Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis.

In vitro and in vivo studies indicate that oxidant stress is implicated in liver fibrogenesis. However, it is still unknown whether, in vivo, oxidant stress directly affects the hepatic cells responsible for fibrogenesis, ie, the hepatic stellate cells (HSCs). This study was aimed at answering this question by assessing the temporal and spatial relationships between oxidant stress and activation of HSCs in an in vivo model of oxidant-stress-associated fibrogenesis. To this purpose, rats were treated with carbon tetrachloride (CCl4) and livers subjected to in situ perfusion with nitroblue tetrazolium, which, in the presence of superoxide ions, is reduced to an insoluble blue-colored formazan derivative and is readily detectable in the tissue by light microscopy. Moreover, various combinations of in situ hybridization and immunocytochemical analyses were performed. An acute dose of CCl4 caused a transient production of superoxide radicals at 24 hours into pericentral necrotic areas, whereas HSC appearance and expression of collagen mRNA were detectable only at 48 and 72 hours. After chronic CCl4 intoxication, higher levels of oxygen radical production in necrotic areas were detectable along with dramatic and sustained activation of HSCs. However, maximal HSC activation was still delayed as compared with superoxide production. Expression of heme oxygenase, a gene responsive to a variety of oxidant stress mediators, was strongly enhanced by chronic CCl4 administration but remained unchanged in HSCs, both in situ and after isolation of pure HSC fractions from control and CCl4-treated animals. In conclusion, during postnecrotic fibrogenesis, oxidant stress anticipates HSC activation. HSCs do not directly face an oxidant stress while engaged in active fibrogenesis.     

Oxidative stress effect on the activation of hepatic stellate cells

Collagen is the most excessive extracellular matrix protein in hepatic fibrosis. Activated, but not quiescent, hepatic stellate cells (HSCs) have a high level of collagen and a smooth muscle actin (alpha SMA) expression. HSCs play a key role in the pathogenesis of hepatic fibrosis. We analyzed a mechanism leading to HSC activation by evaluating the role of oxidative stress and the expression of NFkB. In vitro study HSCs were proliferated (PCNA:2% vs 68%) and activated (alpha SMA: 5% vs 78%) by ascorbate/FeSO4, and HSCs activated by type I collagen were blocked (PCNA: 97% vs 4%, a SMA: 86% vs 9%) by a-tocopherol. In vivo study means of a SMA positive cells in liver at 400 x HPF were 48.3+/-5.2 and 15.2+/-1.8 and [3H]thymidine uptake of HSC was 529.2+/-284.8 cpm and 223.0+/-86.3 cpm in control and a-tocopherol treated group respectively at 32 hours after CCl4 injection. Nuclear extracts from activated, but not from quiescent, HSCs formed a complex with the NFkB cognate oligonucleotidesand alpha-tocopherol inhibited this bindings. This study indicates that oxidative stress plays an essential role through the induction of NFkB on HSC activation.     

Activation of peroxisome proliferator-activated receptor-gamma by curcumin blocks the signaling pathways for PDGF and EGF in hepatic stellate cells

