| Puwei Pill(PWP) is a secret recipe for the treatment of gastric ulcer (Gastric Ulcer, GU) with the advantages of low frequency in using, lasting efficacy, few side reactions and low recurrence rate. Preliminary tests have confirmed that PWP has protective effect for several experimental GU. This experiment further evaluated the action of resisting chronic GU and recurrence and explored the anti-inflammatory mechanisms for protecting gastric mucosa of PWP in ulcer healing and recurrence, which provided the basis for the further study of PWP.Objective:1.To develop GU and recurrence model in rat and then study the action of PWP resisting GU and its recurrence.2. To examine the effect of PWP on the inflammation and inflammatory factor, such as NF-κB, IL-8 and TNF-αprotein expression in gastric moscosa during the process of GU and recurrence so as to reveal the mechanism of PWP for the anti-ulcer and recurrence from the point of inflammation, which provide a theoretical basis for the further study of this drugs.Methods:1 To evaluate PWP's action of resisting GU and its recurrence Rat GU model of acetic acid irritation was developed, then GU recurrence model was preparated by intraperitoneal injection of IL-1β. Ulcer index(UI),ulcer recurrence rate, gastric mucosal pathology and the density of inflammatory cells induced by PWP were all observed so as to study PWP's effect on the resisting of GU and its recurrence.2 To study PWP's anti-inflammatory mechanism in the process of occurrence and recurrence of GU. Immunohistochemistry and Western blot being used to observe PWP's effect on the protein expression of inflammatory factors, such as NF-κB, IL-8, TNF-α.Radioactive immunoassay being performed to detect PWP's effect on the content of plasma IL-8 and TNF-α. As a result, the anti-inflammatory mechanism induced by PWP was studied in the process of occurrence and recurrence of GU.Results:1. PWP'effect on the resisting of GU and its recurrence1.1 PWP'effect on UI and recurrence rate in GU rats After 16d since GU model was developed using acetic acid, UI in model without recurrence (MWR) group was 30.15±6.40. But UI in omeprazole (OMLZ) group and PWP group were 20.32±6.80 and 7.75±2.09, respectively.Compared with MWR group, PWP and OMLZ all markedly reduced the UI (P<0.01). After modeling 92d, there were no ulcer recurrence in normal group, sham group and MWR group. While the ulcer recurrence rate were 83.3%, 33.3% and 10.0% in model with recurrence (MR) group, OMLZ group, and PWP group, which were significant differences comparing with MR group (P<0.01). These results showed that PWP had good effects on resisting of GU and its recurrence.1.2 PWP's effect on the gastric histopathology of the GU and its recurrence.When modeling 16d, HE staining showed that gastric tissue structure was integrity and mucosal glands arranged in order in normal and sham groups.In MWR group, ulcer could be seen.The pathology changes that mucosa being defect, normal structure of mucous glands being disappeared and ulcer being seen in the whole mucosal layer or even in deeper layer showed that necrotic lesions, muscle rupture had occurred. In addition, lymphocytes, monocytes and other inflammatory cells could been found in submucosa. Gland interstitial was edema and glandula expanded along the edge of ulcer in the mucosa. In OMLZ and PWP groups, mucosal defect significantly reduced.Necrosis had not be found. Some regenerative mucosa were migrated to the eara of ulcer, the others were thiner than the mucousa around ulcer. The width of mscle defect was also smaller. Less inflammatory cells were seen. Glands were arranged in order with less expansion.After modeling 92d, there was no ulcer and gastric tissue in normal and sham groups. In addition, there was a small amount of lymphocytes in lamina propria. In MWR group, there was also no ulcer recurrence. But the gastric mucosa became thinner and glands were arranged less regular. The scar tissue could be seen under the mucous membrane and there was infiltration of chronic inflammatory cells in scar mucosa. There was ulcer full of mucosal thickness in MR group. Furthmore, there was