Simiao Yong'an decoction ameliorates murine collagen-induced arthritis by modulating neutrophil activities: An in vitro and in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a common systemic autoimmune disease with high morbidity and disability rate. Currently, there is no effective allopathic treatment for RA, and most of the drugs provoke many adverse effects. Simiao Yong'an decoction (SMYAD) is a traditional Chinese prescription for the treatment of sore and gangrene caused by hot poison. With the development of pharmacology and clinical research, SMYAD has remarkable anti-inflammatory properties and has been used for RA treatments for years. AIM OF THE STUDY: This study aimed to investigate the anti-arthritic effect of SMYAD and further explore the immunopharmacological mechanisms. MATERIALS AND METHODS: Arthritis was induced in DBA/1 mice by two-time immunizations. Collagen-induced rheumatoid arthritis (CIA) mice were divided into 4 groups: control, model, methotrexate (MTX), and SMYAD group (n = 6). The administration groups were given MTX (0.5 mg/kg/3 d) and SMYAD (4.5 g/kg/d) by gavage from day 14. The arthritis index (AI) score was evaluated every 3 days after the second immunization. Hematoxylin and eosin (H&E) staining, Safranin-O fast green staining, Trap staining, and Micro-CT were used to measure the histopathology injuries and bone destruction of joints. Granulocyte changes in the spleen, bone marrow, and period blood were analyzed by flow cytometry. The expression of inflammatory cytokines and chemokines in joints were detected by qRT-PCR. SMYAD-containing serum was obtained from SD rats gavaged with SMYAD. Neutrophils were isolated from peripheral blood and bone marrow for the in vitro experiments of transwell cell assay, apoptosis assay, reactive oxygen species (ROS) generation and neutrophil extracellular traps (NETs) formation. RESULTS: SMYAD significantly relieved arthritis severity in CIA mice. The AI score was significantly decreased in the SMYAD group compared with the model group. Additionally, SMYAD alleviated inflammatory infiltration, cartilage damage, osteoclast formation, and bone damage in the ankle joints. In the flow cytometry assay, SMYAD significantly reduced granulocytes number in the spleen and bone marrow, while increased in peripheral blood. Furthermore, compared with the CIA group, SMYAD suppressed the mRNA levels of inflammatory factors including TNF-α, IL-1ß, IL-6 and chemokines CXCL1, CXCL2, and IL-8 in the inflamed joints. In the in vitro studies, 20% SMYAD-containing serum effectively inhibited the migration of neutrophils, promoted neutrophils apoptosis, reduced ROS production and NETs formation. CONCLUSION: Collectively, our results demonstrated that SMYAD effectively restrained arthritis in CIA mice by modulating neutrophil activities.

[Effect of Huanglian Jiedu Decoction on Monocyte Development in apoE Gene Knockout Mice].