During hepatic fibrogenesis, reduction in the abundance of peroxisome proliferator-activated receptor-gamma (PPARgamma) is accompanied by activation of mitogenic signaling for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in hepatic stellate cells (HSCs), the major effector cells. We previously reported that curcumin, the yellow pigment in curry, interrupted PDGF and EGF signaling, stimulated PPARgamma gene expression, and enhanced its activity, leading to inhibition of cell proliferation of activated HSC in vitro and in vivo. The aim of this study was to elucidate the underlying mechanisms. We hypothesized that the enhancement of PPARgamma activity by curcumin might result in the interruption of PDGF and EGF signaling. Our experiments demonstrated that curcumin, with different treatment strategies, showed different efficiencies in the inhibition of PDGF- or EGF-stimulated HSC proliferation. Further experiments observed that curcumin dose dependently reduced gene expression of PDGF and EGF receptors (ie, PDGF-betaR and EGFR), which required PPARgamma activation. The activation of PPARgamma by its agonist suppressed pdgf-betar and egfr expression in HSC. In addition, curcumin reduced the phosphorylation levels of PDGF-betaR and EGFR, as well as their downstream signaling cascades, including ERK1/2 and JNK1/2. Moreover, activation of PPARgamma induced gene expression of glutamate-cysteine ligase, the rate-limiting enzyme in de novo synthesis of the major intracellular antioxidant, glutathione. De novo synthesis of glutathione was required for curcumin to suppress pdgf-betar and egfr expression in activated HSCs. Our results collectively demonstrated that enhancement of PPARgamma activity by curcumin interrupted PDGF and EGF signaling in activated HSCs by reducing the phosphorylation levels of PDGF-betaR and EGFR, and by suppressing the receptor gene expression. These results provide novel insights into the mechanisms of curcumin in the inhibition of HSC activation and the suppression of hepatic fibrogenesis.     

Loss of discoidin domain receptor 2 promotes hepatic fibrosis after chronic carbon tetrachloride through altered paracrine interactions between hepatic stellate cells and liver-associated macrophages

Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl(4)) to DDR2(+/+) and DDR2(-/-) mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after chronic CCl(4) administration, DDR2(-/-) livers had increased collagen deposition, gelatinolytic activity, and HSC density. Increased basal gene expression of osteopontin, transforming growth factor-β1, monocyte chemoattractant protein-1, and IL-10 and reduced basal gene expression of matrix metalloproteinase-2, matrix metalloproteinase-13, and collagen type I in quiescent DDR2(-/-) HSCs were amplified further after chronic CCl(4). In concordance, DDR2(-/-) HSCs isolated from chronically injured livers had enhanced in vitro migration and proliferation, but less extracellular matrix degradative activity. Macrophages from chronic CCl(4)-treated DDR2(-/-) livers showed stronger chemoattractive activity toward DDR2(-/-) HSCs than DDR2(+/+) macrophages, increased extracellular matrix degradation, and higher cytokine mRNA expression. In conclusion, loss of DDR2 promotes chronic liver fibrosis after CCl(4) injury. The fibrogenic sinusoidal milieu generated in chronic DDR2(-/-) livers recruits more HSCs to injured regions, which enhances fibrosis. Together, these findings suggest that DDR2 normally orchestrates gene programs and paracrine interactions between HSCs and macrophages that together attenuate chronic hepatic fibrosis.     

Curcumin eliminates the inhibitory effect of advanced glycation end-products (AGEs) on gene expression of AGE receptor-1 in hepatic stellate cells in vitro

Diabetes is featured by hyperglycemia, which facilitates the formation of advanced glycation end-products (AGEs). AGEs are a causal factor in development of diabetic complications. AGE receptor-1 (AGE-R1) is responsible for detoxification and clearance of AGEs. Type 2 diabetes mellitus is commonly accompanied by non-alcoholic steatohepatitis, which could cause hepatic fibrosis. Little attention has been paid to effects of AGEs on hepatic fibrogenesis. Curcumin, a phytochemical from turmeric, has been reported to inhibit the activation of hepatic stellate cells (HSCs), the major effectors during hepatic fibrogenesis, and to protect against hepatic fibrogenesis in vitro and in vivo. The current study was designed to evaluate the effects of AGEs on inducing HSC activation, to assess the role of curcumin in diminishing the AGE effects, and to explore the underlying mechanisms. Our results showed that AGEs stimulated HSC activation by inducing cell proliferation and expression of genes relevant to HSC activation, which were abrogated by curcumin. Curcumin induced gene expression of AGE-R1 in passaged HSCs, which might facilitate the attenuation of the stimulatory effects of AGEs on the activation of HSCs. Further experiments revealed that curcumin inhibited the activity of extracellular signal-regulated kinase (ERK), and induced gene expression and the activity of peroxisome proliferator-activated receptor-gamma (PPARγ), leading to the induction of the AGE-R1 gene expression. In summary, AGEs stimulated HSC activation. Curcumin eliminated the AGE effects at least partially by inducing the AGE-R1 gene expression. The process was mediated by inhibiting ERK activity, inducing gene expression of PPARγ and stimulating its transactivity.     