residual regeneration mucosa and a large number of inflammatory cells, mainly neutrophil along the ulcer margine. Neverthless, regenerated mucosa was thicker and mucosa was repaired more completely and arranged in order in PWP group and OMLZ group.1.3 PWP's effect on the density of inflammatory cells in gastric tissue during GU and its recurrence.After modeling 16d, the density of chronic inflammatory cells were 0.30±0.03 and 0.39±0.03 in normal and sham groups, and there were no significant difference between them. The chronic inflammatory cells density was 41.11±2.94 in MWR group and was significantly higher than that in sham group (P<0.01).This showed that inflammatory cells also involved in the occurance of GU. While chronic inflammatory cells density in OMLZ and PWP groups were 21.25±1.81 and 22.58±2.10, respectively, and were both significantly less than that in MR group (P<0.01).These indicated that PWP could significantly reduce the infiltration of inflammatory cells during the process of ulcer.After modeling 92d, in normal and sham groups the densities of inflammatory cells were 0.31±0.03 and 0.41±0.03,respectively,and there were also no significant difference between them. But they were 15.79±1.14 and 60.83±3.26 in MWR group and MR group, respectively, which were significantly higher than that in sham group (P<0.01) and noted that the inflammatory cells change also involved in the ulcer recurrence. The inflammatory cells density in OMLZ and PWP group were 30.51±1.95 and 31.18±2.49, which were significantly less than that in MR group (P<0.01). These showed that the PWP could also significantly reduce the infiltration of inflammatory cells during the ulcer recurrence.Additional, we found that, after modeling 16d, the densities of chronic inflammatory cell was positively associated with UI (r=0.902, P<0.01) through the associated analysis.Samely, after modeling 92d, the densities of inflammatory cells was also correlated with recurrance rate in a positively dependent way(r=0.611, P<0.01). These provided that inflammatory response might participate in the GU and its recurrence, and PWP might resist GU and recurrence by inhibiting the inflammatory response in gastric tissue.2 The study of PWP on the anti-inflammatory mechanisms in the process of GU and its recurrence.2.1 The effect of PWP on the protein expression of NF-κB during GU and its recurrence in rats.2.1.1 The cellular localization of positive cells of NF-κB. There were two states of NF-κB positive cells in cytoplasm and nucleus, namely, inactivation and activation of NF-κB. In normal group and sham group, NF-κB mainly were expressed in the cytoplasm of gastric epithelial cell. After modeling 16d, in MWR group, NF-κB was expressed in both cytoplasm and nucleus in the epithelial cells.Moreover, the expression in nucleus, in OMLZ group and PWP groups decreased more than that in MWR group.After modeling 92d, they mainly expressed in nucleus along ulcer edge, in MR group, and significantly increased than that in MWR group.At the same time, the nucleus of interstitial inflammation cells exhibited strongly positive.Nevertheless, NF-κB were expressed mainly in cytoplasm in OMLZ and PWP group.2.1.2 The comparion of positive cells of NF-κB expression by immunohistochemical method⑴After modeling 16d, few positive cells with NF-κB expressed in normal group and sham group, which averaged 0.41±0.39 and 0.58±0.31.There showed no significant difference between them (P>0.05). Compared with the normal group, MWR significantly increase (11.75±0.43) (P<0.01). Positive cells in OMLZ and PWP groups were 5.09±0.16 and 5.23±0.24, and compared with the MWR, were significantly lower (all P<0.01).