OBJECTIVE: To observe monocyte (Mo) development in wild type C57BL/6 mice and apoE gene knockout (apoE(-/-)) mice, and to evaluate the immuno-regulatory effect of Huanglian Jiedu Decoction (HJD) on peripheral Mo development in apoE(-/-) mice. METHODS: Four, 8, 12, and 16 weeks old female C57BL/6 mice were set up as control groups of different ages, while 4, 8, 12, and 16 weeks old female apoE(-/-) mice were set up as hyperlipidemia groups of different ages. Four-week old female C57BL/6 mice were recruited as a blank group. Four-week old female apoE(-/-) mice were randomly divided into the control group, the Western medicine group, and the Chinese medicine group by paired comparison, 5 in each group. Equivalent clinical dose was administered to mice according to body weight. Mice in the Western medicine group were administered with Atrovastatin at the daily dose of 10 mg/kg by gastrogavage, while those in the Chinese medicine group were administered with HJD at the daily dose of 5 g/kg by gastrogavage. Body weight was detected each week. After 4 weeks blood lipids levels (such as TG, TC, LDL-C, and HDL-C), and the proportions of Mo and Ly6c(hi) were detected. RESULTS: Compared with 4-week-old homogenic mice, the proportion of Mo decreased in 16-week-old C57BL/6 mice (P < 0.05). Levels of TC and TG, and the proportion of Ly6c(hi) subtype increased, but the proportion of Mo de- creased in 8-week-old apoE(-/-) mice (P <0. 05). Levels of TC, TG, and LDL-C increased in 12-week-old apoE(-/-) mice (P < 0.05). Levels of TC, TG, LDL-C, and HDL-C increased in 16-week-old apoE(-/-) mice (P < 0.05, P < 0.01). Compared with 8-week-old homogenic mice, the proportion of Mo decreased in 16-week-old C57BL/6 mice (P < 0.05); levels of TC and LDL-C increased in 12-week-old apoE(-/-) mice (P < 0.05); levels of TC and HDL-C increased in 16-week-old apoE(-/-) mice (P < 0.05, P < 0.01). Compared with C57BL/6 mice of the same age, TC and TG increased, HDL-C decreased (P < 0.01) in 4-and 8-week-old apoE(-/-) mice (P < 0.01); levels of TC, TG, LDL-C increased, and HDL-C level decreased in 12- and 16-week-old apoE(-/-) mice (P < 0.05, P < 0.01); the proportion of Mo increased in 4-week-old apoE(-/-) mice (P < 0.05); proportions of Mo and Ly6c(hi) increased in 8-week-old apoE(-/-) mice (P < 0.05). Compared with the blank control group, levels of TC, TG, and LDL-C, proportions of Mo and Ly6c(hi) increased (P < 0.01, P < 0.05), but HDL-C level decreased (P <0. 01) in the control group after intervention. Compared with the control group, body weight gained less in the Western medicine group and the Chinese medicine group (P < 0.05); the proportion of Ly6c(hi) subtype decreased in the Chinese medicine group (P < 0.05). CONCLUSIONS: In development process blood lipids levels in apoE(-/-) mice are not only associated with age. Blood lipids levels induced growth changes in natural immune system are also correlated with age. In early stage of lipids development HJD intervention could correct this special immune disorder in apoE(-/-) mice.

[Huanglian jiedu decoction regulated and controlled differentiation of monocytes, macrophages, and foam cells: an experimental study].

OBJECTIVE: To observe the effect of Huanglian Jiedu Decoction (HLJDD) in in vivo regulating differentiation of monocytes in an apolipoprotein E knockout (ApoE(-/-)) mouse model, and to observe the effect of HLJDD-containing serum in in vitro regulating differentiation of macrophages and foam cells. METHODS: Fifteen apoE(-/-) mice were randomly divided into the common diet group, the hyperlipidemia group, and the hyperlipidemia +HLJDD treatment group, 5 in each group. Mice in the common diet group were fed with a chow diet. Mice in the hyperlipidemia group were fed with high cholesterol wild diet (WD). Those in the hyperlipidemia +HLJDD treatment group were fed with high cholesterol WD supplemented with HLJDD. All mice were fed for 4 weeks. Five C57BL/6 wild types were recruited as the wild common diet control group. HLJDD was administered to mice in the hyperlipidemia + HLJDD treatment group by gastrogavage at the daily dose of 5 g/kg. Equal volume of purified water was given by gastrogavage to mice in the rest 3 groups. Four weeks later, subtypes of monocytes in the peripheral blood were detected by FACS. HLJDD administered to another 30 SD rats by gastrogavage at the daily dose of 5 g/kg, once for every 12 h for 5 times in total, thereby preparing 5% HLJDD containing serum to intervene the differentiation of in vitro primary bone marrow-derived macrophage (BMDM) and foam cells. The M2 subtype surface receptor CD206 of macrophages and foam cells were detected by FACS. The expression of Nos2 and Arg1 genes were assayed by Real-time PCR. RESULTS: The ratio of inflammatory subset of monocytes (Ly6C(high)) increased in the peripheral blood after ApoE(-/-) mice were fed with high fat diet for 4 weeks. HLJDD significantly decreased the ratio of inflammatory subset of monocytes (P < 0.05). Compared with the vehicle serum, 5% HLJDD containing serum significantly increased differentiation of CD206 + M2 BMDM (P = 0.034). Results of real-time quantitative PCR showed that the expression level of Arg1 mRNA could be up-regulated by HLJDD containing serum (P < 0.05), and that of Nos2 mRNA down-regulated (P = 0.017). ox-LDL induced the differentiation of M2 subtype foam cells from BMDM, and HLJDD containing serum could further elevate the ratio of CD206 + M2 foam cells and increase the Arg1 mRNA expression level (both P < 0.01). HLJDD containing serum could inhibit the inversion of M2 subtype of foam cells to M1 subtype induced by Th1 factors, significantly elevate the Arg1 mRNA expression level, and decrease the Nos2 mRNA expression level (all P < 0.01). CONCLUSIONS: HLJDD could lower hyperlipidemia induced inflammatory monocyte subtype ratios in the peripheral blood of ApoE(-/-) mice. HLJDD containing serum promoted in vitro differentiation of M2 macrophages and foam cells. HLJDD attenuated and inhibited the occurrence and development of atherosclerosis induced by hyperlipidemia possibly through regulating the functional differentiation of monocytes, macrophages, and foam cells.

