Protective Effect Of The Anthraquinone Extract Of Rhubarb On Organ Damage In Sepsis Mice By Modulating Macrophage Polarization

Background: The systemic "inflammatory storm response" mediated by an excessive defense response of immune cells is the key pathological mechanism of sepsis.Macrophages are mainly divided into M1 and M2 types,with the M1 type mediating the pro-inflammatory response by releasing TNF-α,IL-6,etc.CD16/32 and i NOS are its marker proteins.While the marker proteins of the M2 type are CD206 and Arg-1,which mainly resist inflammatory response and repair tissues by releasing IL-4,IL-10,etc.In addition,the marker protein F4/80 symbolizes all activated macrophages.It has been reported that M1 type macrophage shows uncontrolled activation in the early stage of sepsis,which is closely related to the "inflammatory storm response" and the inflammatory damage of organs.Free anthraquinone(FA)and combined anthraquinone(CA)are the main active components of rhubarb,both of which have significant antiinflammatory effects.Previous studies have confirmed that anthraquinones of rhubarb could reverse the "inflammatory storm response" of sepsis and alleviate liver and lung injury.However,studies on whether rhubarb anthraquinones reverse the inflammatory response of sepsis and protect against liver and lung injury by regulating macrophage polarization are lacking and need further confirmation.Objective: Lipopolysaccharide(LPS)intraperitoneal injection was used to establish the sepsis mouse model,and to investigate whether rhubarb FA and CA extracts exert protective effects on acute liver injury and lung injury in sepsis mice through regulating macrophage polarization.Methods: According to the randomization principle,male,healthy balb/c mice were divided into control group(CG group,n=10),sepsis model group(LPS group,n=10),rhubarb free anthraquinone treatment group(FA group,n=10)and combined anthraquinone treatment group(CA group,n=10),a total of 4 groups.Except for the CG group,sepsis models were constructed in the LPS group,rhubarb FA and CA treated mice by the LPS intraperitoneal injection method at a dose of 15 mg/kg,while the CG group was injected with saline only.Rhubarb FA and CA were dissolved and prepared by 0.5% sodium carboxymethylcellulose(CMC-Na)solution with a solubility of 10mg/ml.And rhubarb FA and CA solutions were administered by gavage at a dose of 100mg/kg at 12 h before and 2 h after modeling;while the CG and LPS groups were given0.5% CMC-Na solution by gavage.The general condition of mice in each group was observed,and serum,liver and lung tissues were collected and preserved after 12 h.Hematoxylin-eosin(HE)staining was used to observe the changes in liver and lung tissues in pathological aspects;the expression of cytokines TNF-α and IL-10 in serum,liver and lung tissues was measured by ELISA;the expression of macrophage markers F4/80 and CD206 protein in liver and lung tissues was measured by immunohistochemistry;the expression of two other macrophages markers i NOS and Arg-1 protein in liver and lung tissues was measured by Western blot.Results:1.The general condition of mice in the CG group was normal,while the general condition of mice in the LPS group was worse,showing poor mental health,reduced activity,shortness of breath,loose stools,clumping and vertical hair phenomenon,and more obvious inflammatory pathological damage of liver and lung tissues was observed under the microscope(P < 0.05).Compared with the LPS group,the general condition of mice in the rhubarb FA and CA treatment groups was improved,and the inflammatory pathological damage in liver and lung tissues was significantly alleviated,but worse than in the CG group(P < 0.05).2.Compared with the CG group,the expression levels of TNF-α and IL-10 in the serum of mice in the LPS group were significantly increased(P < 0.05).Compared with the LPS group,rhubarb FA and CA gavage treatment significantly inhibited the expression of TNF-α and promoted the expression of IL-10 in serum(P < 0.05).3.The expression levels of F4/80,i NOS and TNF-α proteins in the liver and lung tissues of mice in the LPS group were significantly higher than those in the CG group,and the expression levels of CD206 and Arg-1 proteins were significantly lower than those in the CG group(P < 0.05),while no significant differences were seen for IL-10,indicating that LPS may induce macrophages to M1 type.Compared with the LPS group,rhubarb FA and CA gavage treatment significantly inhibited the expression of F4/80,i NOS and TNF-α proteins and promoted the expression of CD206,Arg-1 and IL-10 proteins in liver and lung tissues of mice(P < 0.05),indicating that rhubarb FA and CA treatment inhibited M1 type polarization and promoted M2 type polarization.Conclusion: The sepsis mice were accompanied by more obvious inflammatory pathological damage in liver and lung tissues,as well as an increase in the number of activated macrophages in liver and lung tissues,and M1 type macrophages showed overactivation;while both rhubarb FA and CA extracts were able to inhibit M1 type polarization and promote M2 type polarization after treatment,and suppress the LPS induced systemic inflammatory response by regulating the balance of M1/M2 ratio,thus reducing the inflammatory damage in liver and lung tissues of sepsis mice.