Effect of cholinergic denervation on hepatic fibrosis induced by carbon tetrachloride in rats

Various factors involved in the development of liver fibrosis, including hepatic stellate cells (HSCs), cholinergic nervous activity and fibrogenetic cytokines. The present study aims to investigate the role of cholinergic regulation in the promoting of liver fibrogenesis relating to bone morphogenetic protein-6 (BMP-6) and/or transforming growth factor-beta1 (TGFbeta1). We treated carbon tetrachloride (CCl(4)) into rats for eight weeks to induce liver fibrosis and arranged these rats for cholinergic denervation, hepatic branch vagotomy or atropine administration. Acetylcholinesterase (AChE) staining showed the distribution of cholinergic nerve around fibrosis scaring septa. The immunohistochemical staining for alpha smooth muscle actin (alphaSMA) indicated the less HSCs in CCl(4) treated rat liver with cholinergic denervation as compared to the sham-operated CCl(4) treated rats. It seems that cholinergic nerve not only innervates around the fibrosis area but also promotes HSCs. We also detected TGFbeta1 and BMP-6 expressions using RT-PCR and immunohistochemistry. The obtained results show that cholinergic denerveration decreases BMP-6 and TGF-beta1 expressions in CCl(4) induced liver fibrosis of rats. In conclusion, cholinergic nerve may influence HSCs in addition to the lowering of BMP-6 and TGF-beta1 gene expressions to modify liver fibrosis.     

解耦联蛋白2对大鼠肝纤维化形成中星形细胞活化的影响

目的探讨解耦联蛋白2(UCP2)在肝纤维化形成过程中的作用及其发生机制。方法体内实验采用四氯化碳(CCl4)诱导肝纤维化模型,取肝脏观察病理变化,用Western blotting、免疫组织化学和Real-time PCR等方法检测UCP2和p38丝裂素活化蛋白激酶(p38MAPK)的表达水平;体外实验采用UCP2特异性抑制剂京尼平和CCl4刺激星形细胞,检测UCP2和p38MAPK相关蛋白的表达情况。结果与正常组相比,模型组大鼠肝脏α-平滑肌肌动蛋白(α-SMA)和UCP2表达增高(P0.05,n=10);加入CCl4刺激细胞后,星形细胞α-SMA表达增加,p38MAPK及其磷酸化水平增高(P0.05,n=6);而在加入京尼平后,α-SMA表达增加,p38 MAPK及其磷酸化水平明显降低(P0.05,n=6)。结论 UCP2参与了肝纤维化的发生,可能促进了星形细胞的活化及增殖过程。     

CXCL10 promotes liver fibrosis by prevention of NK cell mediated hepatic stellate cell inactivation

