Protective Effect Of Cannabinoid CB₂Receptor Pathway On Endotoxic Shock And The Related Mechanisms

Backgrounds and objectivesSeptic shock, also known as endotoxic shock, is a syndrome caused by infection withpathogens and including mainly fever, shiver, cold clammy limbs, cyanosis, tachycardiaand the low blood pressure. It is a common clinical critical diseases with a high mortalityrate and often complicated by multiple organ dysfunction. It is the most common thatseptic shock infected with gram-negative bacterium in the clinical. Lipopolysaccharide,also called endotoxin, can activate mononuclear-macrophages in the blood and the tissue toproduce many cytokines which initiates the inflammatory cascade to lead to SIRS, multipleorgan failure, and even death. Now there are many treatments of septic shock including anincrease in blood volume, the rectification of acid intoxication, improving themicrocirculation disturbance, the infection control and the cytokines release inhibition.And then the inhibition of cytokine release is available to control systemic inflammatoryresponse and improve the microcirculation disturbance and the metabolic disorder, so thecytokines release inhibition plays an important role in resistance to shock treatment. In aword, it is important to find new drugs for the septic shock treatment.Marijuana was an ancient plant and considered as a narcotic drug to use widespreadlyas early as thousands of years ago. Although the marijuana had a variety ofpharmacological effects including analgesic and sedative, its application was still restrictedseverely due to its addictive property. The cannabinoid was a main active ingredient in themarijuana. In recent years, researches found cannabinoids still had many otherpharmacological effects, such as appetite regulation, lipid metabolism regulation, bloodpressure regulation, immunoregulation and the protective effects of heart and nerve exceptfor the analgesia and the sedation of the marijuana. Researchers also found two kinds ofcannabinoid receptors existed in vivo, namely CB₁receptors and CB₂receptors. The CB₁receptors mainly distribute in the brain, the spinal cord and the peripheral nervous system,and relate to the memory, the cognitive function, the addiction and the motion control. TheCB₂receptors express mainly in the immune organization, mediating immune regulations.The researchers abundantly researched about the immune regulation of cannabinoids,most of which showed that cannabinoids can exert immunosuppressive actions and the fewof which showed cannabinoids can boost immune responses. The precise mechanisms through which cannabinoids mediate immunoregulation is only now beginning to beunderstood and can be broadly categorized into four pathways: induction of apoptosis,inhibition of proliferation, suppression of cytokine and chemokine production andinduction of T regulatory cells （T regs）.Apoptosis is the death that the body maintains homeostasis and to which cellsgenetically controlled independently and orderly go. Apoptosis includes changes from cellsform （such as cells shrink, karorrhexis） and molecular （such as caspases and cytochrome）.THC, a main active ingredient of the marijuana, played a role through the CB₁receptor andthe CB₂receptor. Studies have confirmed THC promotes apoptosis from splenic cell,macrophages and T lymphocyte. Further study found that the apoptosis-promoting of THCwas relative with changes of activities of Bcl-2and caspase-1; The non-selectivecannabinoid receptor agonist cannabidiol could induce apoptosis of CD4+cells andCD8+T cells by enhancing oxide productions and activities of caspase-3and8.The studiesalso found that cannabinoids could inhibite cell apoptosis.The selective CB₂agonistsJWH-015excepted for inhibiting T lymphocytes apoptosis, still could inhibit the Blymphocytes and thymus cell apoptosis, and further researches found that caspase-3,8and9were involved with cell apoptosis. The studies show that cannabinoids could change theactivities of caspases and oxide and adjust apoptosis.The researchers found that anandamide and JWH-015inhibited T and B lymphocyteproliferation, but the researchers also found the small doses of