| BackgroundGypsum fibrosum is an important part of Chinese herbal medicines for clearing away heat and purging fire.It has the effect of clearing heat and purging fire,eliminating irritability and quenching thirst.The Qing Dynasty doctor Zhang Xichun regarded it as"the special medicine for clearing heat from yangming meridian or stomach".18 prescriptions containing gypsum fibrosum were recorded in the book of "Treatise on Febrile and Miscellaneous Diseases",and the gypsum fibrosum exerts different effects through compatibility with different medicines.In modern clinical practice,it was often used to treat various inflammatory and immune related diseases in combination with other drugs,such as exogenous hyperthermia,pneumonia,viral myocarditis,rheumatoid arthritis,and psoriasis.Some studies reported that the solution reached saturation when gypsum fibrosum was used in large doses,increasing the amount of gypsum fibrosum was not beneficial for improving clinical efficacy.However,it is common in clinical practice that different doses of gypsum fibrosum exert different effects.The basis of gypsum fibrosum exerting pharmacodynamic properties is still unclear.ObjectiveFrom the perspective of inorganic elements,the material basis and mechanism of anti-inflammatory regulation of gypsum fibrosum are explored under different inflammatory models,which provide a basis for better use of gypsum fibrosum in clinical practice.Methods1.Determination of inorganic elements in gypsum fibrosum decoction:Concentrations of the inorganic elements in different concentrations of gypsum fibrosum decoction(240g/40mL,480g/40mL,960g/40mL),gypsum fibrosum and glutinous rice decoction(270g/40mL),glutinous rice decoction(30g/40mL)were detected by using ICP-MS method.There were mainly 15 inorganic elements being detected:calcium(Ca),strontium(Sr),iron(Fe),aluminum(Al),nickel(Ni),manganese(Mn),chromium(Cr),copper(Cu),zinc(Zn),arsenic(As),cobalt(Co),selenium(Se),titanium(Ti),potassium(K),magnesium(Mg);differences of inorganic elements in different concentrations of gypsum fibrosum decoction were explored;differences between the gypsum fibrosum decoction and the gypsum fibrosum and glutinous rice decoction were explored.2.In the LPS-induced RAW264.7 cell inflammatory model,the anti-inflammatory effect of gypsum fibrosum decoction was investigated:The RAW264.7 cells were cultured in vitro.The effect of different concentrations of gypsum fibrosum decoction on the viability of RAW264.7 cells was detected by CCK8.Concentrations of gypsum fibrosum decoction which did not affect the viability of RAW264.7 cells were selected for the next step.The RAW264.7 cells in logarithmic growth phase were plated into a 24-well plate at an initial concentration of 1×106 cells/mL,and cultured in a 37?,5%CO2 incubator for 24 hours.Different concentrations of gypsum fibrosum decoction and LPS solution(final concentration was 1?g/mL)were added.The cells were cultured for another 24 hours,then content of NO produced in the cell supernatant were detected.3.In the LPS-induced RAW264.7 cell inflammatory model,the anti-inflammatory effects of gypsum fibrosum decoction,gypsum fibrosum and glutinous rice decoction,aqueous calcium sulfate decoction were investigated:Different concentrations of gypsum fibrosum and glutinous rice decoction,glutinous rice decoction,aqueous calcium sulphate decoction were used to intervene RAW264.7 cells,the cell viability was detected by CCK8.Concentrations of decoction which did not affect the viability of cells were selected for subsequent experiments.The RAW264.7 cells were inoculated into a 24-well plate at 1×106 cells/mL and cultured for 24 hours in a 37?,5%CO2 incubator.Different concentrations of gypsum fibrosum decoction/gypsum fibrosum and glutinous rice decoction,LPS solution(final concentration was 1 ?g/mL)were added.The cells were cultured for another 24 hours,then amount of NO produced in the cell supernatant was detected.Gypsum fibrosum decoction(final concentration 0.75mg/mL),gypsum fibrosum and glutinous rice decoction(final concentration 0.75mg/mL+46.875?g/mL),glutinous rice decoction(final concentration 46.875 ?g/mL),aqueous calcium sulfate decoction(final concentration 0.75mg/mL)was used to intervene the LPS-induced RAW264.7 cell for another 24h,then the amount of NO in the supernatant of each group was detected.4.To investigate the anti-inflammatory effects of gypsum fibrosum decoction,gypsum