Chemokines, such as CXCL10, promote hepatic inflammation in chronic or acute liver injury through recruitment of leukocytes to the liver parenchyma. The CXCL10 receptor CXCR3, which is expressed on a subset of leukocytes, plays an important part in Th1-dependent inflammatory responses. Here, we investigated the role of CXCL10 in chemically induced liver fibrosis. We used carbon tetrachloride (CCl(4)) to trigger chronic liver damage in wildtype C57BL/6 and CXCL10-deficient mice. Fibrosis severity was assessed by Sirius Red staining and intrahepatic leukocyte subsets were investigated by immunohistochemistry. We have further analyzed hepatic stellate cell (HSC) distribution and activation and investigated the effect of CXCL10 on HSC motility and proliferation. In order to demonstrate a possible therapeutic intervention strategy, we have examined the anti-fibrotic potential of a neutralizing anti-CXCL10 antibody. Upon CCl(4) administration, CXCL10-deficient mice showed massively reduced liver fibrosis, when compared to wildtype mice. CXCL10-deficient mice had less B- and T lymphocyte and dendritic cell infiltrations within the liver and the number and activity of HSCs was reduced. In contrast, natural killer (NK) cells were more abundant in CXCL10-deficient mice and granzyme B expression was increased in areas with high numbers of NK cells. Further detailed analysis revealed that HSCs express CXCR3, respond to CXCL10 and secrete CXCL10 when stimulated with IFNγ. Blockade of CXCL10 with a neutralizing antibody exhibited a significant anti-fibrotic effect. Our data suggest that CXCL10 is a pro-fibrotic factor, which participates in a crosstalk between hepatocytes, HSCs and immune cells. NK cells seem to play an important role in controlling HSC activity and fibrosis. CXCL10 blockade may constitute a possible therapeutic intervention for hepatic fibrosis.     

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF(+/+) or HB-EGF-null (HB-EGF(-/-)) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF(+/+) or HB-EGF(-/-) mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF(+/+) and HB-EGF(-/-) mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF(+/+) mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF(-/-) mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF(+/+) mice, TAA-stimulated hepatic mRNA levels in HB-EGF(-/-) mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-β1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF(+/+) mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.     

辣椒素抑制大鼠肝纤维化的实验研究

 目的：探讨辣椒素（capsaicin）对大鼠肝星状细胞（hepatic stellate cells, HSCs）活性的影响及其对实验性肝纤维化的治疗作用。方法：DCFH-DA法检测辣椒素对HSCs中活性氧的影响;CCK-8法检测辣椒素对HSCs增殖的影响;Western blotting检测HSCs α-平滑肌肌动蛋白的表达;RT-PCR检测辣椒素对HSCs纤维化相关基因表达的影响;流式细胞术检测辣椒素对HSCs凋亡的影响;通过腹腔注射四氯化碳建立鼠肝纤维化模型,经腹腔输注辣椒素,检测肝组织病理切片,观察肝纤维化指标的变化。结果：与对照组比较,辣椒素抑制了HSCs中活性氧的产生,明显抑制了HSCs的活化与增殖(P<0.05),促进了HSCs的凋亡(P<0.05),降低了金属蛋白酶组织抑制物1及转化生长因子β 1的表达(P<0.05),降低了肝羟脯氨酸含量及血清Ⅲ型胶原和透明质酸水平(P<005)。结论：辣椒素抑制HSCs增殖、活化并诱导其凋亡。辣椒素对实验性肝纤维化具有一定的抑制作用。     

大鼠原代肝星状细胞活化后组蛋白修饰水平的观察

目的:观察体外培养大鼠肝星状细胞(HSCs)活化过程中组蛋白修饰的改变以及与HSCs活化指标α-平滑肌肌动蛋白(α-SMA)的关系,探讨组蛋白修饰在HSCs活化过程中可能的作用。方法:体外分离、鉴定、培养大鼠HSCs,光镜观察HSCs活化过程中的形态变化,细胞免疫荧光染色和Western blotting检测desmin和α-SMA的表达,比较静止型HSCs和激活型HSCs中H4K12乙酰化、H3K9乙酰化、H3K4二甲基化和H3K9二甲基化的变化。结果:(1)细胞形态学观察结果表明HSCs在培养过程中形态由静息状态向高度分化的肌成纤维细胞转化。细胞免疫荧光染色及Western blotting检测结果显示,分离培养24 h的HSCs有desmin表达,但几乎不表达α-SMA;随着培养时间延长,HSCs内α-SMA和desmin表达逐步增加,至15 d时达到最大。(2)根据HSCs细胞形态变化及HSCs活化标志蛋白检测结果,确定培养24 h的HSCs为静止型HSCs,培养15 d的HSCs为激活型HSCs,分别检测其组蛋白修饰变化。结果显示,与静止型HSCs比较,激活型HSCs中H4K12乙酰化、H3K9乙酰化和H3K9二甲基化修饰水平明显降低(P0.01),而H3K4二甲基化修饰水平明显增加(P0.01),且H3K4二甲基化修饰水平变化与α-SMA表达变化一致。结论:在体外培养HSCs活化过程中,组蛋白修饰发生明显异常,提示组蛋白修饰改变有可能参与了HSCs活化以及肝纤维化的发生。     