⑵After modeling 92d, the positive cells were 0.49±0.23, 0.55±0.33 and 1.40±0.29, respectively,in normal group, sham group and MWR group and there were no significant differences between them (P> 0.05).That in MR group was 8.52±0.95, and significantly increased than that in MWR group (P<0.01). The positive cells were 4.68±0.53 and 4.63±0.32 in OMLZ and PWP groups, and were significantly lower than that in MR group (all P<0.01).Moreover, there was no significant difference between the two groups (P>0.05).These indicated that PWP had anti-inflammatory effect by decreasing the NF-κB in nucleus expression during the process of GU occurrence and its recurrence.2.1.3 The results of protein expression of NF-κB by Western-blotThe protein quantification results from western blot revealed that,⑴After modeling 16d, NF-κB protein expression was 0.50±0.03 in sham group, and compared with normal group (0.40±0.03) had no significant difference (P>0.05). NF-κB protein expression in MWR group was 10.66±1.92 and was significantly higher than that in normal and sham groups (P <0.01).Meanwhile, NF-κB protein expressions were 4.75±0.93 and 4.50±1.00 in OMLZ and PWP groups, which were significantly lower than that in MWR group (all P<0.01).⑵After modeling 92d, NF-κB protein expression was 8.16±0.72 in MR group and significantly higher than that in MWR group (0.61±0.05) (P>0.05).Nevertheless, NF-κB protein expression were 4.26±0.56 and 3.82±0.42 in OMLZ and PWP group,repectively, and were significantly lower comparing with MR group (P<0.01). These proved that NF-κB expression increased and PWP could significantly inhibit NF-κB expression during the process of GU occurrence and its recurrence.2.2 The effect of PWP on IL-8 in gastric tissue and plasma during GU and its recurrence in rats.2.2.1 The cellular localization of positive cells of IL-8 protein expression Positive cells of IL-8 protein expression mainly located in the mucosal epithelial cells, and stained brownish yellow or brownish black. After modeling 16d, a little of IL-8 protein expressed in gastric mucosa and submucosa in normal and sham group rats, and expressed significantly more in MWR group. IL-8 protein expressed less in OMLZ group and PWP group compared with MWR group. After modeling 92d, few and similar IL-8 protein expression in MWR, normal and sham group.Meanwhile, IL-8 protein expression significantly increased in MR group comparing with MWR group, and significantly decreased in OMLZ and PWP group comparing with MR group.2.2.2 The comparion of positive cells of IL-8 expression by immunohistochemical method⑴After modeling 16d, IL-8 positive cells were rare, namely 10.63±0.04 and 10.91±0.71 in normal group and sham group, respectively. Moreover, there showed no significant difference between them (P>0.05). IL-8 positive cells was 41.73±0.04 in MWR group, and significantly increased comparing with normal and sham groups (all P <0.01).Nevertheless, they were 21.65±1.96 and 22.01±0.05, in OMLZ group and PWP group, and significantly lower than that in MWR group (P <0.01).⑵After modeling 92d, IL-8 positive cells were 11.45±0.79,12.15±0.56 and 12.92±0.72 in MWR group, normal group and sham group,respectively. There had no significant difference between them(P> 0.05). Positive cells in MR group significantly increased (61.71±1.43) comparing with that in MWR group(P<0.01). Positive cells in OMLZ group and PWP group were 38.21±3.07 and 37.06±1.82,which significantly decreased than that in MR group (all P<0.01).These showed that the PWP could decrease the positive cells expression of inflammatory cytokines IL-8 to reduce the GU relapse.2.2.3 The results of protein expression of IL-8 by Western-blotAccording to the results by Western blot protein quantification, we found that,⑴After modeling 16d, IL-8 protein expressions were 10.45±1.97 , 10.85±1.23,and 39.63±4.27 in normal group, sham group and MWR group,respectively,and that in MWR group was significantly increased comparing with normal group (P<0.01).Protein expressed 18.33±2.31 and 19.92±3.30 in OMLZ group and PWP group, which significantly decreased than that in MWR group (all P<0.01).