CP55940, WIN55212-2,and Δ9THC promoted B lymphocyte proliferation, and CP55940promoting proliferationcould be suppressed by SR144528, but can’t be blocked by SR141716A. In addition,2-AGcan induce HL-60cell differentiating into macrophages cell, and SR144528could restrainthe differentiation of2-AG. These studies suggested that cannabinoids could have two-wayadjustment of immune cell proliferation and differentiation with dependent concentration,and CB₂receptor may mediate the immune adjustment.Cannabinoids regulate the production of a variety of inflammatory cytokines in manystudies. THC, a main active ingredient of the marijuana, suppressed B cells to produceIL-8and MIP-1, NK cells to produce TNF-α, GM-CSF, IFN-γ,MIP-1α,MIP-1β and IL-10,Eosnophils to produce IL-8、MIP-1α and MIP-1β. THC also inhibited Th1cells to produceIFN-γ and IL-12, and promoted Th2cells to produce IL-4and IL-10, which led to Th cellsdifferentiated into Th2cells and promoted humoral immunity from Th2cells in theinfection models of Legionella pneumophilas. The non-selective cannabinoid receptor agonist WIN55212-2reduced IL-6production and abated the fever from LPS. However, inprevious reports cannabinoids do not consistently inhibit inflammation. THC promotedinflammatory cytokines in the acute phase in mice infected with Legionella pneumophilas.So, there were questions on the cytokine regulation.Although cannabinoids had a variety of pharmacological effects, its application wasstill restricted severely due to its addictive property. The selective cannabinoid receptortype-2agonist can combine selectively CB₂receptors, avoiding induction of addiction. Theselective cannabinoid receptor type-2agonist HU-308could not only inhibit the expressionof Chemokines MCP-1, TNF-α, IL-1β and ICAM-1in kidney tissues to reduce kidneydamage from cisplatin, but also inhibit to produce TNF-α, MCP-1α and MCP-2in theblood and the kidney tissue to prevent the kidney ischemiaThe studies above showed that cannabinoids can regulate the production of cytokinesin the inflammatory process. Cytokines had complex relationships due to their mutualadjustment, control and influence. Some researches showed cannabinoids inhibited theproduction of cytokines, but other researches showed cannabinoids promoted theproduction of cytokines. So debates on the effect of cannabinoids on the induction ofcytokines still exist. The purpose of this study is to investigate protective effect ofcannabinoid CB₂receptor pathway on endotoxic shock and the related mechanisms.Methods1In vivo experimentMale wild type mice （C57BL/6mice） and male CB₂-/-mice, the weight about20²5g, were intraperitoneally injected with LPS to build endotoxin shock model and investigate24h survival rate of wild type mice and CB₂-/-mice in the endotoxin shock model2In vitro experiment（1） Isolation and culture of peritoneal macrophagesEach C57BL/6mouse was intraperitoneally injected with2ml/day of3%broth.Three days later, the mice were killed. And peritoneal macrophages were collected understerile conditions. Then the density of the cells was adjusted to106/ml in the6wellculture plates or2×106/ml in the96well culture plates. After incubated in the37°C,5％ CO2incubator for2h, peritoneal macrophages were washed with PBS and incubatedwith new culture medium.（2） Treatment of peritoneal macrophagesOn the fifth day, peritoneal macrophages were washed with PBS and was added thenew culture medium. And then those cells treated with WIN55212-2or HU-308.15minlater,10ng/ml LPS were added to those cells. The group without any treatmens wasregarded as a negative control group; the group with LPS was regarded as a positivecontrol group.