fibrosum and glutinous rice decoction,aqueous calcium sulfate decoction on imiquimod-induced psoriasis mice:42 mg of imiquimod cream were applied to the back of BALB/c mouse for 7 days to establish the psoriasis model.Gypsum fibrosum decoction(36g/kg),gypsum fibrosum and glutinous rice decoction(38.25g/kg),glutinous rice decoction(2.25g/kg),aqueous calcium sulfate decoction(36g/kg)were gavaged to psoriasis mice for 7 days.On the 8th day of the experiment,the back skin lesions of the mice were evaluated by PASI score.The effects of each drug-administered group on the pathological structure of the lesions in psoriasis mice were observed by HE staining.The serum TNF levels in each group were detected.5.Anti-inflammatory effects of different concentrations of gypsum fibrosum decoction on imiquimod-induced psoriasis mice:42 mg of imiquimod cream were applied to the back of BALB/c mouse for 7 days to establish the psoriasis model.Different concentrations of gypsum fibrosum decoction(36g/kg,9g/kg)were given to the psoriasis mice for 7 days.On the 8th day of the experiment,the back skin lesions of the mice were evaluated by PASI score.HE staining was used to observe the effect of gypsum fibrosum decoction on the pathological structure of the lesions in psoriasis mice.6.Study on the anti-inflammatory effect and mechanism of gypsum fibrosum,aqueous calcium sulfate,gypsum fibrosum and glutinous rice decoction in LPS-induced systemic inflammatory response syndrome mice:The model of systemic inflammatory response syndrome in mice was established.The mice were administered with dexamethasone solution(10mg/kg),gypsum fibrosum decoction(36g/kg),gypsum fibrosum and glutinous rice decoction(38.25g/kg),glutinous rice decoction(2.25g/kg),aqueous calcium sulfate decoction(36g/kg)0.5h before the model establishment.After modeling for 4h,blood was taken from the eyeballs,and levels of proinflammatory factors TNF-a and IL-6 in the serum were detected.The effect of each administration on lung tissue of mice was observed by HE staining.The mRNA expressions of IL-1?,TNF-a and IL-6 in lung tissue of mice were detected by RT-PCR.The protein expressions of TLR4,MyD88,IKBa and phospho-NF-?B p65 were detected by western blot.Results1.Gypsum fibrosum decoction contained inorganic elements such as Ca,Mg,Ti,and Sr.In the gypsum fibrosum decoction,content of the inorganic element Ca was the highest.In different concentration of gypsum fibrosum decoctions,the concentration of inorganic element Ca increased in 240g-480g/40mL gypsum fibrosum decoction,and the Ca concentration was significantly correlated with the quality of gypsum fibrosum in decoction(P<0.05).The concentration of Ca in the 480g/40mL,960g/40mL gypsum fibrosum decoction did not change much;the concentration of inorganic elements Ti and Mg did not change significantly in 240g/40mL,480g/40mL,960g/40mL gypsum fibrosum decoction;the concentration of inorganic element Sr in 240g/40mL,480g/40mL,960g/40mL gypsum fibrosum decoction increased gradually,and its concentration was significantly correlated with the quality of gypsum fibrosum in decoction(P<0.05).Gypsum fibrosum fried with glutinous rice could increase the content of Ca,Mg and other elements in the decoction.2.The cell viability of RAW264.7 cells was not affected with the gypsum fibrosum decoction in the concentration range of 1.5ng/mL-15mg/mL(P>0.05);Compared with the LPS model group,NO content in the supernatant of the cells in the 15mg/mL,1.5mg/mL gypsum fibrosum decoction group was significantly reduced(P<0.01).3.The amount of NO produced in the supernatant of LPS-induced RAW264.7 cells increased with the decrease of the concentration of the gypsum fibrosum decoction in the range of 1.5 mg/mL-0.375 mg/mL.Compared with the LPS model group,the production of NO in the supernatant of cells in the(1.5mg/mL+93.75?g/mL),(150?g/mL+9.375?g/mL),(15?g/mL+937.5ng/mL)gypsum fibrosum and glutinous rice decoction group was significantly reduced(P<0.01).4.Compared with the LPS model group,the production of NO in the supernatant of gypsum fibrosum group,gypsum fibrosum and glutinous rice group,aqueous calcium sulphate group and glutinous rice group was significantly decreased(P<0.01);compared with gypsum fibrosum and glutinous rice group,the production of NO in the supernatant of cells in the gypsum fibrosum group increased significantly(P<0.01).Compared with the aqueous calcium sulfate group,the