Ultrastructural characteristics of the respective forms of hepatic stellate cells in chronic hepatitis B as an example of high fibroblastic cell plasticity. The first assessment in children

Purpose

Activation of hepatic stellate cells (HSCs), mainly responsible for extracellular matrix synthesis, is assumed to be central event in the process of liver fibrogenesis. The major objective of the research was to analyze the ultrastructural profile of activated HSCs in children with chronic hepatitis B (chB), with respect to fibrosis intensity.

The interruption of the PDGF and EGF signaling pathways by curcumin stimulates gene expression of PPARgamma in rat activated hepatic stellate cell in vitro

Activation of hepatic stellate cells (HSC), the major effector in hepatic fibrogenesis, is coupled with sequential alterations in expression of genes, including the upregulation of platelet-derived growth factor-beta receptor (PDGF-betaR) and epidermal growth factor receptor (EGFR), as well as the down-regulation of the peroxisome proliferator-activated receptor-gamma (PPARgamma). However, the relationship among the alterations in expression of the genes and the activation of their signaling in activated HSC remains obscure. We recently showed that curcumin, the yellow pigment in curry, inhibited cell growth and induced gene expression of endogenous PPARgamma in activated HSC in vitro. The present study is to elucidate the underlying mechanisms, focusing on the impacts of PDGF and EGF signaling. It is hypothesized that the interruption of the PDGF and EGF signaling pathways by curcumin might stimulate gene expression of PPARgamma in activated HSC. Our results in this report indicate that the activation of PDGF or EGF signaling by exogenous PDGF or EGF inhibits PPARgamma gene expression in passaged HSC. Curcumin interrupts PDGF and EGF signaling demonstrated by inhibiting tyrosine phosphorylation of PDGF-betaR and EGFR and by reducing the levels of phosphorylated phosphatidylinositol-3 kinase (PI-3K/AKT), extracellular signal-regulated kinase (ERK) and the Jun N-terminal kinase (JNK). The blockade of PI-3K/AKT, ERK or JNK signaling negatively regulates PPARgamma gene expression in activated HSC, leading to the reduction in cell growth, including inducing cell arrest and apoptosis. Our results collectively demonstrate that the interruption of the PDGF and EGF signaling pathways by curcumin stimulates gene expression of PPARgamma in activated HSC. These results provide novel insights into the mechanisms of curcumin in the induction of PPARgamma gene expression in activated HSC.     

Amelioration of liver fibrogenesis by dual inhibition of PDGF and TGF-beta with a combination of imatinib mesylate and ACE inhibitor in rats

Both platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are known to be pivotal cytokines in liver fibrosis development. The aim of our current study was to elucidate the effects of dual inhibition of PDGF and TGF-beta by combination of the clinically used imatinib mesylate (STI-571) and perindopril (an ACE-inhibitor; ACE-I), respectively, on ongoing liver fibrosis development in rats. The effects of STI-571 and ACE-I at clinically comparable low doses were examined in a rat model of CCl4-induced liver fibrogenesis. Treatment with both STI-571 and ACE-I inhibited liver fibrogenesis and suppressed activation of hepatic stellate cells (HSCs). Administration of both agents exerted a more potent inhibitory effect than administration of either single agent. Our in vitro study demonstrated that STI-571 and ACE-I suppressed PDGF receptor (PDGFR) phosphorylation and TGF-beta expression in activated HSCs, respectively. Dual suppression of PDGF and TGF-beta with a combination of clinically comparable low doses of STI-571 and ACE-I exerted a significant inhibitory effect on ongoing liver fibrosis development. Since both agents are widely used in clinical practice, this combination therapy may provide a new strategy against liver fibrosis in the future.     