[Protection of huanglian jiedu decoction on livers of hyperlipidemia mice].

OBJECTIVE: To observe the protection of Huanglian Jiedu Decoction (HJD) on high fat diet induced liver damage mice [hyperlipidemic mice lacking apolipoprotein E (ApoE(-/-))]. METHODS: Wild type mice were divided into the wild common food group and the wild hyperlipidemia group. ApoE(-/-) mice were divided into the ApoE(-/-) common food group, the ApoE(-/-) hyperlipidemia group, and the ApoE(-/-) hyperlipidemia plus HJD group, 5 in each group. In the present study, wild type mice and homozygous apoE(-/-) mice were fed with a chow diet or a high cholesterol Western diet for 4 weeks. HJD at the daily dose of 5 g/kg was given to mice in the ApoE(-/-) hyperlipidemia plus HJD group by gastrogavage. The plasma levels of total cholesterol (TC), triglyceride (TG), low density cholesterol protein (LDL-C) were detected. The pathohistological changes of the liver were observed by Eosin and Hematoxylin (HE) staining. The liver macrophages and their subtype ratios, as well as macrophage surface receptor CD206 and CD36 were detected by flow cytometry. RESULTS: Typical pathological changes of simple fatty liver were manifested in the ApoE(-/-) hyperlipidemia group, TC, TG, and LDL-C increased, the macrophage ratio increased, the expression level of macrophage surface receptor CD206 decreased, showing statistical difference when compared with the ApoE(-/-) common food group (P < 0.01, P < 0.05). The ratio of alternatively activated macrophages (M2) subpopulations was lower in the ApoE(-/-) hyperlipidemia group than in the wild common food group (P < 0.05). There was no obvious change in the expression level of CD36. After intervened by HJD for 4 weeks, there was no obvious improvement in blood lipids. But the ratio of CD206+ M2 macrophages was significantly improved, when compared with the ApoE(-/-) hyperlipidemia group (P < 0.05). The pathological changes of fatty liver were significantly attenuated. CONCLUSIONS: The liver protection effect of HJD might be associated with immunoregulation of M2 macrophage subpopulations and injured tissue repairmen. Its immunoregulation and liver protection were independent from lipids lowering.

[Protection of huanglian jiedu decoction on systemic and vascular immune responses of high fat induced apoE(-/-) mice].