NO production in the supernatant of the gypsum group increased(P<0.01).5.Gypsum fibrosum decoction,gypsum fibrosum and glutinous rice decoction,glutinous rice decoction,aqueous calcium sulphate decoction could significantly reduce the PASI score of the back skin lesions in mice with psoriasis.There was no significant difference in the PASI score between the calcium sulfate group and the gypsum fibrosum group.Compared with the gypsum fibrosum group,the PASI score of the back skin lesions of the gypsum glutinous rice group was significantly lower(P<0.01).The results of HE staining showed:gypsum fibrosum decoction,gypsum gypsum fibrosum and glutinous rice decoction,glutinous rice decoction and aqueous calcium sulphate decoction could alleviate the epidermal thickening of the mice,reduce the epidermis acanthosis,reduce the hyperkeratosis or keratinocytes,and reduce the inflammatory cell infiltration.The pathological changes of skin lesions in the gypsum fibrosum and glutinous rice group were most obvious,followed by gypsum fibrosum group,tripterygium group,aqueous calcium sulfate group and glutinous rice group.Serum proinflammatory factor TNF test results showed:compared with normal group,the content of TNF in the psoriasis model group was significantly increased(P<0.01).The serum levels of TNF in the tripterygium group,gypsum fibrosum group,gypsum fibrosum and glutinous rice group,calcium sulfate group were lower than those in the model group(P<0.05);in each administration group,content of TNF in the gypsum fibrosum and glutinous rice group was the lowest,followed by the positive drug group,aqueous calcium sulfate group,gypsum fibrosum group,glutinous rice group.6.Gypsum fibrosum decoction could significantly reduce the PASI score of the back skin lesions in mice with psoriasis induced by imiquimod.HE staining showed that gypsum fibrosum decoction could reduce the degree of back skin lesions in psoriasis mice,epidermis symptoms were reduced,hyperkeratosis or parakeratosis was reduced,inflammatory cell infiltration was reduced;the high dose gypsum fibrosum decoction was more effective than the lower dose gypsum fibrosum decoction in improving the erythema color and skin infiltration hypertrophy in mice with psoriasis.7.Compared with the LPS model group,the levels of TNF-a and IL-6 in the serum of dexamethasone group,gypsum fibrosum group,gypsum fibrosum and glutinous rice group,glutinous rice group and aqueous calcium sulphate group were significantly decreased(P<0.01),the lung tissue structure disorder,inflammatory cell infiltration of these groups were also improved.Among these groups,the improvement effect of dexamethasone group was the most obvious,followed by gypsum fibrosum and glutinous rice group,gypsum fibrosum group,hydrous calcium sulphate group,glutinous rice group.8.Compared with the LPS model group,gypsum fibrosum decoction could significantly reduce the expression of IL-1?,TNF-a and IL-6 in lung tissue of LPS-induced systemic inflammatory response syndrome mice(P<0.05);when gypsum fibrosum was combined with glutinous rice,the expression of IL-1?,TNF-a and IL-6 in lung tissue of mice was lower than that in gypsum fibrosum group(P<0.05);hydrous calcium sulfate could significantly reduce the expression of TNF-a and IL-6 in lung tissue(P<0.01),but had little effect on the expression of IL-1?.9.Compared with the LPS model group,gypsum fibrosum decoction,aqueous calcium sulfate decoction and gypsum fibrosum and glutinous rice decoction could significantly reduce the expression of TLR4,MyD88 and phospho-NF-?B p65 protein(P<0.01),and reduce LPS-induced IKBa protein degradation(P<0.05),but the expression of TLR4/NF-?B pathway protein in gypsum fibrosum and glutinous rice group was not significantly different from that in gypsum fibrosum group.Conclusions1.Gypsum fibrosum decoction contained inorganic elements such as Ca,Mg,Ti,and Sr.2.Gypsum fibrosum decoction has certain anti-inflammatory effects on LPS-induced RAW264.7 cell inflammatory response model,imiquimod-induced psoriasis model,and LPS-induced systemic inflammatory response syndrome mouse model.Gypsum fibrosum decoction may partially exert its anti-inflammatory effect by inhibiting the activation of the TLR4/NF-?B pathway.3.The material basis of gypsum fibrosum decoction to exert anti-inflammatory effects is Ca,elements of Mg and Mn may also included. |
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