| Lupus nephritis(LN)is a immune inflammatory disease,in which systemic lupus erythematosus(SLE)is accompanied with different types of autoimmune kidney damage,speically with severe kidney damage.The information on mechanism of pathogenesis and treatment way is lacking.Recent reports showed that Kruppel-like factor 15(KLF15)can alter the Nuclear Factor-? B(NF-?B),and activate NF-?B signaling.It cause inflammation and immune reponse and glomerular lesions,play an important role in the pathogenesis of LN.Currently,hydroxychloroquine(HCQ)has been widely used for treating SLE with good efficacy.It also showed that HCQ play a protective role in lupus associated-clinical manifestations or medical complications,such as kidney damage.However,HCQ can result in many adverse reactions,it is the limitation for long-term use.Artemisinin(ART)can depress inflammation and immunity.Meanwhile,ART has fewer side effects and is suitable for long-term use.The combination of ART and HCQ(AH)may not only guarantee the pharmacodynamic effects,but also reduce the toxic side effects,which can achieve new breakthroughs in the clinical treatment.Objective:1)To investigate whether AH can reduce the dose of hydroxychloroquine,and reduce the level of adverse events.2)To investigate the effect of AH on LN murine model induced by formaldehyde of campylobacter jejuni plus adjuvant and allogeneic lymphocytes.3)To investigate the expression of KLF15 and NF-?B in the LN model mice induced by formaldehyde of campylobacter jejuni plus adjuvant and allogeneic lymphocytes.Methods:1)The effect of AH on immune function in murine models.The various concentration(ART:HCQ=1:3,1:land 3:1)were carried out by orally in murine models in the present study.The rational ratio of AH will be collected for the following experiments.After the lymphocytes were co-inculcated with PHA-P,we observed lymphocyte transformation rate by different ratio of AH;Using chicken red blood cells as immunogen for hemolysin assay,we observed the effects of different ratio of AH on the content of serum hemolysin in murine;In order to detecte phagocytosis of macrophage throughout carbon particle clearance method,we observed the influence of different ratio of AH on phagocytic function of mononuclear macrophage in mice,and evaluated the inhibition immunity of AH.2)Based on the data from above experiments,the rational ratio of AH(1:3)was collected.In the next experiment,according to the dose of HCQ(33.3 mg · kg-1),the dose of AH were divided in to three groups(16.7?33.3?66.6 mg · kg-1).(A)Modeli LN murine models caused by formaldehyde of campylobacter jejuni plus adjuvantThe LN murine models caused formalized campylobacter jejuni plus adjuvant by were successfully established through plantar injected with jejuni adjuvant mixture,campylobacter jejuni suspension injection on day 21.ii Allogeneic lymphocytes induced LN animal model in miceAfter incubated with ConA,spleen cells were injected by subcutaneous way into KM mouse,in order to LN murine models caused by allogeneic lymphocytes.(B)DetectionIn the following,different dose of AH were used to treat LN murine models.The urine protein were detected by Bradford method;the expression of serum creatinine(SCr),blood usea nitrogen(BUN),albumin(ALB),anti double stranded DNA(anti ds-DNA),antinuclear antibodies(ANA),Immunoglobulin G(IgG),interferon y(IFN-?),tumor necrosis factor-?(TNF-a)and transforming growth factor-?1(TGF-?1)were detected by ELISA method;pathological changes on kidney and spleen tissues were also analysed,the effect of AH on pharmacodynamics evaluation in LN murine models was detected;the mRNA expression of KLF15,NF-? B in LN murine models treated with different ration of AH was investigated by RT-PCR method,in order to explore the mechanism of AH in LN murine models.Results:1)The effect of AH on immune function in mice(A)When compared with control group,lymphocyte transformation rate in all ratio of AH group(1:3,1:1,and 3:1)was significantly decreased(P<0.01).(B)When compared with control group,the content of serum hemolysin in ratio of AH group(1:3 and 1:1)was significantly decreased(P<0.01).