MOH group,ADSCs+MOH group.Normal group and model group were filled with normal saline;Atrial mesenchymal stem cells injected into the tail vein of ADSCs group;MOH group administered with Morinda officinalis extract;ADSCs+MOH group for combination therapy for a total of 8 weeks.Record the weight and general condition of the mice;ELISA was used to detect the concentration of anti-dsDNA antibody in serum and the expression levels of tumor necrosis factor-a,interleukin 17,interleukin-10 and interleukin-6 in renal tissue homogenate.Coomassie blue method was used to detect the change of urine protein concentration;HE staining was used to observe the histopathological changes of kidney.Result1.10 adult C57BL/6J mice were isolated to obtain 10 dishes of primary ADSCs,When the primary cells are cultured,the cells are short,round,oval,and triangular;After 24 hours,a small amount of cells adhered to the culture dish.After 48 hours,the number of adherent cells increased greatly,and the cells climbed out of the adipose tissue,and the cells became a pan-shaped and bundled shape.At 4 to 5 days,the cell fusion can reach 80%to 90%,and the cells are densely arranged in a bundle-like interlaced or spiral-shaped shape,and the cell morphology is long fusiform.The growth curve of mouse adipose-derived mesenchymal stem cells is a typical "S" type.The cell proliferation process has experienced latency,growth and plateau.The fastest cell proliferation rate on the 3 to 4 day is logarithmic growth phase.6 to 7 days into the platform period.This indicates that the adipose-derived mesenchymal stem cells have stable biological characteristics,strong vigor,fast regeneration rate,and easy to expand and culture in vitro.2.Immunofluorescence identification of ADSCs showed that CD29 expression was strongly positive,CD105 expression was weakly positive,and CD45 and CD34 expression were negative.The adipogenic induction solution was induced to 14 days,and the oil red "O" staining was carried out.It was found that many lipid droplets of different sizes were stained into red in the cytoplasm,indicating that ADSCs were successfully induced into adipocytes.When the osteogenic induction was carried out to 21 days,the cell structure was severely deformed under the microscope,and the body structure was formed.There was a residue of calcium nodules.The alizarin red staining showed that the calcium nodules were stained red;indicating that the osteogenic induction of ADSCs was successful.3.The results of CCK-8 test showed that MOH could increase the survival rate of ADSCs in the range of 20～200ug.mL-1(P<0.05).The survival rate of ADSCs decreased significantly when the concentration reached 500ug.mL-1(P<0.05).This indicates that Morinda officinalis How can promote the proliferation of ADSCs and increase cell viability in a certain concentration range.4.General observation of mice:The model group had severe hair loss and poor mental state,and 2 mice died;The hair and mental state of the ADSCs group improved,no mouse death;MOH group,ADSCs+MOH group B6.MRL/lpr mice gained significantly weight(P<0.05),hair luster,good mental state,no mouse death.In terms of experimental parameters,anti-dsDNA antibody,urinary protein,TNF-a,IL-17,IL-10 and IL-6 were significantly increased in the model group compared with the normal group(P<0.05;P<0.01).Compared with the model group,the anti-dsDNA antibody,urine protein,TNF-a,IL-17,IL-6 decreased in the ADSCs group(P<0.05;P<0.01),and the IL-10 difference was not statistically significant(P>0.05).In the MOH group,anti-dsDNA antibody,urine protein,IL-17,IL-10 and IL-6 decreased(P<0.05;P<0.01),and the difference of TNF-a was not statistically significant(P>0.05).ADSCs+MOH group:anti-dsDNA antibody,urine protein,TNF-a,IL-17,IL-10,IL-6 decreased(P<0.05;P<0.01).Renal pathological examination showed that adipose-derived mesenchymal stem cells,Morinda officinalis How and combination therapy can improve the renal pathological structure of B6.MRL/1pr mice,improve the proliferation of glomerular cells,and reduce the volume,while adipose-derived mesenchymal stem cells Combined with Morinda officinalis How treatment effect is is better.ConclusionThe ADSCs cultured in this experiment are stable in morphology,active,and proliferative,and can differentiate into adipocytes and osteoblasts.The expression of CD29 in ADSCs was strongly positive by immunofluorescence,the expression of CD105 was weakly positive,and the expression of CD45 and CD34 were negative.Morinda officinalis can promote the proliferation of ADSCs.ADSCs,Morinda officinalis How and combination therapy can improve the pathological structure of kidney in B6.MRL/lpr mice,and has a certain regulatory effect on some immunoinflammatory factors in renal tissue,but combined treatment is better.Which indicates that adipose-derived mesenchymal stem cells,Morinda officinalis How combined with the treatment of lupus mice have a synergistic effect;Chinese herbal medicine Morinda officinalis How can promote the prolifera-tion of ADSCs to improve the renal injury of B6.MRL/lpr lupus mice,and regulate the related immune inflammatory factors.Morinda officinalis How has a certain therapeutic effect on lupus mice,which can reduce Anti-dsDNA antibody,urine protein,IL-17,IL-10,IL-6 in lupus mice,and increase the weight of lupus mice.