⑵After modeling 92d, IL-8 protein expression in MWR group had no significant difference comparing with that in normal and sham group (all P>0.05),and was 50.22±3.05 in MR group, compared with MWR group (12.53±3.50), which significantly increased (P<0.01). IL-8 protein expression were 32.23±3.49 and 31.06±3.28 in OMLZ group and PWP group, which were significantly lower than that in MR group (all P<0.01). This manifested that PWP had role of resisting GU and its recurrence through reducing the expression of IL-8 protein.2.2.4 The effect of PWP on inflammatory factors IL-8 in plasma during GU recurrence in rats.Radioimmunoassay showed that plasma IL-8 was 0.48±0.07 ng/ml when GU was recurred and was significantly higher comparing with that in MWR group (0.14±1.18 ng/ml) (P<0.01). Plasma IL-8 were 0.36±0.43 ng/ ml and 0.34±0.03 ng/ml in OMLZ group and PWP group, and were significantly lower than that in MR group (P<0.01),which showed that PWP played a role of anti-GU relapse by reducing plasma IL-8.2.3 The effect of PWP on TNF-αin gastric tissue and plasma during GU and its recurrence in rats.2.3.1 The cellular localization of positive cells of TNF-αprotein expression After modeling 16d, none or small amount of TNF-αpositive cells located in cytoplasm at the bottom of gastric mucosa or in submucosa in normal group and sham group rats. TNF-αprotein expression in MWR group was significantly higher than that in normal group and sham group.In OMLZ group and PWP group they decreased than that in MWR group. At the same time, after modeling 92d, TNF-αprotein expressed similar in MWR group, normal group and sham roup. TNF-αprotein expressed mainly in the cytoplasm of mucosal ulcers and expressed more in MR group than in MWR group.Nevertheless, TNF-αprotein expressed less in OMLZ group and PWP than MR group.2.3.2 The comparion of positive cells of TNF-αexpression by immunohistochemical method⑴After modeling 16d, only a small amount of TNF-αpositive cells in normal group and sham group, which were 3.90±0.18 and 0.40±0.04 and it showed no significant difference between them(P>0.05). Compared with normal group, TNF-αpositive cells in MWR group significantly increased (21.59±4.58) (P<0.01), and compared with MWR group, that in OMLZ group and PWP group were 14.05±2.55 and 13.67±3.12 and significantly decreased (all P<0.01).⑵After modeling 92d, TNF-αpositive cells were no significant differences between MWR group, normal group and sham group (all P>0.05). Positive cells in MR group was 40.13±8.48, and compared with MWR group (0.44±0.05), significantly increased (P <0.01). Positive cells were 19.92±3.72 and 18.88±4.17 in OMLZ group and PWP group, respectively,and were significantly lower than that in MR group (all P<0.01). These showed that the PWP could decrease positive cells of the inflammatory cytokines TNF-αexpression toresisting GU and its recurrence.2.3.3 The results of protein expression of TNF-αby Western-blot The results of Western blot showed that:⑴After modeling 16d, TNF-αprotein expression was 19.80±4.09 in MWR group, and compared with normal and sham group, significantly increased (all P<0.01). TNF-αprotein expression ,in OMLZ group and the PWP group, were 13.02±2.58 and 12.36±2.49,respectively,and compared with that in MWR, was significantly lower (P<0.01).⑵After modeling 92d, TNF-αprotein expression protein had no significant difference among MWR group, the normal and sham group (P>0.05). TNF-αprotein expression was 34.64±5.36 in MR group and significantly increased than that in MWR group (0.38±0.04) (P<0.01). Meanwhile, TNF-αprotein expression in OMLZ group and PWP group were 17.43±2.35 and 16.96±3.30,which significantly decreased comparing with that in MR group (all P<0.01). This showed that PWP had role of resisting GU and its recurrence through reducing the expression of TNF-αprotein.2.3.4 The effect of PWP on inflammatory factors TNF-αin plasma during GU recurrence in rats.The plasma TNF-αwas 23.74±2.70 fmol/ml in MR group when GU recurred,and compared with MWR group (12.07±1.73 fmol/ml) ,was significantly higher (P<0.01). The plasma TNF-αin OMLZ group and PWP group were 14.00±2.02 fmol/ml and 13.55±1.95 fmol/ml, and were significantly lower than that in MR group (all P<0.01). This provided that PWP played a role of resisting GU relapse by reducing plasma TNF-α.Conclusions:1. PWP has good role of resisting GU and its recurrence,which is related with the model of acetic.2. PWP plays the role of resisting GU and its recurrence by inhibiting the activation or the expression of some inflammatory cytokines, such as NF-κB, IL-8 and TNF-α.
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