OBJECTIVE: To observe the effect of Huanglian Jiedu IJecoction (HJU) on systemic and vascular immune responses of high fat diet fed apoE deficient (apoE(-/-)) mice. METHODS: Eight wild type C57BL6 mice were recruited as the wild type common food group. Totally 24 apoE(-/-) mice were randomly divided into the ApoE'common food group, the ApoE(-/-) hyperlipidemia group, and the ApoE(-/-) hyperlipidemia plus HJD group, 8 in each group. In the present study, the common food mice and high fat fed mice were fed with a chow diet or a high cholesterol diet for 4 weeks. HJD was given to mice in the ApoE(-/-) hyperlipidemia plus HJD group at the daily dose of 5 g/kg by gastrogavage, while equal volume of pure water was given to mice in the rest groups by gastrogavage. Four weeks later, the plasma levels of blood lipids, the ratio of peripheral blood mononuclear cells, and expressions of Toll-like receptor 4 (TLR-4) and CD36 on the monocytes were detected. The pathological changes and expressions of cytokines in local aorta were detected. The plasma cytokine levels in response to lipopolysaccharide (LPS) were analyzed. Results (1) Compared with the wild type common food group, TO, TG, and LDL-O significantly increased in the ApoE(-/-) common food group (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, TC and LDL-C significantly increased in the hyperlipidemia group (P < 0. 05). There was no statistical difference in each index between the ApoE(-/-) hyperlipidemia group and the ApoE(-/-) hyperlipidemia plus HJD group (P > 0.05). (2) Compared with the wild type common food group, no obvious change of the ratio of peripheral blood mononuclear cells happened, the TLR4 expression level significantly increased in the ApoE'common food group (P < 0. 05). Compared with the ApoE common food group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly increased in the ApoE' hyperlipidemia group (P < 0.05). Compared with the ApoE(-/-) hyperlipidemia group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly decreased. Besides, the CD36 expression level also significantly decreased (P<0.05). (3) After stimulated by LPS for 3 h, compared with the wild type common food group, plasma TNF-ct and IL-b expressions significantly increased in the ApoE(-/-) common food group (P < 0.05). Compared with the ApoE(-/-) common food group, plasma expressions of IL-12, TNF-alpha, MCP-1, and IL-10 increased, but with no statistical difference in the ApoE(-/-) hyperlipidemia group (P > 0.05). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, the MCP-1 expression was significantly down-regulated, while the IL-10 expression significantly increased, showing statistical difference (P < 0.05). Compared with the wild type common food group, mRNA expression levels of IFN-gamma, MCP-1 , TNF-alpha, IL-10, and IL-1beta significantly increased (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, not only mRNA expression levels of IFN-gamma, MCP-1, TNF-alpha, and IL-1beta, further significantly increased, but also IL-12, IL-10, and TGF-beta significantly increased (P < 0. 05, P < 0. 01). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, mRNA expression levels of MCP-1, TNF-alpha, IL-1beta, and IL-12 significantly decreased in the ApoE(-/-) hyperlipidemia plus HJD group (P < 0.05, P < 0.01). CONCLUSIONS: High fat diet induced systemic reaction and inflammatory reactions of local vessels. The local inflammatory response of vessels exceeded systemic inflammatory response. Intervention of HJD could attenuate inflammatory response, especially in local arteries. Meanwhile, it enhanced systemic anti-inflammatory reactions.

[Impact of qutan huayu jiedu herbs on monocyte subpopulations abnormality in patients with hyperlipidemia of phlegm-stagnancy obstruction syndrome pattern].