Hepatic stellate cells and liver fibrosis

Substantial evidence now exists to recognize hepatic stellate cells (HSCs) as the main matrix-producing cells in the process of liver fibrosis. Liver injury of any etiology will ultimately lead to activation of HSCs, which undergo transdifferentiation to fibrogenic myofibroblast-like cells. Quantitative analysis of HSC activation by immunohistochemistry has been shown to be useful in predicting the rate of progression of liver fibrosis in some clinical situations. In the activation process, transforming growth factor beta is thought to be the main mediator of fibrogenesis and platelet-derived growth factor is the major inducer of HSC proliferation. Different platelet-derived growth factor and transforming growth factor beta inhibitors have been shown to effectively prevent liver fibrosis in animal models and represent promising therapeutic agents for humans.     

Mineralocorticoid receptor antagonist spironolactone prevents pig serum-induced hepatic fibrosis in rats

Mineralocorticoid receptor (MR) antagonist spironolactone (SPL) is an effective agent for prevention of cardiovascular injury. However, whether and how SPL ameliorates hepatic fibrosis in rats is unknown. Pig serum (PS) (0.5 mL, twice a week, ip) or vehicle-administered rats for 12 weeks were used as rats with hepatic fibrosis or control rats, respectively. Rats given PS were treated with SPL (50 mg/kg/day, sc) for 12 weeks. Hepatic fibrosis, using picro-sirius red staining and determination of hydroxyproline content, immunohistochemistries of alpha-smooth muscle actin (alpha-SMA)-positive hepatic stellate cells (HSCs), Na/H exchange isoform-1 (NHE-1) protein, CYP11B2 aldosterone synthase protein for liver tissues, and plasma aldosterone concentrations were compared among the 3 groups of rats. Rats given PS alone exhibited hepatic fibrosis as well as increases in the number of the alpha-SMA-positive HSCs and NHE-1 protein expression in HSCs and hepatocytes, all of which were suppressed by SPL. Rats given PS alone revealed increased CYP11B2 protein expression in HSCs and hepatocytes, which was not inhibited by SPL. Plasma aldosterone concentrations were significantly greater in rats given PS and SPL than in control rats and rats given PS alone, although they were not different between control rats and rats given PS alone. PS-induced hepatic fibrosis together with HSC activation and NHE-1 protein expression occurs via MRs, and SPL ameliorates hepatic fibrosis presumably via the inhibition of HSC activation and NHE-1 protein expression in PS-induced liver injuries. The aldosterone produced in the injured liver contributes to the PS-induced hepatic fibrosis.     

结缔组织生长因子在人及大鼠肝纤维化组织中表达增强

目的观察结缔组织生长因子(CTGF)在人及大鼠肝纤维化组织中的表达。方法雄性SD大鼠32只,皮下注射CCl4后1、4、8周收集肝组织标本;44例人肝组织,其中包括12例正常肝组织、32例慢性病毒性肝炎和肝硬化组织。用免疫组化方法检测CTGF的表达及分布。结果CTGF主要表达于大鼠肝星状细胞及肝细胞胞质中。注射CCl4后,大鼠肝组织中CTGF呈时间依赖性表达增强(P<0.01或P<0.05)。CTGF在人肝纤维化组织中的表达与大鼠相类似,表达水平显著高于正常人(P<0.01)。结论CTGF作为一种促纤维化因子,其过表达可促进肝星状细胞的增殖活化,促进细胞外基质的形成,从而促进肝纤维化的发生、发展。