Effect And Molecular Mechanisms Of D-lactate In Regulation Of Inflammation

Lactate is not simply considered as a metabolite in the process of anaerobic glycolysis,but a crusial source of tricarboxylic acid cycle and energy metabolism.Furthermore,lactate could act as a signaling molecule through the lactate’s receptor(G protein-coupled receptor81,GPR81)to guide metabolic activities.D-lactate is usually considered as the product that causes metabolic burden of the body due to the lack of D-lactate dehydrogenase(DLDH)in mammals,thus the World Health Organization advocates the use of L-lactate instead of DL-lactate in foods.And therein lies the paradox for nutritionist,it is necessary to strengthen studies on the physiological activities of D-lactate,which can provide scientific evidence and theoretical basis for directed selection and individualized application of probiotics,significant for precision nutrition and medicine.The present study tended to investigate whether or how D-lactate affects the body’s immunity based on discussing the in-vivo biological differences between D-and L-lactates.The objective of this study is to evaluate the potential of D-lactate in host immune regulation and clarify its underlying mechanism of actions.There are several major findings as below:1.The study explored the safety and effectiveness of D-lactate as an immune regulator for food.(1)Both the two configurations promoted cell proliferation and damage repair at 40 mM,while they did not negatively affect the body weight,indexes for immune organ,indexes for the kidney function(represented by BUN and CRE),and indexes for the liver function(represented by ALP,ALT,and AST)in mice at the highest 2,000 mg/kg.(2)With respect to the immunomodulatory activity,lymphocyte transformation test and delayed hypersensitivity test were applied to assess the effects of different lactate configurations on cell immune function;hemolytic plaque assay was used to assess their effects on humoral immune function;and microsphere phagocytosis assay was adopted to evaluate their effects on non-specific immune functions.As analyzed,D-lactate enhanced the immunity of the body in a dose-dependent manner by increasing immune organ index,ear swelling index,the ratio of lymphocyte transforming,hemolysis ratio,and phagocytosis ratio of macrophages,exhibiting stronger immunoregulatory activity than L-lactate.(3)In terms of the difference in pharmacokinetics between the two configurations,pharmacokinetic approach and HPLC column for chiral separation were applied.We found that the D-lactate had a plasm terminal half-life(T1/2)9-fold longer than L-lactate,which maintained high D-lactate concentration in the plasm and was conducive for D-lactate to exerting biological effects.2.Two representative models of acute inflammation,including a DSS-induced injury-type inflammation model and a LPS-induced systemic inflammation model,were selected to assess the effect of D-and L-lactates on the occurrence and development of inflammation with pharmacologic approaches.(1)Administration of D-lactate alleviated DSS-induced acute ulcerative colitis by decreasing the decline in body weight and DAI index,reducing the shortening of colon length and the ulcerated area within the colon.In addition,preventive administration of D-lactate accelerated the recovery of mice from ulcerative colitis.(2)D-lactate was also effective in alleviating the LPS-induced systemic inflammation,conducive to suppressing the enlargement of immune organs(represented by the spleen),inflammatory cytokines storm(represented by a surge in peripheral TNF-α and IL-6),and the systemic activity of neutrophils.3.The mechanism of the anti-inflammatory activity of D-lactate was explored using the primary peritoneal macrophages isolated from mice deficient in GPR81 gene.(1)Expression analysis revealed that GPR81 gene deficiency augmented the LPS-induced expression of macrophage M1 markers(TNF-α,IL-6,and NOS-2)but inhibited the IL-4-mediated expression of M2 markers(Arg1 and YM1),suggesting a critical role of the GPR81 gene in macrophage M1 polarization.(2)GPR81 gene deficiency was beneficial for the phosphorylation of MAPK kinases(JNK and p38),Ik B degradation,TRAF6-dependent K63-linked ubiquitination,and expression of pro-inflammatory proteins(COX-2 and i NOS).(3)AKT inhibitor Wortmannin blocked the promoting effect of GPR81 on phosphorylation of GSK-3β,and silencing A20 gene attenuated the anti-inflammatory effects exerted by activated GPR81.These results demonstrated that GPR81 could increase the stability of A20 gene and inhibit the TRAF6-dependent ubiquitination via the AKT-GSK3β pathway,thereby improving the inflammatory cascade.(4)D-lactate could suppress primary peritoneal macrophage M1 polarization and alleviate the endotoxin shock and acute inflammatory responses in wild-type mice,which indicated that D-lactate played its anti-inflammatory activity dependent on GPR81.4.the immunoregulatory function of the D-lactate-GPR81 axis was validated in older mice with age-related immune disorder.(1)Intestinal barrier dysfunction and disordered macrophage polarization were results of aging,while aging was accompanied by decrease in receptor GPR81 expression.(2)The GPR81 gene deficiency exacerbated the age-related immune disorders and enhanced the sensitivity of older mice to the DSS-induced intestinal barrier disruption by increasing intestinal permeability and degree of bacterial translocation,and decreasing expression of intestinal tight junction proteins related m RNA(ZO-1,Occludin)and mucosal proteins related m RNA(Muc-1,Muc-2).(3)With the GPR81-A20 axis,D-lactate improved the age-related disorders in intestinal barrier function and macrophage polarization,and it also reduced the sensitivity of older mice to the DSS-induced intestinal barrier disruption,leading to decreased intestinal permeability and degree of bacterial translocation,increased expression of intestinal tight junction proteins and mucosal proteins,and attenuated DSS-induced exacerbation of colonic histopathology.In all,the present study highlighted the housekeeping function of the D-lactate in inflammatory regulation,providing scientific evidence and theoretical basis for future directed selection and individualized application of probiotics.