(C)When compared with control group,the carbon clearance index K and phagocytic index a in ratio of AH group(1:3)was significantly decreased(P<0.01).The results show that,the rational ratio of AH(1:3)can reduced the rate of lymphocyte transformation,reduce the content of serum hemolysin,reduce the carbon clearance index K and phagocytic index a,it has a good inhibitory effect of immune.2)The effect of AH on LN murine model(A)The effect of AH on LN murine model induced by formaldehyde of campylobacter jejuni plus adjuvantCompared with the control murine models,the mental state,diet,hair color,activity state in murine models treated with low,medium,and high dose of AH is better;and the content of urinary protein were significantly decreased(P<0.01);the content of SCr in serum in murine model treated with medium and high dose was significantly decreased(P<0.01 and P<0.05,respectively);whereas BUN in serum in murine models treated with low,medium,and high dose of AH was not significantly changed(P>0.05);the serum ALB level significantly increased in high dose group of mice(P<0.05);the serum anti ds-DNA level significantly decreased in low and medium dose groups(P<0.05);the serum ANA in medium dose group was significantly decreased(P<0.01);the serum IgG in low,medium and high dose groups mice were significantly decreased(P<0.01,P<0.05,respectively);the serum IFN-y and TNF-? in low,medium and high dose groups were significantly decreased(P<0.01,P<0.05,P<0.01,respectively);the serum TGF-?1 in high dose group was significantly increased(P<0.05);In all AH treated groups,mice glomerular size and shape recovered,the number of mesangial cells were decreased,mesangial area gradually became narrow,especially in the medium and high dose group;interstitial edema in spleen cells were improved,splenic corpuscle hyperplasia were reduce,especially in the medium and high dose group;the mRNA expression of KLF15 in low,medium and high dose groups were significantly increased(P<0.01);the mRNA expression of NF-?B in the low,medium,high dose groups were significantly decreased(P<0.01).(B)The effect of AH on LN murine model induced by allogeneic lymphocytesCompared with the control murine models,the mental state,diet,hair color,and activity state in models treated low,medium and high dose groups(AH)is better;the content of urinary protein were significantly decreased in low,medium and high dose groups(P<0.01);the content of SCr and BUN in models treated with low,medium and high dose were significantly decreased(P<0.01);the ALB level in models treated with medium dose significantly increased(P<0.05);the serum anti ds-DNA level was significantly decreased in low,medium and high dose groups(P<0.01);the serum ANA in low,medium,and high dose groups mice was significantly decreased(P<0.01);the serum IgG in low,medium,and high dose groups were significantly decreased(P<0.01);the serum IFN-? in low,medium,and high dose groups were significantly decreased(P<0.01);the serum TNF-a in low,medium,and high dose groups were decreased(P<0.01,P<0.05);the serum level of TGF-?1 in low,medium,and high dose groups were significantly increased(P<0.01,P<0.05);glomerular size and shape in all AH treated groups were recovered;the number of mesangial cells was decreased,mesangial area gradually narrow,especially in the medium and high dose groups;interstitial edema in spleen cells were improved,splenic corpuscle hyperplasia reduced,especially in the medium and high dose groups;the mRNA expression of KLF15 in the low,medium,high dose groups,were significantly increased(P<0.01);the mRNA expression of NF-?B were significantly reduced in the low,medium,high dose groups(P<0.01).Conclusion:1)Our data indicated that AH can inhibit cellular immunity,humoral immunity and nonspecific immunity,especially at the ratio of 1:3(AH).AH can not only reduce the hydroxychloroquine dosage,but also reduce adverse reactions while maintaining efficacy.2)AH has a good therapeutic effect in LN models induced by formaldehyde of campylobacter jejuni plus adjuvant and allogeneic lymphocytes,in which serum SCr,BUN,anti ds-DNA,ANA,IgG,IFN-y,and TNF-? were reduce,whereas serum ALB,and TGF-?1 were increased,and kidney and spleen lesions were improved.3)AH may increase the mRNA expression of KLF15 in kidney tissue of LN murine model,and reduce the mRNA expression of NF-?B. |
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