| Schistosomiasis, a serious parasitic diseases, is impact human health social and economic development and prevalent in 76 countries. It has long been looking for a method to effectively control and eradication of schistosomiasis, but the geographical or economic conditions in some areas restricted to carry out the eradication of snail and universal vaccination. Therefore, chemical drugs are the primary means of control of schistosomiasis. Currently, schistosomiasis control is to reduce the worldwide incidence. Chemical drug praziquantel(PZQ) is highly effective against all species of schistosomes pathogenic to humans and has been designated as the best schistosomicidal agent by WHO. In recent years, however, the potential tolerance or resistance problem of schistosomes to PZQ has become an issue, which necessitates the searching for alternatives.Pulsatilla is the dried root of Pulsatilla chinensis(Bge.) Regel, which exhibits “blood-cooling”, detoxification activities, anti-tumor, anti-inflammatory, anti-pathogenic microorganisms, and anti-parasitic effects. It used for the clinical management of amoebic dysentery, gynecological inflammation and other diseases. Pulsatilla saponins have the antiparasitic effects on Schistosoma eggs, miracidia and cercariae and can reduce the glycogen, protein and enzymes related of metabolism of Schistosoma japonicum. This paper is aim to study the effect of Pulsatilla saponin extracts Hederacolchiside A(HSA) against S. japonicum and S. mansoni of different developmental stages in vivo, the influence on the skin and metabolism of adult S. japonicum and the immunomodulatory effect on mice infected with S. japonicum and S. mansoni, providing theoretical and experimental basis for HSA becomes new antischistosomal drug.First, the mice were infected by S. japonicum by direct skin contact through exposure to 55 ± 5 cercariae per mouse. These mice were administered with different medications in different developmental stages of S. japonicum(1d, 7d, 14 d, 21 d, 28 d, 35d), as follows, 8 mg/kg HSA or 6 mg/kg HSA by intraperitoneal injection of 5 consecutive days, 300 mg/kg PZQ or 300 mg/kg ART by oral. To detect the effects of different drugs on different developmental stages of S. japonicum, the mice were sacrificed on the 7 weeks after infection. Different doses of HSA were administered with different periods showed mortality effects on the S. japonicum in vivo. 8mg/kg HSA given at 1d, 7d after infection, the total worm reduction rate was 97.2% and 92.9%, respectively; the female worm reduction rate was 94.7% and 89.5%, respectively; and the egg reduction rate of 99.3% and 98.7%, respectively. The schistosomicidal effects of HSA were superior to the same period of the positive control PZQ and artesunate(ART) treatment group. Although the schistosomicidal effects of HSA administrated on the other period is less than the positive control group PZQ or ART, the schistosomicidal effects of HSA on the total worm burdens, female worm burdens and the egg burdens in liver were significantly reduced compared to the control group.Second, the mice were infected by 45 ± 5 S. mansoni cercariae per mouse. These mice were administered with different medications in different developmental stages of S. mansoni(1d, 7d, 21 d, 42 d, 49d), as follows, 8 mg/kg HSA by intraperitoneal injection of 5 consecutive days, 300 mg/kg PZQ or 300 mg/kg ART by oral. To detect the effects of different drugs on different developmental stages of S. mansoni, the mice were sacrificed on the 8 weeks after infection. HSA were administered with different periods showed mortality effects on the S. mansoni in vivo. 8mg/kg HSA given at 1d, 7d after infection, the total worm reduction rate was 88.6% and 80.7%, respectively; the female worm reduction rate was 92.9% and 85.0%, respectively; and the egg reduction rate of 98.6% and 95.3%, respectively. The schistosomicidal effects of HSA were superior to the same period of the positive control PZQ and ART treatment group. Although the schistosomicidal effects of HSA administrated on the other period is less than the positive control group PZQ or ART, the schistosomicidal effects of HSA on the total worm burdens, female worm burdens and the egg burdens in liver were significantly reduced compared to