OBJECTIVE: To investigate the distribution of monocyte subpopulations and the changes of Toll-like receptor 4 (TLR4), CD163 and CD36 expressions in the peripheral blood of patients with hyperlipidemia of phlegm-stagnancy obstruction syndrome pattern (HLE-PSO), and to evaluate the intervening effects of Qutan Huayu Jiedu (QHJ) herbs on these parameters. METHODS: Monocytes in the peripheral blood were sorted using flow cytometry into 3 subpopulations: the CD14high CD16- (Mo1), the CD14high CD16+ (Mo2a) and the CD14low CD16+ (Mo2b) subpopulation. The percentages of various monocyte subpopulations in 83 patients and 42 matched healthy controls were determined and the levels of their surface receptors TLR4, CD163 and CD36 expressions were assayed with flow cytometer. Furthermore, patients allocated in the tested group (10 patients) and the control group (11 patients) were treated with QHJ Herbs and Qutan Huayu (QH, removing Phlegm and dissolving stagnancy but without detoxication) herbs respectively. The changes of monocyte subpopulations percentage and TLR4, CD163 and CD36 expressions were determined 4 weeks after they received treatment. RESULTS: Percentage of Mo2a subpopulation was significantly higher in HLE-PSO patients than the normal range (5.35 +/- 2.57 vs. 3.09 +/- 2.38, P < 0.01), but the deviation of the other two subpopulations in percentage was insignificant. The TLR4 expression on Mo1 monocyte in patients was lower than normal (50.73 +/- 24.45 vs. 69.92 +/- 21.06, P < 0.01), while CD163 and CD36 expressions of all three subpopulations in patients were similar to those in healthy persons respectively. After 4 weeks of treatment, a significant lowered Mo2a proportions were observed in the tested group (3.73 +/- 1.05 vs. 5.50 +/- 2.06, P = 0.043); but not in the control group (4.20 +/- 1.81 vs. 5.65 +/- 1.89, P = 0.097), while the levels of TLR4, CD163 and CD36 were not significantly altered after treatment in both groups. CONCLUSIONS: The elevated proportions of Mo2a subpopulation and the lowered TLR4 expression in Mol subpopulation are the characteristic changes in HLE-PSO patients, which might be related with the hyperlipidemia caused immune injury in patients. QHJ herbs could effectively improve the disproportion of Mo2a subpopulation.

[Relationship between plasma protein expression profiles and states of Zang-Fu organs in patients with phlegm or blood stagnation syndromes due to hyperlipidemia and atherosclerosis].

OBJECTIVE: To investigate the relationship between the plasma biomarker proteins and the states of Zang-Fu organs in patients with phlegm or blood stagnation syndromes due to hyperlipidemia and atherosclerosis. METHODS: The states of Zang-Fu organs in 146 patients with hyperlipidemia and atherosclerosis were diagnosed by syndrome differentiation of traditional Chinese medicine. The plasma proteins from these patients were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Differential protein profiling was established by Image Master 6.0 software, and the differential proteins were analyzed by quadrupole time of flight mass spectrometry (Q-TOF-MS). The association between the plasma biomarker proteins and the states of Zang-Fu organs was analyzed by graphical models. RESULTS: The biomarker proteins such as fibrinogen gamma chain, albumin and apolipoprotein AI (precursor) in discrimination of the patients with phlegm syndrome from phlegm accumulating with stagnation syndrome were correlated with the deficiency of kidney-qi, heart-qi and spleen-qi. Among the four biomarker proteins in discrimination of the patients with phlegm syndrome from blood stagnation syndrome, albumin, adrenomedullin binding protein (precursor) and haptoglobin (precursor) were correlated with the deficiency of kidney-qi and heart-qi, but complement component C4 was independent of the deficient Zang-Fu organs. The biomarker albumin was associated with the deficiency of kidney-qi, heart-qi and spleen-qi, and adrenomedullin binding protein (precursor) was correlated with the deficiency of spleen-qi in discrimination of the patients with blood stagnation syndrome from phlegm accumulating with stagnation syndrome. As the potential biomarker proteins in discrimination of the patients with non-phlegm and non-stagnation syndrome from phlegm accumulating with stagnation syndrome, the fibrinogen beta chain was related with the deficiency of kidney-qi, and apolipoprotein AI (precursor) was correlated with both the deficiency of kidney-qi and heart-qi. CONCLUSION: There exists inherent correlation between the states of Zang-Fu organs and the plasma probable biomarker proteins in the patients with different phlegm or blood stagnation syndromes due to hyperlipidemia and atherosclerosis.