（3） Analysis the expression of cytokines at gene levelPeritoneal macrophages with above treatment were incubated in in the37°C,5％CO2incubator for3h. And RNA were extracted from peritoneal macrophages, then RNA wasreversely transcripted into cDNA.RT-PCR and qRT-PCR were measured cytokines TNF-αand IL-6generated by LPS-challenged peritoneal macrophages（4） Measurement of the amounts of cytokines at protein levelPeritoneal macrophages with above treatment were incubated in in the37°C,5％CO2incubator for24h. IL-6assay were measured cytokines IL-6in the supernate.Results（1） The wild-type mice and CB₂-/-mice in the LPS-induced endotoxin shock modelshowed the typical shock symptoms, such as eye secretions increased, diarrhea andsluggish movement, and shock symptoms of CB₂-/-mice were more serious. CB₂-/-micebigan to die within1h when CB₂-/-mice were intraperitoneally injected with LPS（15mg/kg）, and the survival rate of CB₂-/-mice is46.7%. The wild-type mice all lived, andthe survival rate of the wild-type mice is100%.This show CB₂receptor was beneficial tothe resistance of endotoxic shock.（2） The results of RT-PCR showed that3μM WIN55212-2could significantly reducethe LPS-activated peritoneal macrophages to express the TNF-α mRNA and IL-6mRNA;0.3μM WIN55212-2could significantly promote the LPS-activated peritonealmacrophages to express the TNF-α mRNA and IL-6mRNA. The results of qRT-PCR further showed that the expression of TNF-α mRNA in3μM WIN55212-2group was1/2times that in the positive control group; the expression of TNF-α mRNA in0.3μMWIN55212-2group was less than twice that in the positive control group; and the resultshowed WIN55212-2regulate bidirectionally the LPS-activated peritoneal macrophages toexpress the TNF-αmRNA. the expression of IL-6mRNA in3μM WIN55212-2group was1/3times that in the positive control group; the expression of IL-6mRNA in0.3μMWIN55212-2group was about2.5times that in the positive control group; the expressionof IL-6mRNA in1μM WIN55212-2group was about2times that in the positive controlgroup; and the result showed WIN55212-2regulate bidirectionally the LPS-activatedperitoneal macrophages to express the IL-6mRNA.（3） The results of ELISA showed that3μM WIN55212-2could significantly reduce theLPS-activated peritoneal macrophages to produce IL-6, and the production of IL-6in3μMWIN55212-2group was1/3times that in the positive control group;0.3μM�'�1μMWIN55212-2could significantly promote the LPS-activated peritoneal macrophages toproduce IL-6, and the production of IL-6in0.3μM�'�1μM WIN55212-2group wasrespectively4and2times that in the positive control group. and the result showedWIN55212-2regulate bidirectionally the LPS-activated peritoneal macrophages to produceIL-6.（4） The effects of HU-308on inflammatory cytokines the TNF-αmRNA and IL-6mRNA generated by LPS-challenged peritoneal macrophages was identical toWIN55212-2, The results of RT-PCR showed showed that3μM�'�10μM HU-308couldsignificantly reduce the LPS-activated peritoneal macrophages to express the TNF-αmRNA and IL-6mRNA;0.3μM�'�1μM HU-308could significantly promote theLPS-activated peritoneal macrophages to express the TNF-α mRNA and IL-6mRNA. Theresults of qRT-PCR further showed that the expression of TNF-α mRNA in3μM and10μM HU-308group was lower than that in the positive control group, the expression ofTNF-α mRNA in3μM and10μM HU-308group was respectively1/2and1/3times thatin the positive control group, the expression of TNF-α mRNA in0.3μM HU-308groupwas3times that in the positive control group, and the result showed HU-308regulatebidirectionally the LPS-activated peritoneal macrophages to express the TNF-α mRNA. theexpression of IL-6mRNA in3μM and10μM HU-308group was lower than that in thepositive control group, the expression of IL-6mRNA in3μM and10μM HU-308groupwas1/4times that in the positive control group, the expression of IL-6mRNA in0.3μM HU-308group was2times that in the positive control group, and the result showedHU-308regulate bidirectionally the LPS-activated peritoneal macrophages to express theIL-6mRNA.（5） The results of ELISA showed that0.3μM HU-308could significantly promote theLPS-activated peritoneal macrophages to produce IL-6, and the production of IL-6in0.3μM HU-308group was nearly5times that in the positive control group;3μM and10μMHU-308could significantly reduce the LPS-activated peritoneal macrophages to produceIL-6, and the production of IL-6in3μM and10μM HU-308group was1/4times that inthe positive control group.Conclusions1CB₂receptor passway was beneficial to the resistance of endotoxic shock.2Cannabinoid receptor agonists regulate bidirectionally the LPS-activated peritonealmacrophages to express the TNF-α and IL-6, and CB₂receptor were related with this role.