the control group. The excellent schistosomicidal effects of HSA against S. japonicum and S. mansoni at in 1d and 7d affter infection, which were superior to PZQ and ART, is expected to become a new anti-schistosome drug.To further explore the effect of HSA on inflammation and granuloma caused by schistosomiasis, we detected the body weight, spleen weight and spleen/body weight of the mice the expression levels of immunoglobulin(Ig G) and tumor necrosis factor(TNF)-α、interleukin(IL)-4, IL-17 a in mice and liver morphology, size of the granuloma and impact of liver fibrosis in mice which were administered with different medications in different developmental stages of S. japonicum and S. mansoni. The results showed that HSA treatment can significantly improve the body weight of mice, significantly reduced spleen/body weight of mice and significantly reduced the expression levels of TNF-α and IL-17 a at 1-21 d after S. japonicum infection. Low dose of HSA therapy can significantly decrease the expression of IL-4 at 1-35 d after S. japonicum infection and the expression of Ig G at 7d, 14 d, 21 d after S. japonicum infection. HSA treatment can significantly improve the body weight of mice and significantly reduced spleen/body weight of mice infected S. mansoni. HSA treatment can significantly reduce the expression levels of IL-4 at 1-21 d after S. mansoni infection. 1d, 7d, 21 d, 42 d after infection given HSA treatment can significantly decrease the expression of TNF-α, and 1d, 7d, 21 d, 49 d after infection given HSA treatment significantly inhibited the expression of IL-17 a. However, HSA treatment can significantly reduce the expression levels of immunoglobulin Ig G at 42 d or 49 d after S. mansoni infection. The earlier HSA treatment to give the more obvious effect on granuloma in mice infected with S. japonicum and S. mansoni. HSA treatment with different doses can significantly reduce the average diameter of the granuloma in all infected mice. By administration of HSA at days 1 or 7, the number of inflammatory cells in the infected liver was significantly reduced, the hepatic lobular architecture restored its normal organization and most hepatocytes showed normal appearance. By administration of HSA at days 1 or 7, low expression of transforming growth factor-β1(TGF-β1), transforming growth factor-β1(TIMP-1) and collagen typeⅠwere observed in mice infected with S. japonicum and S. mansoni. Early administration of HSA on mice infected with S. japonicum and S. mansoni can significantly reduce the granulomatous inflammation and inhibit fibrosing inflammation, which implies that HSA may be hope to become the new drug which is prevention and adjuvant therapy of schistosomiasis.Finally, we detected the effects of HSA on adult S. japonicum in vitro, and observed schistosome epidermis morphological changes using a scanning electron microscope scanning. Mice, infected S. japonicum for 42 d, treated with 8 mg/kg HSA, and then killed after intraperitoneal injection 24 h. The changes of glycogen, protein, acidic phosphatase, alkaline phosphatase and superoxide dismutase in adult S. japonicum treated with HSA were detected to explore the mechanism of effects of HSA on S. japonicum. The results showed that the drug sensitivity of parasites in a concentration-dependent and time-dependent manner. The 100% lethal concentration of HSA for 12 h was 0.5 μg/ml, however, The 100% lethal concentration of PZQ for 12 h required 30 μg/ml. The 100% lethal concentration of HSA is less than the positive control group PZQ at the same time. Adult female and male S. japonicum incubated with HSA for 4h, the epidermis sponge-like structure arranged confusion, the inter-tubercle tegumental regions showed extensive erosion and disorganization and the peeling of damaged tegument resulted in the exposure of subtegument tissues. Glycogen, protein, alkaline phosphatase, acid phosphatase and superoxide dismutase of S. japonicum were significantly decreased after treated with HSA for 24 h compared with the untreated control group. These results demonstrated that HSA may have some effects on the metabolism and antioxidant capacity of S. japonicum, and ultimately been killed through the host’s immune defenses. |
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